2000年

2000

Wheat germ translation initiation factor eIF4B affects eIF4A and eIFiso4F helicase activity by increasing the ATP binding affinity of eIF4A.

Goss, Dixie J.; Bi, Xiping; Ren, Jianhua

Biochemistry, VOL. 39, NO. 19, May 16, 2000, PP. 5758-5765

It has been proposed that, during translational initiation, structures in the 5' untranslated region of mRNA are unwound. eIF4A, a member of the DEAD box family of proteins (those that contain a DEAD amino acid sequence), separately or in conjunction with other eukaryotic initiation factors, utilizes the energy from ATP hydrolysis to unwind these structures. As a step in defining the mechanism of helicase activity in the wheat germ protein synthesis system, we have utilized direct fluorescence measurements, ATPase assays, and helicase assays. The RNA duplex unwinding activity of wheat germ eIF4A is similar to other mammalian systems; however, eIF4F or eIFiso4F is required, probably because of the low binding affinity of wheat germ eIF4A for mRNA. Direct ATP binding measurements showed that eIF4A had a higher binding affinity for ADP than ATP, resulting in a limited hydrolysis and procession along the RNA in the helicase assay. The addition of eIF4B resulted in a change in binding affinity for ATP, increasing it almost 10-fold while the ADP binding affinity was approximately the same. The data presented in this paper suggest that eIF4F or eIFiso4F acts to position the eIF4A and stabilize the interaction with mRNA. ATP produces a conformational change which allows a limited unwinding of the RNA duplex. The binding of eIF4B either prior to or after hydrolysis allows for increased affinity for ATP and for the cycle of conformational changes to proceed, resulting in further unwinding and processive movement along the mRNA.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *translation initiation factor eIFiso4F --analysis; *translation initiation factor eIFiso4F --eukaryotic; *translation initiation factor eIFiso4F --helicase activity; *translation initiation factor eIF4A --analysis; *translation initiation factor eIF4A --eukaryotic; *translation initiation factor eIF4A --ATP binding affinity; *translation initiation factor eIF4B --analysis; *translation initiation factor eIF4B --wheat germ; *translation initiation factor eIF4B --RNA duplex unwinding activity; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *helicase assay --bioassay method; *helicase assay --Bioassays/Physiological Analysis; *ATPase assay --bioassay method; *ATPase assay --Bioassays/Physiological Analysis; *Spex Fluorolog 2 spectrofluorometer --equipment

2000

Three-Dimensional Fluorescence Microscopy of Endothelial Cells Labeled with Coumarins.

Geoffroy-Chapotot, C., Carre, M,C., Baros, F., Muller, S., Dumas, D., and Stoltz, J. F.;

Journal of Fluorescence, (June, 2000), Volume 10, Number 2, pp. 203-207.

2000

Thermodynamics of substrate binding to the chaperone SecB.

Varadarajan, Raghavan; Panse, Vikram G.; Surolia, Avadhesha; Swaminathan, Chittoor P.

Biochemistry, VOL. 39, NO. 9, March 7, 2000, PP. 2420-2427

The thermodynamics of binding of unfolded polypeptides to the chaperone SecB was investigated in vitro by isothermal titration calorimetry and fluorescence spectroscopy. The substrates were reduced and carboxamidomethylated forms of RNase A, BPTI, and alpha-lactalbumin. SecB binds both fully unfolded RNase A and BPTI as well as compact, partially folded disulfide intermediates of alpha-lactalbumin, which have 40-60% of native secondary structure. The heat capacity changes observed on binding the reduced and carboxamidomethylated forms of alpha-lactalbumin, BPTI, and RNase A were found to be -0.10, -0.29, and -0.41 kcal mol-1 K-1, respectively, and suggest that between 7 and 29 residues are buried upon substrate binding to SecB. In all cases, binding occurs with a stoichiometry of one polypeptide chain per monomer of SecB. There is no evidence for two separate types of binding sites for positively charged and hydrophobic ligands. Spectroscopic and proteolysis protection studies of the binding of SecB to poly-L-Lys show that binding of highly positively charged peptide ligands to negatively charged SecB leads to charge neutralization and subsequent aggregation of SecB. The data are consistent with a model where SecB binds substrate molecules at an exposed hydrophobic cleft. SecB aggregation in the absence of substrate is prevented by electrostatic repulsion between negatively charged SecB tetramers.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Escherichia coli (Enterobacteriaceae); *Bacteria; *Eubacteria; *Microorganisms; *SecB --analysis; *SecB --molecular chaperone; *SecB --substrate binding thermodynamics; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: CB; *isothermal titration calorimetry --analytical method; *isothermal titration calorimetry --Analysis/Characterization Techniques: CB; *proteolysis reaction --chemical method; *proteolysis reaction --Synthesis/Modification Techniques; *JASCO 720 spectropolarimeter --equipment; *Microcal Omega Titration Calorimeter --equipment; *Spex Fluorolog-2 fluorimeter --equipment

2000

The effect of sterol structure on membrane lipid domains reveals how cholesterol can induce lipid domain formation.

London, Erwin .; Xu, Xiaolian

Biochemistry, VOL. 39, NO. 5, Feb. 8,. 2000, PP. 843-849

Detergent-insoluble membrane domains, enriched in saturated lipids and cholesterol, have been implicated in numerous biological functions. To understand how cholesterol promotes domain formation, the effect of various sterols and sterol derivatives on domain formation in mixtures of the saturated lipid dipalmitoylphosphatidylcholine (DPPC) and a fluorescence quenching analogue of an unsaturated lipid was compared. Quenching measurements demonstrated that several sterols (cholesterol, dihydrocholesterol, epicholesterol, and 25-hydroxycholesterol) promote formation of DPPC-enriched domains. Other sterols and sterol derivatives had little effect on domain formation (cholestane and lanosterol) or, surprisingly, strongly inhibit it (coprostanol, androstenol, cholesterol sulfate, and 4-cholestenone). The effect of sterols on domain formation was closely correlated with their effects on DPPC insolubility. Those sterols that promoted domain formation increased DPPC insolubility, whereas those sterols that inhibit domain formation decreased DPPC insolubility. The effects of sterols on the fluorescence polarization of diphenylhexatriene incorporated into DPPC-containing vesicles were also correlated with sterol structure. These experiments indicate that the effect of sterol on the ability of saturated lipids to form a tightly packed (i.e., tight in the sense that the lipids are closely packed with one another) and ordered state is the key to their effect on domain formation. Those sterols that promote tight packing of saturated lipids promote domain formation, while those sterols that inhibited tight packing of saturated lipids inhibited domain formation. The ability of some sterols to inhibit domain formation (i.e., act as "anti-cholesterols") should be a valuable tool for examining domain formation and properties in cells..

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques.; *androstenol; *cholestane; *cholesterol; *cholesterol sulfate; *coprostanol; *dihydrocholesterol; *dipalmitoylphosphatidylcholine; *epicholesterol; *lanosterol; *membrane lipid domains; *sterol.; *25-hydroxycholesterol; *4-cholestenone; *fluorescence emission spectrometry; *quenching measurements; *thin-layer chromatography.; *Beckman 650 spectrophotometer; *Spex 212 Fluorolog fluorimeter; *lipid domain formation; *protein structure

2000

The Amino Terminus of the Fourth Cytoplasmic Loop of Rhodopsin Modulates Rhodopsin-Transducin Interaction.

Marin, Ethan, P., Krishna, A. Gopala, Zvyaga, Tatyana A., Isele, Juergen, Siebert, Friedrich, and Sakmar Thomas P.;

The Journal of Biological Chemistry, Vol. 275, No, 3, Issue of January 21, pp. 1930-1936, 2000

2000

Structural transition at actin's N-terminus in the actomyosin cross-bridge cycle.

Reisler, Emil; Hansen, James E.; Marner, Joyce; Pavlov, Dimitry; Rubenstein, Peter A.

Biochemistry, VOL. 39, NO. 7, Feb. 22, 2000, PP. 1792-1799

Force and motion generation by actomyosin involves the cyclic formation and transition between weakly and strongly bound complexes of these proteins. Actin's N-terminus is believed to play a greater role in the formation of the weakly bound actomyosin states than in the formation of the strongly bound actomyosin states. It has been the goal of this project to determine whether the interaction of actin's N-terminus with myosin changes upon transition between these two states. To this end, a yeast actin mutant, Cys-1, was constructed by the insertion of a cysteine residue at actin's N-terminus and replacement of the C-terminal cysteine with alanine. The N-terminal cysteine was labeled stoichiometrically with pyrene maleimide, and the properties of the modified mutant actin were examined prior to spectroscopic measurements. Among these properties, actin polymerization, strong S1 binding, and the activation of S1 ATPase by pyrenyl-Cys-1 actin were not significantly different from those of wild-type yeast actin, while small changes were observed in the weak S1 binding and the in vitro motility of actin filaments. Fluorescence changes upon binding of S1 to pyrenyl-Cys-1 actin were measured for the strongly (with or without ADP) and weakly (with ATP and ATPgammaS) bound acto-S1 states. The fluorescence increased in each case, but the increase was greater (by about 75%) in the presence of MgATP and MgATPgammaS than in the rigor state. This demonstrates a transition at the S1 contact with actin's N-terminus between the weakly and strongly bound states, and implies either a closer proximity of the pyrene probe on Cys-1 to structural elements on S1 (most likely the loop of residues 626-647) or greater S1-induced changes at the N-terminus of actin in the weakly bound acto-S1 states.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *actin's N-terminus --assay; *actin's N-terminus --labeling; *electron microscopy --microscopy method; *electron microscopy --microscopy: CB; *motility assay --analytical method; *motility assay --Analysis/Characterization Techniques: CB; *pyrene maleimide labeling --labeling method; *pyrene maleimide labeling --Detection/Labeling Techniques; *spectrofluorometry --analytical method; *spectrofluorometry --fluorophotometry: CB; *ATPase assay --analytical method; *ATPase assay --Analysis/Characterization Techniques: CB; *Spex Fluorolog spectrofluorometer --equipment

2000

Spectroscopic Characterization of Porphyrin-Doped Sol-Gel-Derived Matrices.

Hungerford, G., Ferreira, M. I C, Pereira, M. R., Ferreira, J, A., and Coelho, A. F.;

Journal of Fluorescence, (September, 2000), Volume 10, Number 3, pp. 283-290.

2000

Solvent Exchange in Excited-State Relaxation in Mixed Solvents.

Koti, A. S., and Periasamy, N.;

Journal of Fluorescence, (June, 2000), Volume 10, Number 2, pp. 177-184.

2000

Solvation and Solvent Relaxation in Swellable Copolymers as Studied by Time-Resolved Fluorescence Spectroscopy.

Egelhaaf, H.-J., Lehr, B., Hof, M., Hafner, A., Fritz, H, Schneider, F. W., Bayer, E. and Oelkrug, D.;

Journal of Fluorescence, (December, 2000), Volume 10, Number 4, pp. 383-392.

2000

Solid-matrix luminescence of heterocyclic aromatic amines in glucose glasses prepared from a glucose melt.

Hurtubise, Robert J.; Mendonsa, Shaun D.

Applied Spectroscopy, VOL. 54, NO. 3, March, 2000, PP. 456-459

NO-ABSTRACT

DESCRIPTOR(S)- *Chemistry; *Methods and Techniques; *Pollution Assessment Control and Management; *Toxicology; *glucose --analysis; *glucose glasses --analysis; *heterocyclic aromatic amines --analysis; *solid-matrix luminescence spectrometry --analytical method; *solid-matrix luminescence spectrometry --applications; *solid-matrix luminescence spectrometry --Spectrum Analysis Techniques; *Spex Fluorolog 2 spectrometer --equipment; *Spex Fluorolog 2 spectrometer --uses; *Spex Fluorolog 2 spectrometer --Instruments SA Inc.; *environmental toxicology

2000

Simultaneous Registration of Contraction and Cytosolic Calcium ([Ca2+] I) of Smooth Muscle Strips Using Front-Surface Fluorometry.

Ferreira, Alice T., Neri, Rogerio, Oshiro, Maria E, M., and Kanaide Hideo;

Journal of Fluorescence, (September, 2000), Volume 10, Number 3, pp. 223-228.

2000

Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin.

Glasgow, Ben J.; Abduragimov, Adil R.; Gasymov, Oktay K.; Yusifov, Taleh N.

Protein Science, VOL. 9, NO. 2, Feb., 2000, PP. 325-331

The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loopand show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is held in a static position in the calyx, but rather suggest that the ligand is in motion. The combination of site-directed tryptophan fluorescence with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and provides dynamic information that is not attainable by X-ray crystallography.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *lipocalin --analysis; *lipocalin --purification; *tryptophan; *circular dichroism --analytical method; *circular dichroism --spectroscopic techniques: CB; *column purification --purification method; *column purification --Isolation/Purification Techniques: CB; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --fluorescence spectrometry; *site-directed mutagenesis --genetic engineering method; *site-directed mutagenesis --protein engineering; *EPR spectra --analytical method; *EPR spectra --optical analysis; *Jasco 600 spectropolarimeter --equipment; *Jobin Yvon-SPEX Fluorolog-3 spectrofluorometer --equipment

2000

Permeability of yeast cell envelope to fluorescent galactosylated telomers derived from THAM.

Bonaly, R.; Contino, C.; Coulon, J.; Polidori, A.; Pucci, B.; Thiebault, F.

Bioconjugate Chemistry, VOL. 11, NO. 4, July-August, 2000, PP. 461-468

The work reported herein deals with the study of cellular recognition and permeability phenomena in yeasts. Various galactosylated organic telomers derived from trishydroxymethyl-aminomethane (THAM) and bearing fluorescent moieties were synthesized in order to measure their ability to cross the yeast cell envelope. Grafting fluorescent probes on the organic telomer backbone allowed us to study their specific behaviors toward the yeasts by fluorescence microscopy. Yeasts belonging to the genera Kluyveromyces and Saccharomyces were used for this study. With Saccharomyces yeast cells bearing mannose-specific lectins or lectin-like proteins, on their outer surface, all the galactosylated or nongalactosylated organic telomers passed through the cell envelope and invaded the cytoplasm. With Kluyveromyces yeast cells bearing galactose-specific lectins, the galactosylated organic telomers were blocked at the outer surface while the nongalactosylated derivatives crossed the cell envelope. Moreover, preincubation of Kluyveromyces yeasts with galactose or methylgalactose inhibited the cell surface anchorage of the organic telomers and allowed their penetration into the cytoplasm. When assays were performed on spheroplasts of both Kluyveromyces and Saccharomyces yeasts, no fixation on the surface could be observed, and all the derivatives went through the membrane and invaded the cytoplasm.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Membranes (Cell Biology); *Methods and Techniques; *Kluyveromyces bulgaricus yeast (Ascomycetes); *Kluyveromyces lactis yeast (Ascomycetes); *Saccharomyces cerevisiae yeast (Ascomycetes); *Fungi; *Microorganisms; *Nonvascular Plants; *Plants; *cell envelope; *spheroplasts; *galactose; *galactosylated telomers --fluorescent moieties; *lectin-like proteins; *mannose-specific lectins; *methylgalactose; *trishydroxymethyl-aminomethane; *fluorescence microscopy --analytical method; *fluorescence microscopy --confocal laser microscopy: CB; *fluorescence microscopy --light microscopy: CB; *fluorescence microscopy --light microscopy: CT; *growth assay --analytical method; *growth assay --Bioassays/Physiological Analysis; *spectrophotometer --laboratory equipment; *spectrophotometer --Perkin-Elmer; *toxicity assay --analytical method; *toxicity assay --Bioassays/Physiological Analysis; *Axioscope 50 microscope --laboratory equipment; *Axioscope 50 microscope --Zeiss; * Fluorolog II spectrofluorimeter --laboratory equipment; * Fluorolog II spectrofluorimeter --SPEX; *MC80 camera --laboratory equipment; *MC80 camera --Zeiss; *cellular recognition

2000

On the role of the carboxyl-terminal helix of RXR in the interactions of the receptor with ligand.

Budhu, Anuradha S.; Noy, N

Biochemistry, VOL. 39, NO. 14, April 11, 2000, PP. 4090-4095

The retinoid X receptor (RXR), a ligand-inducible transcription factor that is activated by 9-cis-retinoic acid, is a member of the superfamily of nuclear hormone receptors. The ligand-induced transcriptional activity of nuclear receptors is coordinated by their C-terminal region termed the ligand-binding domain. Structural analyses of several nuclear receptors showed that the most dramatic ligand-induced conformational change in these proteins involves a positional shift in the receptors' C-terminal helix, termed helix 12. Consequently, in the liganded state, helix 12 is folded over the entrance to the ligand-binding pocket where it serves as a lid, and it has been proposed that this region functions to stabilize ligand binding by at least some nuclear receptors. Here, to examine the possible role of helix 12 in contributing to the association of RXR with its ligand, the equilibrium and kinetic parameters of the interactions of 9-cis-retinoic acid with RXR and with a deletion mutant lacking helix 12 were measured. Deletion of the region did not significantly alter the ligand-binding affinity of RXR at equilibrium. However, both the rate of dissociation and the rate of association of the RXR-9-cis-retinoic acid complex were significantly slower in the absence of helix 12. Taken together, these observations suggest that helix 12 of RXR facilitates both the entry and the exit of the ligand from the binding pocket without affecting the equilibrium ligand-binding affinity. The results thus point at a previously unsuspected function for this region.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *dioleoylphosphatidylcholine --Avanti Polar Lipids; *retinoid X receptor --carboxyl-terminal helix; *retinoid X receptor --ligand-inducible transcription factor; *9-cis-retinoic acid; *fluorescence titrations --analytical method; *structural analysis --analytical method; *structural analysis --Molecular Biology Techniques and Chemical Characterization; * Fluorolog-2 spectrofluorometer --laboratory equipment; * Fluorolog-2 spectrofluorometer --SPEX Industries; *PCR amplification polymerase chain reaction amplification --amplification method; *PCR amplification polymerase chain reaction amplification --Molecular Biology Techniques and Chemical Characterization; *receptor-ligand interaction

2000

On the Intramolecular Eximer Formation of 1,10-Bis(1-Pyrene)Decane in Organized Media.

Vasilescu, Marilens, Almgren, Mats, and Angelescu, Daniel;

Journal of Fluorescence, (December, 2000), Volume 10, Number 4, pp. 339-346.

2000

On the improvement of solid-phase extraction room-temperature phosphorimetry for the analysis of polycyclic aromatic hydrocarbons in water samples.

Campiglia, Andres D.; Arruda, Andrea F.; Hagestuen, Erik D.

Talanta, VOL. 52, NO. 4, 31 July, 2000, PP. 727-737

This article presents considerable improvements in SPE-RTP methodology for the analysis of polycyclic aromatic hydrocarbons in water samples. Bulky glassware and vacuum pump were removed from the previous extraction procedure (E.D. Hagestuen, A.D. Campiglia, Talanta 49 (1998) 547) by processing 10 ml of sample with syringes and stainless steel filter holders. The oven-drying step was substituted by air sample drying, which employed positive pressure to remove water excess from the extraction membrane. As a result, analysis times from 8 to 10 min per sample were obtained. No costs in levels of detection were observed. Limits of detection at the pg ml-1 level were estimated for phenanthrene, pyrene, benzo(k)fluoranthene, benzo(a)pyrene, and chrysene. The feasibility of identifying single components in PAH mixtures was investigated. Binary and tertiary mixtures were resolved by appropriate choice of excitation and emission wavelengths. The advantage of using excitation-emission matrix analysis was demonstrated for a six-component mixture.

DESCRIPTOR(S)- *Chemistry; *Methods and Techniques; *Pollution Assessment Control and Management; *benzo(a)pyrene --analysis; *benzo(a)pyrene --extraction; *benzo(k)fluoranthene --analysis; *benzo(k)fluoranthene --extraction; *chrysene --analysis; *chrysene --extraction; *phenanthrene --analysis; *phenanthrene --extraction; *polycyclic aromatic hydrocarbons --analysis; *polycyclic aromatic hydrocarbons --extraction; *model FL3-11 Fluorolog-3 spectrofluorimeter --equipment; *model FL3-11 Fluorolog-3 spectrofluorimeter --ISA; *solid-phase extraction room-temperature phosphorimetry --analytical method; *solid-phase extraction room-temperature phosphorimetry --extraction method; *solid-phase extraction room-temperature phosphorimetry --Bioassays/Physiological Analysis; *solid-phase extraction room-temperature phosphorimetry --Extraction; *solid-phase extraction room-temperature phosphorimetry --Isolation; *solid-phase extraction room-temperature phosphorimetry --Purification and Separation Techniques; *water samples

2000

Octamers of mitochondrial creatine kinase isoenzymes differ in stability and membrane binding.

Schlattner, Uwe; Wallimann, Theo,

Journal of Biological Chemistry, VOL. 275, NO. 23, June 9, 2000, PP.17314-17320

Octamer stability and membrane binding of mitochondrial creatine kinase (MtCK) are important for proper functioning of the enzyme and were suggested as targets for regulatory mechanisms. A quantitative analysis of these properties, using fluorescence spectroscopy, gel filtration, and surface plasmon resonance, revealed substantial differences between the two types of MtCK isoenzymes, sarcomeric (sMtCK) and ubiquitous (uMtCK). As compared with human and chicken sMtCK, human uMtCK showed a 23-34 times slower octamer dissociation rate, a reduced reoctamerization rate and a superior octamer stability as deduced from the octamer/dimer ratios at thermodynamic equilibrium. Octamer stability of sMtCK increased with temperature up to 30 degreeC, indicating a substantial contribution of hydrophobic interactions, while it decreased in the case of uMtCK, indicating the presence of additional polar dimer/dimer interactions. These conclusions are consistent with the recently solved x-ray structure of the human uMtCK (Eder, M., Fritz-Wolf, K., Kabsch, W., Wallimann, T., and Schlattner, U. (200) Proteins 39, 216-225). When binding to 16% cardiolipin membranes, sMtCK showed slightly faster on-rates and higher affinities than uMtCK. However, human uMtCK was able to recruit the highest number of binding sites on the vesicle surface. The observed divergence of ubiquitous and sarcomeric MtCK is discussed with respect to their molecular structures and the possible physiological implications.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Membranes (Cell Biology); *Methods and Techniques; *mitochondrial creatine kinase isoenzymes --analysis; *mitochondrial creatine kinase isoenzymes --membrane binding; *mitochondrial creatine kinase isoenzymes --separation; *sarcomeric mitochondrial creatine kinase --analysis; *sarcomeric mitochondrial creatine kinase --membrane binding; *sarcomeric mitochondrial creatine kinase --separation; *ubiquitous mitochondrial creatine kinase --analysis; *ubiquitous mitochondrial creatine kinase --membrane binding; *ubiquitous mitochondrial creatine kinase --separation; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: CB; *gel filtration chromatography --column chromatographic techniques; *gel filtration chromatography --separation method; *surface plasmon resonance --analytical method; *surface plasmon resonance --membrane binding assay; *surface plasmon resonance --Spectrum Analysis Techniques; *BIAcore 2000 instrument --equipment; *SPEX Fluorolog-2 instrument --equipment

2000

Nanosecond dynamics of tryptophans in different conformational states of apomyoglobin proteins.

Tcherkasskaya, Olga; Knutson, Jay R.; Ptitsyn, Oleg B.

Biochemistry, VOL. 39, NO. 7, Feb. 22, 2000, PP. 1879-1889

Fluorescence anisotropy kinetics were employed to quantify the nanosecond mobility of tryptophan residues in different conformational states (native, molten globule, unfolded) of apomyoglobins. Of particular interest is the similarity between the fluorescence anisotropy decays of tryptophans in the native and molten globule states. We find that, in these compact states, tryptophan residues rotate rapidly within a cone of semiangle 22-25degree and a correlation time of 0.5 ns, in addition to rotating together with the whole protein with a correlation time of 7-11 ns. The similar nanosecond dynamics of tryptophan residues in both states suggests that the conformation changes that distinguish the molten globule and native states of apomyoglobins originate from either subtle, slow rearrangements or fast changes distant from these tryptophans.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *apomyoglobin --analysis; *tryptophan; *fluorescence spectra --analytical method; *fluorescence spectra --optical analysis; *spectrofluorometry --analytical method; *spectrofluorometry --fluorophotometry: CB; *SPEX Fluorolog-2 spectrofluorometer --equipment

2000

Nanosecond dynamics of the single tryptophan reveals multi-state equilibrium unfolding of protein GB1.

Tcherkasskaya, Olga; Bowley, Sara A.; Frank, M. Kirsten; Gronenborn, Angela M.; Knutson, Jay R.

Biochemistry, VOL. 39, NO. 39, September 19, 2000, PP. 11216-11226

Unfolding of the immunoglobulin binding domain B1 of streptococcal protein G (GB1) was induced by guanidine hydrochloride (GdnHCl) and studied by circular dichroism, steady-state, and time-resolved fluorescence spectroscopy. The fluorescence methods employed the single tryptophan residue of GB1 as an intrinsic reporter. While the transitions monitored by circular dichroism and steady-state fluorescence coincided with each other, the transitions followed by dynamic fluorescence were markedly different. Specifically, fluorescence anisotropy data showed that a relaxation spectrum of tryptophan contained a slow motion with relaxation times of 9 ns in the native state and 4 ns in the unfolded state in 6 M GdnHCl. At intermediate GdnHCl concentrations of 3.8-4.2 M, however, the slow relaxation time increased to 18 ns. The fast nanosecond motion had an average time of 0.8 ns and showed no dependence on the formation of native structure. Overall, dynamic fluorescence revealed two preliminary stages in GB1 folding, which are equated with the formation of local structure in the beta3-strand hairpin and the initial collapse. Both stages exist without alpha-helix formation, i.e., before the appearance of any ordered secondary structure detectable by circular dichroism. Another stage in GB1 folding might exist at very low (apprx1 M) GdnHCl concentrations.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *guanidine hydrochloride --reagent; *guanidine hydrochloride --U.S. Biochemicals Inc.; *G protein --analysis; *G protein --immunoglobulin binding domain B1; *G protein --multi-state equilibrium unfolding; *G protein --nanosecond dynamics; *G protein --tryptophan residue; *circular dichroism spectroscopy --analytical method; *circular dichroism spectroscopy --Spectrum Analysis Techniques; *steady-state spectroscopy --analytical method; *steady-state spectroscopy --Spectrum Analysis Techniques; *time-resolved fluorescence spectroscopy --analytical method; *time-resolved fluorescence spectroscopy --Spectrum Analysis Techniques; *J-720 spectropolarimeter --equipment; *J-720 spectropolarimeter --JASCO; *SPEX Fluorolog-2 spectrofluorometer --equipment

2000

Monochromator Wavelength Calibration Standards Extending into the Near-Infrared Using Second and Third-Order Emission Lines from Mercury Vapor Lamps.

Bare, William D. and Demas, J. N.;

Journal of Fluorescence, (December, 2000), Volume 10, Number 4, pp. 317-324.

2000

Monitoring the Phase Transition of C12E5/Water/Alkane Microemulsions Through Eximer Formation.

Real Oliveira, M. E. C D., Hungerford, Graham, Castanheira, E. M. S., Miguel, M. da G and Burrows,H.D.;

Journal of Fluorescence, (December, 2000), Volume 10, Number 4, pp. 347-353.

2000

Modulation of pig kidney Na+/K+-ATPase activity by cholesterol: Role of hydration.

Sotomayor, Carlos P.; Aguilar, Luis F.; Cuevas, Francisco J.; Helms, Michael K.; Jameson, David M.

Biochemistry, VOL. 39, NO. 35, September 5, 2000, PP. 10928-10935

Cholesterol is known to affect the activity of membrane-bound enzymes, including Na+/K+-ATPase. To gain insight into the mechanism of cholesterol's effect, we have used various hydrophobic fluorescent probes which insert into different regions of the membrane bilayer and report on the degree of hydration of their environment. Specifially, we have measured the generalized polarization of Laurdan and the lifetime of DPH and derivatives of DPH inserted into membranes from pig kidneys enriched in Na+/K+-ATPase. Spectral measurements were also carried out on these membranes after modification of their cholesterol content. The generalized polarization of Laurdan increased with increasing cholesterol, showing an abrupt modification at the native cholesterol content. The fluorescence lifetimes of DPH and the DPH derivatives were analyzed using a distribution model. The center value of these lifetime distributions and their widths also changed with increasing cholesterol. One DPH derivative, DPH-PC, showed a minimum value for the lifetime center at the native cholesterol concentration, whereas the other derivatives showed a maximum value for the lifetime center at that cholesterol concentration. DPH-PC is known to sense the protein-lipid interface, whereas the other derivatives sense the bulk lipid phase. These data suggest that hydration at the protein-lipid interface is maximal at the native cholesterol concentration as is the enzymatic activity. Hydration at the protein-lipid interface is therefore proposed to be required for activity. These results are in agreement with current models of membrane dynamics and thermodynamics of protein function.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Urinary System (Chemical Coordination and Homeostasis); *pig (Suidae); *Animals; *Artiodactyls; *Chordates; *Mammals; *Nonhuman Mammals; *Nonhuman Vertebrates; *Vertebrates; *sodium-potassium ATPase --activity; *sodium-potassium ATPase --analysis; *sodium-potassium ATPase --cholesterol modulation; *sodium-potassium ATPase --kidney; *2-3(diphenylhexatr- ienyl)propanyl-1-hexadecanoyl-sn-glycero-3-phospho-choline DPH-PC --reagent; *2-3(diphenylhexatr- ienyl)propanyl-1-hexadecanoyl-sn-glycero-3-phospho-choline DPH-PC --Molecular Probes; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *Spex Fluorolog photon counting spectrofluorometer --equipment; *protein-lipid interface --hydration degree

2000

Mechanisms of Fluorescence Quenching in Donor-Acceptor Labeled Antibody-Antlgen Conjugates.

Schobel, Uwe, Egelhaaf, Hans-Joachim, Frohlich, Dieter, and Brecht, Andreas;

Journal of Fluorescence, (June, 2000), Volume 10, Number 2, pp. 147-154.

2000

Measurements of cysteine reactivity during protein unfolding suggest the presence of competing pathways.

Udgaonkar, Jayant B.; Ramachandran, S.; Rami, Bhadresh R.

Journal of Molecular Biology, VOL. 297, NO. 3, March 31, 2000, PP. 733-745

Evidence that proteins may unfold utilizing complex competing pathways comes from a new pulse-labeling protocol in which the change in reactivity of a single cysteine residue in a protein during unfolding is measured, making use of its easily monitored reaction with the Ellman reagent, dithionitrobenzoic acid. The kinetics of unfolding of two single cysteine-containing mutant forms of the small protein barstar, C82A, which contains only Cys40, and C40A, which contains only Cys82, have been studied. The data suggest that unfolding occurs via two parallel pathways, each forming competing intermediates. In one of these early intermediates, Cys40 and Cys82 are already as reactive as they are in the fully unfolded protein, while in the other intermediate, the Cys thiol groups are unreactive. One more long-lived intermediate also needs to be included on the pathway defined by the early intermediate with unreactive Cys thiol groups to account for the difference in the rates of fluorescence change and of change in Cys40 reactivity. The demonstration of multiple intermediates and pathways for unfolding indicates that protein unfolding reactions can be as complex as protein folding reactions.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *cysteine; *dithionitrobenzoic acid --Ellman reagent; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --photometry: CB; *kinetic analysis --activity assays; *kinetic analysis --analytical method; *protein purification --purification method; *protein purification --Extraction; *protein purification --Isolation; *protein purification --Purification and Separation Techniques; *pulse-labeling protocol --detection method; *pulse-labeling protocol --detection/labeling techniques; *Abbe type refractometer --laboratory equipment; *Abbe type refractometer --Milton Roy; *Biologic SFM-3 stopped-flow machine --laboratory equipment; *Biologic SFM-3 stopped-flow machine --Biologic; *Biologic SFM-4 stopped-flow machine --laboratory equipment; *Biologic SFM-4 stopped-flow machine --Biologic; *Genesys spectrophotometer --laboratory equipment; *Genesys spectrophotometer --Milton Roy; *Spex DM3000 FLuorolog spectrofluorimeter --laboratory equipment; *Spex DM3000 FLuorolog spectrofluorimeter --Spex; *gene mutation; *protein unfolding

2000

Local interactions and the role of the 6-120 disulfide bond in alpha-lactalbumin: Implications for formation of the molten globule state

Raleigh, Daniel P. .; Demarest, Stephen J.; Fairman, Robert; Moriarty, Daniel F.; Robblee, James

Biochimica et Biophysica Acta, VOL. 1476, NO. 1, Jan. 3,. 2000, PP. 9-19,

Molten globule states are partially folded states of proteins which are compact and contain a high degree of secondary structure but which lack many of the fixed tertiary interactions associated with the native state. A set of peptides has been prepared in order to probe the role of local interactions in the vicinity of the Cys6-Cys120 disulfide bond in stabilizing the molten globule state of human alpha-lactalbumin. Peptides derived from the N-terminal and C-terminal regions of human alpha-lactalbumin have been analyzed using nuclear magnetic resonance, circular dichroism, fluorescence spectroscopy and sedimentation equilibrium experiments. A peptide corresponding to the first helical region in the native protein, residues 1-13, is only slightly helical in isolation. Extending the peptide to include residues 14-18 results in a modest increase in helicity. A peptide derived from the C-terminal 12 residues, residues 112-123, is predominantly unstructured. Crosslinking the N- and C-terminal peptides by the native disulfide bond results in almost no increase in structure and there is no evidence for any significant cooperative structure formation over the range of pH 2.2-11.7. These results demonstrate that there is very little enhancement of local structure due to the formation of the Cys6-Cys120 disulfide bond. This is in striking contrast to peptides derived from the region of the Cys28-Cys111 disulfide..

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques.; *alpha-lactalbumin --analysis; *alpha-lactalbumin --molten globule state.; *alpha-lactalbumin --6-120 disulfide bond; *circular dichroism spectroscopy --analytical method; *circular dichroism spectroscopy --spectroscopic techniques: CB; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: CB; *high performance liquid chromatography --liquid chromatography; *high performance liquid chromatography --purification method; *matrix-assisted laser/desorption ionization-time-of-flight-mass spectrometry MALDI-time-of-flight-MS --analytical method; *proton NMR spectroscopy --analytical method; *proton NMR spectroscopy --spectroscopic techniques: CB.; *Aviv 62DS instrument --equipment; *ISA Fluorolog spectrometer FL3-21 --equipment; *Varian Unity INOVA spectrometer --equipment

2000

Ligand-induced conformational change in the minimized insulin receptor.

Kaarsholm, Niels C.; Dunn, Michael F.; Havelund, Svend; Kristensen, Claus; Schlein, Morten

Journal of Molecular Biology, VOL. 303, NO.2, 20 October, 2000, PP. 161-169.

Within the class of insulin and insulin-like growth factor receptors, detailed information about the molecular recognition event at the hormone-receptor interface is limited by the absence of suitable co-crystals. We describe the use of a biologically active insulin derivative labeled with the NBD fluorophore (B29NBD-insulin) to characterize the mechanism of reversible 1:1 complex formation with a fragment of the insulin receptor ectodomain. The accompanying 40% increase in the fluorescence quantum yield of the label provides the basis for a dynamic study of the hormone-receptor binding event. Stopped-flow fluorescence experiments show that the kinetics of complex formation are biphasic comprising a bimolecular binding event followed by a conformational change. Displacement with excess unlabeled insulin gave monophasic kinetics of dissociation. The rate data are rationalized in terms of available experiments on mutant receptors and the X-ray structure of a non-binding fragment of the receptor of the homologous insulin-like growth factor (IGF-1).

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Endocrine System (Chemical Coordination and Homeostasis); *Methods and Techniques; *insulin --fluorescent label; *insulin receptor --conformational change; *insulin-like growth factor-1; *B29NBD-insulin; *circular dichroism spectroscopy --analytical method; *circular dichroism spectroscopy --spectroscopic techniques: CB; *free fat cell assay --activity assays; *free fat cell assay --analytical method; *receptor binding assay --analytical method; *receptor binding assay --binding assays; *receptor fragment purification --purification method; *receptor fragment purification --Isolation/Purification Techniques: CB; *stopped-flow fluorescence method --analytical method; *stopped-flow fluorescence method --Analysis/Characterization Techniques: CB; *Applied Photophysics microvolume SF.17MV rapid-mixing stopped-flow instrument --equipment; *B29NBD-insulin preparation --sample preparation method; *B29NBD-insulin preparation --specimen preparation techniques; *Perkin-Elmer LS-50B Luminescence Spectrometer --equipment; *Spex Fluorolog II spectrofluorimeter --equipment; *hormone receptor dynamics; *stopped-flow kinetics

2000

Interaction of the gonococcal porin P.IB with G- and F-actin.

Rubenstein, Peter A.; Apicella, Michael A.; Blake, Milan S.; Edwards, Jennifer; Giardina, Peter C.; Wen, Kuo-Kuang

Biochemistry, VOL. 39, NO. 29, July 25, 2000, PP. 8638-8647

The invasion of epithelial cells by N. gonorrheae is accompanied by formation of a halo of actin filaments around the enveloped bacterium. The transfer of the bacterial major outer membrane protein, porin, to the host cell membrane during invasion makes it a candidate for a facilitator for the formation of this halo. Western analysis shows here that gonococcal porin P.IB associates with the actin cytoskeleton in infected cells. Using the pyrene-labeled Mg forms of yeast and muscle actins, we demonstrate that under low ionic strength conditions, P.IB causes formation of filamentous actin assemblies, although they, unlike F-actin, cannot be internally cross-linked with N,N'-4-phenylenedimaleimide (PDM). In F-buffer, low porin concentrations appear to accelerate actin polymerization. Higher P.IB concentrations lead to the formation of highly decorated fragmented F-actin-like filaments in which the actin can be cross-linked by PDM. Co-assembly of P.IB with a pyrene-labeled mutant actin, S265C, prevents formation of a pyrene excimer present with labeled S265C F-actin alone. Addition of low concentrations of porin to preformed F-actin results in sparsely decorated F-actin. Higher P.IB concentrations extensively decorate the filaments, thereby altering their morphology to a state like that observed when the components are copolymerized. With preformed labeled S265C F-actin, P.IB quenches the pyrene excimer. This decrease is prevented by the F-actin stabilizers phalloidin and to a lesser extent beryllium fluoride. P.IB's association with the actin cytoskeleton and its ability to interact with and remodel actin filaments support a direct role for porin in altering the host cell cytoskeleton during invasion.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Neisseria gonorrheae (Neisseriaceae) --pathogen; *Bacteria; *Eubacteria; *Microorganisms; *cytoskeleton; *beryllium fluoride; *porin P.IB; *F-actin; *G-actin; *N,N'-4-phenylenedimaleimide; *autoradiography --analytical method; *autoradiography --detection/labeling techniques; *electron microscopy --imaging method; *electron microscopy --microscopy: CB; *electron microscopy --microscopy: CT; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *immunoprecipitation --analytical method; *immunoprecipitation --precipitation techniques; * Fluorolog 3 instrument --laboratory equipment; * Fluorolog 3 instrument --Spex; *Western analysis --analytical method; *Western analysis --Molecular Biology Techniques and Chemical Characterization; *Western blotting --analytical method; *Western blotting --detection/labeling techniques

2000

Green up-conversion emission from Er/sup 3+/:LiNbO/sub 3/ sol-gel powder

Cheng, S. D.; Kam, C. H.; Zhou, Y.; Lam, Y. L.; Chan, Y. C.; Xiang, Q.; Que, W. X.; Zhang, H. X.; Zhou, B.; Buddhudu, S.

Conference on Lasers and Electro-Optics (CLEO 2000). Technical Digest. Postconference Edition. TOPS Vol.39 (IEEE Cat. No.00CH37088) , 2000, PP. 384-5

Summary form only given. Lithium niobate (LiNbO/sub 3/) exhibits excellent electro-optical, acousto-optical and nonlinear optical characteristics, and therefore, it has been widely used for both bulk and integrated optics. As no work has so far been reported on the up-conversion emission study of sol-gel derived Er/sup 3+/ doped LiNbO/sub 3/ we have therefore taken up the present work for the first time. Photoluminescence spectra in the visible wavelength range were measured on a Fluorolog system attached with a phosphorimeter by using xenon flash cw lamp. A strong upconversion emission at 543 nm has been observed.

2000

Functional AT1-, and alpha1-adrenoceptors in cultured smooth muscle cells from WKY and SHR rats.

Lee, R. M. K. W.; Kim, James J. H.; Werstiuk, Eva S.

Biomedical Research (Aligarh) , VOL. 11, NO. 2, May-August, 2000, PP. 163-171

Altered regulation of intracellular Ca2+ is associated with the development of hypertension. We analysed the effects of angiotensin II (Ang II, 2X10-7M) and norepinephrine (NE,1X10-6M) treatment on the intracellular Ca2+ concentrations ((Ca2+)i) in cultured aortic smooth muscle cells (SMC) from normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) at three stages of hypertension development: at 3-4-, 10-12-, and 28-30-weeks. In the 10-12 week-old group we also evaluated the effect of alpha1-adrenoceptors activation on the intracellular distribution of the Ca2+-dependent isoforms of the enzyme protein kinase C (PKC). (Ca2+)i was measured using the fluorescent Ca2+ indicator, Fluo-3, and monitoring the excitation and emission wavelength at 490 and 530 nm with a SPEX Fluorolog 112. Intracellular distribution of PKC was determined in cytosolic and membrane fractions of cultured SMC from 10-12-week-old WKY and SHR, by SDS-PAGE electrophoresis and Western immunoblotting, using a monoclonal antibody against the alpha, beta, and gamma-isoforms of PKC. (Ca2+)i under basal, unstimulated conditions was similar in SMC from both strains of rats at all three age groups measured. Treatment of SMC with Ang II led to a significant increase in (Ca2+)i in the 3-4-week-old WKY and in both strains of SMC of the two older age groups, confirming the expression of functional AT1 receptors in these cells. AT1 receptor function, however was similar in WKY and SHR SMC at all three ages. NE treatment of cultured SMC induced elevations in (Ca2+)i in SMC from both strains at 10-12 weeks and 28-30 weeks, but not at 3-4 weeks, thus affirming the presence of functional alpha1-adrenoceptors in the two older age groups. alpha1-adrenoceptor function did not differ between WKY and SHR cells. In the 10-12-week-old WKY SMC treatment with NE or PE induced a translocation of PKC from the cytoplasmic to the membrane fraction, demonstrating enzyme activation consequent to alpha1-adrenoceptor simulation. No such translocation of PKC was detected in the 10-12-week-old SHR SMC, indicating that the Ca2+-dependent PKC isoforms are not expressed in the hypertensive cells. Our results are consistent with the expression of functional AT1 receptors in cultured SMC from the older WKY and SHR. They further support the expression of functional alpha1-adrenoceptors in SMC from both strains of rats in the older age groups but not in the 3-4-week-old group.

DESCRIPTOR(S)- *Cardiovascular System (Transport and Circulation); *Muscular System (Movement and Support); *rat (Muridae); *Animals; *Chordates; *Mammals; *Nonhuman Mammals; *Nonhuman Vertebrates; *Rodents; *Vertebrates; *aorta --circulatory system; *smooth muscle cells --cultured; *smooth muscle cells --muscular system; *hypertension --vascular disease; *alpha-1 adrenoceptors --activation; *angiotensin II; *calcium; *norepinephrine; *protein kinase C; *AT-1 adrenoceptors --expression; *AT-1 adrenoceptors --function; *SDS-PAGE electrophoresis --analytical method; *Western blot --analytical method; *Western blot --detection/labeling techniques; *Western blot --gene mapping; *disease development; *enzyme activity; *Hypertension (MeSH)

2000

Forster distances between green fluorescent protein pairs.

Barisas, B. George; Patterson, George H.; Piston, David W.

Analytical Biochemistry, VOL. 284, NO. 2, September 10, 2000, PP. 438-440

NO-ABSTRACT

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *green fluorescent protein; *DsRed --coral protein; *DsRed --Clontech Laboratories; *spectroscopy --analytical method; *spectroscopy --photometry: CB; *spectroscopy --photometry: CT; *SPEX 1681 Fluorolog spectrofluorometer --laboratory equipment; *SPEX 1681 Fluorolog spectrofluorometer --SPEX; *fluorescence resonant energy transfer; *Forster distances; *Note

2000

Fluorescence-Labeled Pyrenesulfonamide response for Characterizing Polymeric Interfaces in Composite Materials.

Gonzalez-Benito, J., Aznar, A. J., Lima, J., Bahia, F., Macanita, A. L., and Baselga, J.;

Journal of Fluorescence, (June, 2000), Volume 10, Number 2, pp, 141-146.

2000

Fluorescence Studies of Melanin by Stepwise Two-Photon Femtosecond Laser Excitation.

Teuchner, Klaus, Ehlert, Jurgen, Freyer, Wolfgang, Leupold, Dieter, Altmeyer, Peter, Stucker, Markus, and Hoffmann, Klaus;

Journal of Fluorescence, (September, 2000), Volume 10, Number 3, pp. 275-281.

2000

Fluorescence of tetrols, tetrols complexed with DNA, and benzo(a)pyrene-DNA adducts in methanol/water solutions.

Hurtubise, Robert J.; Steinbach, Paul B.

Applied Spectroscopy, VOL. 54, NO. 2, February, 2000, PP. 287-293

Several solution fluorescence parameters were acquired for the four tetrol hydrolysis products of benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts, tetrols complexed with DNA, and BPDE-DNA adducts in several methanol/water solvents. The relative polarity of the environment for the tetrols and BPDE-DNA adducts was determined by using a modified definition of the R value that is commonly employed for pyrene. The R values for the tetrols and BPDE-DNA adducts were calculated by obtaining the ratios of the intensities of the two major fluorescence emission bands at 380 and 400 nm (I380/I400). The positions of the hydroxyl groups on the hydroaromatic ring of the tetrols were compared in reference to the R values and the changes in the R values as a function of methanol/water composition. This approach resulted in a method for determining whether the hydroxyl groups in the 9 and 10 positions were on the same side or opposite sides of a hydroaromatic ring. The tetrols intercalated between the DNA bases showed quite different fluorescence spectra compared to tetrols not complexed with DNA. Also, the quasi-intercalated BPDE-DNA adducts gave significant changes in the R values with an increase in methanol in the solvent, and excitation spectra showed large shifts and changes in shape with an increase in methanol. The approaches developed provide unique structural and polarity information on tetrols and BPDE-DNA adducts.

DESCRIPTOR(S)- *Chemistry; *Methods and Techniques; *Pollution Assessment Control and Management; *benzo a pyrene --analysis; *benzo a pyrene-DNA adducts --analysis; *methanol/water solutions --analysis; *tetrol-DNA complexes --analysis; *tetrols --analysis; *DNA --analysis; *fluorescence spectrometry --analytical method; *fluorescence spectrometry --fluorophotometry: CB; * Fluorolog spectrofluorometer --equipment; * Fluorolog spectrofluorometer --Instruments SA; *fluorescence reactions --analysis; *fluorescence reactions --applications

2000

Endogenous Skin Fluorescence is a Good Marker for Objective Evaluation of Comedolysis

Gonzales, Salvador, Zonios, George, Nguyen, Bach Cuc, Gillies, Robert and Kollias, Nikiforos;

The Society of Investigative Dermatology, (2000), Vol. 115, No. 1, pp. 100-103.

2000

Direct eye visualization of Cy5 fluorescence for immunocytochemistry and in situ hybridization.

Ferri, Gian-Luca; Berger, Peter; Giro, Gabriele; Isola, Jorma

Journal of Histochemistry and Cytochemistry, VOL. 48, NO. 3, March, 2000, PP. 437-444

Cyanine 5.18 (or Cy5) is a fluorochrome emitting in the long-red/far-red range, usually regarded as unsuitable for direct observation by the human eye. We describe here the optimization of a direct visualization approach to Cy5 labeling, based on a standard fluorescence microscope with mercury light excitation and applicable to both immunocytochemistry and fluorescent in situ hybridization. Crucial factors were (a) an excitation path in the microscope not absorbing light in the orange-red range, up to 640 nm, (b) a 588-640-nm excitation filter range, distinctly below the excitation optimum for Cy5, (c) a 650-700-nm emission filter range, transmitting the low-wavelength portion of Cy5 emission, and (d) high-efficiency filter set components allowing a narrow gap between excitation and emission ranges without visible cross-talk of excitation light in the emission path.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *human (Hominidae); *Animals; *Chordates; *Humans; *Mammals; *Primates; *Vertebrates; *lymphocytes --blood and lymphatics; *lymphocytes --immune system; *cyanine 5.18 Cy5 --direct eye visualization; *cyanine 5.18 Cy5 --fluorochrome; *fluorescence in situ hybridization --analytical method; *fluorescence in situ hybridization --fluorescence detection; *fluorescence in situ hybridization --in situ hybridization; *fluorescence microscopy --confocal laser microscopy: CB; *fluorescence microscopy --light microscopy: CB; *fluorescence microscopy --light microscopy: CT; *fluorescence microscopy --microscopy method; *immunocytochemistry --analytical method; *immunocytochemistry --Immunohistochemical/Immunocytochemical Techniques; *in situ hybridization --analytical method; *in situ hybridization --nucleic acid labeling; *spectrofluorometry --analytical method; *spectrofluorometry --photometry: CB; *spectrophotometer Lambda 9 --laboratory equipment; *spectrophotometer Lambda 9 --Perkin-Elmer; *spectrophotometry --analytical method; *spectrophotometry --photometry: CB; *Cy5 labeling --detection method; *Cy5 labeling --detection/labeling techniques; * Fluorolog-2 spectrofluorometer --laboratory equipment; * Fluorolog-2 spectrofluorometer --Spex Industries; *Olympus BX-50 microscope --laboratory equipment; *Olympus BX-50 microscope --Olympus Italia; *Olympus BX-60 microscope --laboratory equipment; *Olympus BX-60 microscope --Olympus Italia; *VarioCam CCD camera --laboratory equipment; *VarioCam CCD camera --PCO Computer Optics

2000

Development of a mechanism-based, DNA staining protocol using SYTOX orange nucleic acid stain and DNA fragment sizing flow cytometry.

Marrone, Babetta L.; Cordek, Julia M.; Habbersett, Robert C.; Jett, James H.; Nolan, John P.; Yan, Xiaomei; Yoshida, Thomas M.

Analytical Biochemistry, VOL. 286, NO. 1, November 1, 2000, PP. 138-148

Accurate measurement of single DNA fragments by DNA fragment sizing flow cytometry (FSFC) depends upon precise, stoichiometric DNA staining by the intercalating dye molecules. In this study, we determined the binding characteristics of a commercially available 532 nm wavelength-excitable dye and used this information to develop a universal DNA staining protocol for DNA FSFC using a compact frequency-doubled Nd:YAG laser excitation source. Among twelve 532 nm wavelength-excitable nucleic acid staining dyes tested, SYTOX Orange stain showed the highest fluorescence intensity along with a large fluorescence enhancement upon binding to double-stranded DNA (apprx450-fold). Furthermore, using SYTOX Orange stain, accurate fragment-size-distribution histograms were consistently obtained without regard to the staining dye to base pair (dye/bp) ratio. A model describing two binding modes, intercalation (primary, yielding fluorescence) and external binding (secondary, involving fluorescence quenching), was proposed to interprete the performance of the dyes under different dye/bp ratios. The secondary equilibrium dissociation constant was found to be the most critical parameter in determining the sensitivity of each fluorophore to the staining dye/bp ratio. The measurements of both equilibrium dissociation constants provided us with a theoretical framework for developing a universal protocol which was successfully demonstrated over a wide range of DNA concentrations on a compact flow cytometer equipped with a frequency-doubled, diode-pumped, solid-state Nd:YAG laser for rapid and sensitive DNA fragment sizing.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *DNA fragments --Promega Co.; *SYTOX Orange Nucleic Acid --stain; *SYTOX Orange Nucleic Acid --Inc.; *SYTOX Orange Nucleic Acid --Molecular Probes; *compact continuous-wave, diode-pumped, solid state neodymium-YAG laser --laboratory equipment; *compact continuous-wave, diode-pumped, solid state neodymium-YAG laser --model 4612-020-1000; *compact continuous-wave, diode-pumped, solid state neodymium-YAG laser --Uniphase; *mechanism-based, DNA staining protocol --staining method; *DNA fragment sizing flow cytometry --analytical method; *DNA fragment sizing flow cytometry --cytophotometry: CB; *DNA fragment sizing flow cytometry --cytophotometry: CT; *DNA fragment Sizing Flow Cytometry --analytical method; *DNA fragment Sizing Flow Cytometry --Molecular Biology Techniques and Chemical Characterization; *FLuorescence Spectrometry --analytical method; *FLuorescence Spectrometry --fluorophotometry: CB; *FLuorescence Spectrometry --fluorophotometry: CT; *SPEX Fluorolog-2 spectrofluorometer --laboratory equipment; *SPEX Fluorolog-2 spectrofluorometer --Inc.; *SPEX Fluorolog-2 spectrofluorometer --Sienco

2000

Chemiluminescent determination of Ce(IV) using Cypridina luciferin analog.

Osman, Ahmed Mohamud; Hilhorst, Riet; Laane, Colj

Analytica Chimica Acta, VOL. 442, NO. 1, October 6th, 2000, PP. 81-87

A cerium(IV)-induced Cypridina luciferin analog (CLA) chemiluminescence is reported. This Ce(IV) triggered luciferin analog CL is enhanced about 5-fold by 0.5% (v/v) Tween 20. Using this enhanced luciferin analog-dependent chemiluminescence, a lower limit detection of 25 nM was determined for Ce(IV). Ce(III) could not generate this luciferin analog-dependent chemiluminescence. Oxidising agents such as potassium permanganate and potassium dichromate were also without effect. While the tested scavengers of reactive species such as mannitol, superoxide dismutase and catalase had no specific effect on the luminescence, this Ce(IV)-elicited luciferin analog chemiluminsecent reaction was inhibited when the reaction was performed under anaerobic condition, indicating that molecular oxygen participates in the reaction mechanism leading to the formation of the light-emitting product. A mechanism is proposed for this chemiluminescent reaction which is consistent with the presented data.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *catalase --reagent; *catalase --Boehringer Mannheim; *mannitol --reagent; *potassium dichromate --reagent; *potassium permanganate --reagent; *superoxide dismutase --reagent; *superoxide dismutase --Boehringer Mannheim; *2-methyl-6-phenyl-3,7-dihydroimidazo 1,2-a pyrazin-3-one --cerium(IV)-induced; *2-methyl-6-phenyl-3,7-dihydroimidazo 1,2-a pyrazin-3-one --Cypridina luciferin analog; *2-methyl-6-phenyl-3,7-dihydroimidazo 1,2-a pyrazin-3-one --Tokyo Kasei Kogyo Co.; *chemiluminescence --detection method; *chemiluminescence --detection/labeling techniques; * Fluorolog 3-22 spectrofluorometer --equipment

2000

Backbone dynamics of Tet repressor alpha8Xalpha9 loop.

Chabbert, Marie; Alberti, Patrizia; Bombarda, Elisa; Doglia, Silvia M.; Hillen, Wolfgang; Kintrup, Martin; Lami, Hans; Piemont, Etienne; Vergani, Benedetta

Biochemistry, VOL. 39, NO. 10, March 14, 2000, PP. 2759-2768

A set of single Trp mutants of class B Tet repressor (TetR), in which Trp residues are located from positions 159 to 167, has been engineered to investigate the dynamics of the loop joining the alpha-helices 8 and 9. The fluorescence anisotropy decay of most mutants can be described by the sum of three exponential components. The longest rotational correlation time, 30 ns at 10 degreeC, corresponds to the overall rotation of the protein. The shortest two components, on the subnanosecond and nanosecond time scale, are related to internal motions of the protein. The initial anisotropy, in the 0.16-0.22 range, indicates the existence of an additional ultrafast motion on the picosecond time scale. Examination of physical models for underlying motions indicates that librational motions of the Trp side chain within the rotameric chi1 X chi2 potential wells contribute to the picosecond depolarization process, whereas the subnanosecond and nanosecond depolarization processes are related to backbone dynamics. In the absence of inducer, the order parameters of these motions, about 0.90 and 0.80 for most positions, indicate limited flexibility of the loop backbone. Anhydrotetracycline binding to TetR induces an increased mobility of the loop on the nanosecond time scale. This suggests that entropic factors might play a role in the mechanism of allosteric transition.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Tet repressor --alpha helix-8; *Tet repressor --alpha helix-9; *Tet repressor --analysis; *Tet repressor --characterization; *Tet repressor --class B; *Tet repressor --fluorescence anisotropy decay; *Tet repressor --loop joining dynamics; *Tet repressor --tryptophan mutant; *genetic engineering --genetic method; *genetic engineering --molecular genetics/genetic engineering; *pulse fluorometry technique --analytical method; *pulse fluorometry technique --Analysis/Characterization Techniques: CB; *spectrophotometry --analytical method; *spectrophotometry --photometry: CB; *spectrophotometry --photometry: CT; *Cary 4 spectrophotometer --equipment; * Fluorolog 1680 spectrofluorometer --equipment; * Fluorolog 1680 spectrofluorometer --Spex; *MPF66 spectrofluorometer --equipment; *MPF66 spectrofluorometer --Perkin-Elmer

2000

ATPase cycle of an archaeal chaperonin.

Gutsche, Irina; Baumeister, Wolfgang; Mihalache, Oana

Journal of Molecular Biology, VOL. 300, NO. 1, 30 June, 2000, PP. 187-196

Recent structural data imply differences in allosteric behavior of the group I chaperonins, typified by GroEL from Escherichia coli, and the group II chaperonins, which comprise archaeal thermosome and eukaryotic TRiC/CCT. Therefore, this study addresses the mechanism of interaction of adenine nucleotides with recombinant alpha-only and native alphabeta-thermosomes from Thermoplasma acidophilum, which also enables us to analyze the role of the heterooligomeric composition of the natural thermosome. Although all subunits of the alpha-only thermosome seem to bind nucleotides tightly and independently, the native chaperonin has two different classes of ATP-binding sites. Furthermore, for the alpha-only thermosome, the steady-state ATPase rate is determined by the cleavage reaction itself, whereas, for the alphabeta-thermosome, the rate-limiting step is associated with a post-hydrolysis isomerisation into a non-covalent ADP*Pi species prior to the release of the gamma-phosphate group. After half-saturation with ATP, a negative cooperativity in hydrolysis is observed for both thermosomes. The effect of Mg2+ and K+ nucleotide cycling is documented. We conclude that archaeal chaperonins have unique allosteric properties and discuss them in the light of the mechanism established for the group I chaperonins.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Escherichia coli (Enterobacteriaceae); *Thermoplasma acidophilum (Cell Wall-less Archaeobacteria); *Archaeobacteria; *Bacteria; *Eubacteria; *Microorganisms; *adenine nucleotides; *archeal thermosome; *eukaryotic TRiC/CCT; *group I chaperonins --allosteric behavior; *group I chaperonins --structure; *group I chaperonins --ATP-binding sites; *group II chaperonins --ATP-binding sites; *magnesium ion; *native alpha-beta-thermosome; *potassium ion; *recombinant alpha-only thermosome; *ATP; *ATPase; *Escherichia coli GroEL; *fluorescence --analytical method; *fluorescence --Molecular Biology Techniques and Chemical Characterization; *ultrafiltration --filtration techniques; *ultrafiltration --purification method; *ATPase assay --analytical method; *ATPase assay --Molecular Biology Techniques and Chemical Characterization; *Spex Fluorolog-3 spectrofluorimeter --laboratory equipment; *Spex Fluorolog-3 spectrofluorimeter --Spex

2000

Analysis of the RNA-editing reaction of ADAR2 with structural and fluorescent analogues of the GluR-B R/G editing site.

Beal, Peter A.; Stephens, Olen M.; Yi-Brunozzi, Hye Young

Biochemistry, VOL. 39, NO. 40, October 10, 2000, PP. 12243-12251

ADARs are adenosine deaminases responsible for RNA editing reactions that occur in eukaryotic pre-mRNAs, including the pre-mRNAs of glutamate and serotonin receptors. Here we describe the generation and analysis of synthetic ADAR2 substrates that differ in structure around an RNA editing site. We find that five base pairs of duplex secondary structure 5' to the editing site increase the single turnover rate constant for deamination 17-39-fold when compared to substrates lacking this structure. ADAR2 deaminates an adenosine in the sequence context of a natural editing site >90-fold more rapidly and to a higher yield than an adjacent adenosine in the same RNA structure. This reactivity is minimally dependent on the base pairing partner of the edited nucleotide; adenosine at the editing site in the naturally occurring AcntdotC mismatch is deaminated to approximately the same extent and only 4 times faster than adenosine in an AcntdotU base pair at this site. A steady-state rate analysis at a saturating concentration of the most rapidly processed substrate indicates that product formation is linear with time through at least three turnovers with a slope of 13 cntdot 1.5 nMcntdotmin-1 at 30 nM ADAR2 for a kss = 0.43 +- 0.05 min-1. In addition, ADAR2 induces a 3.3-fold enhancement in fluorescence intensity and a 14 nm blue shift in the emission maximum of a duplex substrate with 2-aminopurine located at the editing site, consistent with a mechanism whereby ADAR2 flips the reactive nucleotide out of the double helix prior to deamination.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *ADAR2 --analysis; *ADAR2 --substrate specificity; *ADAR2 --RNA-editing reaction; *GluR-B R/G editing site --analysis; *GluR-B R/G editing site --fluorescent analogs; *GluR-B R/G editing site --structural analogs; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; * fluorolog-3 spectrophotometer --equipment; * fluorolog-3 spectrophotometer --Instruments S.A. Inc.

2000

Amphiphilic derivatives of sodium alginate and hyaluronate: Synthesis and physico-chemical properties of aqueous dilute solutions.

Dellacherie, E.; Hubert, P.; Lapicque, F.; Payan, E.; Pelletier, S.

Carbohydrate Polymers, VOL. 43, NO. 4, December, 2000, PP. 343-349

This paper reports on the synthesis and the physico-chemical characterisation of various amphiphilic derivatives of two natural polysaccharides, sodium alginate and sodium hyaluronate, in which a rather small proportion of the carboxylic groups (ltoreq10% mol) was esterified by long alkyl chains (C12H25 or C18H37). The derivatives thus prepared were characterised by gas chromatography, 1H and 13C n.m.r. spectroscopy and size exclusion chromatography coupled to a multi-angle laser light scattering detection. The tendency of these water-soluble compounds to hydrophobic association in aqueous solutions was evidenced firstly in dilute regime using capillary viscometry as well as fluorescence spectroscopy in the presence of a molecular probe, 1,1-dicyano-(4'-N,N-dimethylaminophenyl)-1,3-butadiene.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *amphiphilic sodium alginate derivatives --analysis; *amphiphilic sodium alginate derivatives --aqueous dilute solution; *amphiphilic sodium alginate derivatives --physico-chemical properties; *amphiphilic sodium alginate derivatives --synthesis; *amphiphilic sodium hyaluronate derivatives --analysis; *amphiphilic sodium hyaluronate derivatives --aqueous dilute solution; *amphiphilic sodium hyaluronate derivatives --physico-chemical properties; *amphiphilic sodium hyaluronate derivatives --synthesis; *sodium alginate --Sigma; *sodium hyaluronate --Acros Organics; *capillary viscometry --analytical method; *capillary viscometry --Molecular Biology Techniques and Chemical Characterization; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: Cb; *gas chromatography --analytical method; *gas chromatography --chromatographic techniques; *size exclusion chromatography-multi-angle laser light scattering detection --analytical method; *size exclusion chromatography-multi-angle laser light scattering detection --liquid chromatography; *Bruker AC 200P NMR spectrometer --laboratory equipment; *Bruker AC 200P NMR spectrometer --Bruker; *NMR spectroscopy --analytical method; *NMR spectroscopy --carbon-13; *NMR spectroscopy --proton; *NMR spectroscopy --spectroscopic techniques: CB; * Spex-fluorolog-2 spectrometer --laboratory equipment; * Spex-fluorolog-2 spectrometer --Spex; *Viscologic TI.1 Ostwald-type automatic capillary viscometer --laboratory equipment; *Viscologic TI.1 Ostwald-type automatic capillary viscometer --Sematech

2000

Alteration around the active site of rhodanese during urea-induced denaturation and its implications for folding.

Horowitz, Paul; Bhattacharyya, Anusri Mitra

Journal of Biological Chemistry, VOL. 275, NO. 20, May 19, 2000, PP. 14860-14864

The enzyme rhodanese contains two globular domains connected by a tether region and associated by strong hydrophobic interactions. The protein has proven to be very difficult to refold without assistance to prevent oxidation and aggregation. For this study, the active site cysteine 247, near the interdomain region, was modified with the environmentally sensitive fluorescent probe, 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS), to yield a derivative that reversibly unfolds. Structural transitions during urea unfolding/refolding were complex and multiphasic. Increasing urea concentrations increased the IAANS fluorescence intensity and polarization. Both values reached maxima at apprxeq4 M urea, where there is a concomitant large exposure of hydrophobic sites as reported by both IAANS and the noncovalent fluorescent probe, bis-ANS. The exposure of the hydrophobic sites arises from the decrease in strong interaction between the domain interfaces, which lead to their partial separation. This correlates with the loss of activity of the unlabeled enzyme. Above 4.5 M urea, there is progressive loss of rigid, hydrophobic surfaces, and both fluorescence and polarization of IAANS decrease, with accompanying loss of secondary structure. These results are consistent with a folding model in which there is an initial, rapid hydrophobic collapse of the denatured form to an intermediate with native like secondary structure, with exposed interdomain, hydrophobic surfaces. This step is followed by adjustment of the domain-domain interactions and the proper positioning of reduced cysteine 247 at the active site.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *mitochondrial enzymes; *rhodanese EC 2.8.1.1 --active site; *urea; *circular dichroism --analytical method; *circular dichroism --spectroscopic techniques: CB; *fluorescence microscopy --confocal laser microscopy: CB; *fluorescence microscopy --light microscopy: CB; *fluorescence microscopy --microscopy method; *rhodanese assay --activity assays; *rhodanese assay --analytical method; * Fluorolog-3 --equipment; * Fluorolog-3 --Jobin Yvon-Spex; *enzyme kinetics; *protein denaturation; *protein folding

2000

A step toward the prediction of the fluorescence lifetimes of tryptophan residues in proteins based on structural and spectral data

Engelborghs, Yves .; Diaz, Jose Fernando; Sillen, Alain

Protein Science, VOL. 9, NO. 1, Jan.,. 2000, PP. 158-169

A method is presented that allows the calculation of the lifetimes of tryptophan residues on the basis of spectral and structural data. It is applied to two different proteins. The calcium binding protein from the sarcoplasm of the muscles of the sand worm Nereis diversicolor (NSCP) changes its conformation upon binding of Ca2+ or Mg2+. NSCP contains three tryptophan residues at position 4, 57, and 170, respectively. The fluorescence lifetimes of W57 are investigated in a mutant in which W4 and W170 have been replaced. The time resolved fluorescence properties of W57 are linked to its different microconformations, which were determined by a molecular dynamics simulation map. Together with the determination of the radiative rate constant from the wavelength of maximum intensity of the decay associated spectra, it was possible to determine an exponential relation between the nonradiative rate constant and the distance between the indole CE3 atom and the carbonyl carbon of the peptide bond reflecting a mechanism of electron transfer as the main determinant of the value for the nonradiative rate constant. This result allows the calculation of the fluorescence lifetimes from the protein structure and the spectra. This method was further tested for the tryptophan of Ha-ras p21 (W32) and for W43 of the Tet repressor, which resulted in acceptable values for the predicted lifetimes..

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques.; *Nereis diversicolor sand worm (Polychaeta).; *Animals; *Annelids; *Invertebrates.; *muscle --muscular system; *sarcoplasm --muscular system.; *proteins --characterization; *proteins --tryptophan residue fluorescence lifetime prediction.; *Ha-ras p21 --characterization; *Nereis diversicolor calcium binding protein --characterization; *Tet repressor --characterization; *molecular dynamics simulations --mathematical method; *mutagenesis --molecular genetics/genetic engineering; *mutagenesis --protein modification method; *spectrofluorimetry --analytical method; *spectrofluorimetry --spectroscopic techniques: cb.; *SPEX Fluorolog 1691 spectrofluorimeter --laboratory equipment; *SPEX Fluorolog 1691 spectrofluorimeter --Spex Industries