Delineation of fucosyltransferase activities with thiol reagents.

The thiol reagent dithiothreitol inhibits the activity of a core GDP-fucose-N-acetylglucosaminide alpha-6-fucosyltransferase in plasma and blood-cell homogenates, while promoting the activity of alpha-2- and alpha-3-fucosyltransferases. The latter enzymes catalyse transfer of fucose on to terminal galactose and subterminal N-acetylglucosamine residues respectively. A thiol-blocking reagent N-ethylmaleimide does not affect the activity of the alpha-6-fucosyltransferase, but inhibits the other two enzymes. These results indicate the presence of a critical disulphide linkage in the alpha-6-fucosyltransferase, and provide a means of delineation of different fucosyltransferases.     

Evaluation of Six Fluorescent Protein Stains for Use in Flow Microfluorometry

Flow microfluorometric (FMF) analysis of stained cells has provided protein distribution histograms for large populations of cells. Spectral data and staining protocols were evaluated for six fluorescent protein dyes suggested for staining cells in liquid suspension. The requirements for dyes and/or staining protocol included minimal cell clumping and cell loss, near-optimal dye excitation at existing laser wavelengths, and tenacity of the dye/protein interaction. These criteria were best satisfied by fluoresoein isothiocyanate (FITC) and rho-damine B isothiocyanate (RITC). Both fluoreseamine and 8-aniline-1-naphthakne sulfonic acid (ANSA) showed potential applicability for use in systems where excitation wavelengths in the ultraviolet range are available. Protein staining with fluorescamine was extremely rapid. Brilliant sulfaflavine and 1-dimethyl-aminonaphthalene-5-sulfonyl chloride (DANSYL) WCR found unsatisfactory in these studies, since the former dye tended to diffuse from the all., while the latter induced excessive all clumping and cell loss. These techniques have application to immunofluoregcence analysis and can also be profitably employed in dual-parameter analysis systems in connection with double-staining techniques for simultaneous DNA and protein analysis.     

Affinity chromatography of heavy meromyosin subfragment-1 reacted with thiol reagents.

Separation of heavy meromyosin subfragment-1 treated with N-ethyl maleimide (MalNEt) into native -SH1- and -(SH1, SH2)-blocked protein populations could be achieved by affinity chromatography on agarose-ATP columns in the presence of Mg2+ or Ca2+. Covalent bridging of the two -SH groups by p-phenylenedimaleimide gave a product which has the same affinity of binding to ATP columns as the doubly blocked MalNEt preparation. Treatment with p-phenylenedimaleimide abolished binding to immobilized F-actin columns, whereas modifications by MalNEt did not affect adsorption by this chromatographic medium. Affinity chromatography on immobilized nucleotide and actin columns is suggested as an analytical tool in the study of the involvement of thiol groups in the myosin active site and its conformation.     

Evaluation of Six Fluorescent Protein Stains for Use in Flow Microfluorometry

Flow microfluorometric (FMF) analysis of stained cells has provided protein distribution histograms for large populations of cells. Spectral data and staining protocols were evaluated for six fluorescent protein dyes suggested for staining cells in liquid suspension. The requirements for dyes and/or staining protocol included minimal cell clumping and cell loss, near-optimal dye excitation at existing laser wavelengths, and tenacity of the dye/protein interaction. These criteria were best satisfied by fluoresoein isothiocyanate (FITC) and rho-damine B isothiocyanate (RITC). Both fluoreseamine and 8-aniline-1-naphthakne sulfonic acid (ANSA) showed potential applicability for use in systems where excitation wavelengths in the ultraviolet range are available. Protein staining with fluorescamine was extremely rapid. Brilliant sulfaflavine and 1-dimethyl-aminonaphthalene-5-sulfonyl chloride (DANSYL) WCR found unsatisfactory in these studies, since the former dye tended to diffuse from the all., while the latter induced excessive all clumping and cell loss. These techniques have application to immunofluoregcence analysis and can also be profitably employed in dual-parameter analysis systems in connection with double-staining techniques for simultaneous DNA and protein analysis.     

Modification of the mitochondrial sulfonylurea receptor by thiol reagents.

The purpose of this study was to investigate the effects exerted by thiol-modifying reagents on themitochondrial sulfonylurea receptor. The thiol-oxidizing agents (timerosal and 5, 5'-dithio-bis(2-nitrobenzoic acid)) were found to produce a large inhibition (70% to 80%) of specific binding of [(3)H]glibenclamide to the beef heart mitochondrial membrane. Similar effects were observed with membrane permeable (N-ethylmaleimide) and non-permeable (mersalyl) thiol modifying agents. Glibenclamide binding was also decreased by oxidizing agents (hydrogen peroxide) but not by reducing agents (reduced gluthatione, dithiothreitol and the 2,3-dihydroxy-1,4-dithiolbutane). The results suggest that intact thiol groups, facing the mitochondrial matrix, are essential for glibenclamide binding to the mitochondrial sulfonylurea receptor.     

Reactions of cellulose isothiocyanates with thiol and amino compounds.

A cellulose isothiocyanate has been prepared by treatment of cellulose with 2,4-di-isocyanatotoluene followed by hydrolysis and reaction of the resulting amine with thiophosgene. The cellulose isothiocyanate was characterized by its binding capacity with respect to [14C]-glycine, [131 I]-human serum albumin, and 2-mercaptoethanol. An analytical method for binding capacity, based on reaction with [35 S]-alpha-toluenethiol, was developed. Because of the aromatic character of the NCS group of the cellulose isothiocyanate, the covalently bonded thiol can be quantitatively liberated.     

Reactions of d-glyceraldehyde 3-phosphate dehydrogenase with chromophoric thiol reagents

The kinetics of the reaction of d-glyceraldehyde 3-phosphate dehydrogenase with 5,5'-dithiobis-(2-nitrobenzoic acid) show that NAD(+) dissociates from the enzyme before the reaction. In contrast 2-chloromercuri-4-nitrophenol reacts with the holoenzyme without prior dissociation of NAD(+). These studies and observations on the dissociation constant of NAD(+) to the lobster enzyme show that NAD(+) must dissociate from sites modified by substrates during the reductive dephosphorylation of 1,3-diphosphoglycerate. All four sites per tetramer of the apoenzyme are acylated by 1,3-diphosphoglycerate. Hydrolysis of the acyl-enzyme occurs at a significant rate even in the absence of NAD(+), which may explain previous estimates that only two sites per tetramer can readily be acylated.     

Effect of thiol reagents on extractability of protein from yeast.

The effect of soluble thiol reagents on the extractability of protein from yeast cells was studied. The incubation of yeast cells with dithiothreitol, 2-mercaptoethanol, or monothioglycerol markedly stimulated the release of soluble carbohydrates into the medium. There was a concomitant improvement (over twofold) in the extractability of protein from the yeast cells. The thiol reagents activated the proteolytic enzymes of the yeast cells. Unless inactivated, these enzymes hydrolyze the extracted protein.     

Anomalous reaction of 4-chloro-7-nitrobenzofurazan with thiol compounds.

The kinetics of reaction of 4-chloro-7-nitrobenzofurazan with thiol groups at pH values above 5 cannot be accounted for solely on the basis of formation of a single product, the 4-thio derivative. Spectroscopic observations indicate that, in addition to the 4-thio derivative, at least two other products are formed. One of these, referred to as P1, is most likely a reversible complex of thiol compound and 4-chloro-7-nitrobenzofurazan of the Meisenheimer type. The other product, P2, which forms primarily when thiol compound is in a large excess, does not appear to result from direct reaction of thiol group and 4-chloro-7-nitrobenzofurazan, but may be a reaction of product P1 and thiol compound. The coloured product, P2, will react further with proteins, such as bovine serum albumin and Escherichia coli RNA polymerase. This reaction irreversibly destroys the catalytic activity of RNA polymerase. The implications of these observations for utilization of 4-chloro-7-nitrobenzofurazan as a protein-modifying agent are discussed.     

Microfluorometry of cell membrane dynamics.

Membranes of living cells are characterized by a combination of conventional and total internal reflection fluorescence microscopy (TIRFM) using the membrane marker laurdan. In the first case, all cellular membranes are assessed simultaneously, whereas in the second case, the plasma membrane is excited selectively by the evanescent electromagnetic field of a laser beam. A spectral shift depending on the phase of membrane lipids is used to characterize membrane stiffness, which decreases with temperature and increases with the amount of cholesterol. Spectral properties are evaluated and displayed as microscopic images.     

Labeling of a thiol residue in sarcoplasmic reticulum ATPase by pyrene maleimide. Solvent accessibility studied by fluorescence quenching

Sarcoplasmic reticulum ATPase was specifically labeled by the fluorescent probe N-(1-pyrene)maleimide which modified 1 mol of a highly reactive thiol residue per mol of ATPase under appropriate conditions, when the probe concentration was varied in the range 0.1-1.5 microM. Addition of inorganic phosphate to the labeling medium increased both the rate of labeling and the number of modified thiol residues. Addition of ATP gave a marked kinetic protection from labeling, suggesting that the label was attached to a protein domain which is sensitive to changes at the catalytic site. Quenching of pyrene fluorescence emission of labeled ATPase by acrylamide and cesium chloride gave linear Stern-Volmer plots. The Stern-Volmer quenching constants of pyrene-ATPase fluorescence were 10 times lower than the constant obtained for acrylamide quenching of the fluorescent adduct of pyrene-maleimide-cystein used as a control, indicating that the pyrene moiety of the probe was considerably shielded from the medium solvent when covalently attached to the ATPase. The efficiency of quenching of pyrene-ATPase fluorescence increased by a significant amount upon addition of 100 microM Ca2+, when compared to the quenching in the presence of a Ca2+ chelator. It suggests that occupancy of the high affinity Ca2+ sites of the ATPase increases the accessibility of medium solvent into hydrophobic domains of the enzyme. The fluorescence lifetime of the solubilized pyrene-ATPase emission was 144-149 ns. The fluorescence polarization of pyrene-ATPase solubilized by nonionic detergent C12E8 was rho = 0.10 and it increased with an increase in the viscosity of the medium yielding a linear Perrin plot. The rotational correlation time for the soluble ATPase was 532 ns, corresponding to the overall rotation of a detergent-pyrene-ATPase particle with radius of 87A.     

Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide.

The reactivities of glutathione, cysteine, cysteamine, penicillamine, N-acetylcysteine, dithiothreitol and captopril with superoxide generated from xanthine oxidase and hypoxanthine, and with reagent hydrogen peroxide, have been investigated. Rates of thiol loss on adding hydrogen peroxide, and superoxide-dependent thiol loss and oxygen uptake were measured. The relative reactivities of the different thiols with both oxidants were inversely related to the pK of the thiol group, such that at pH 7.4, penicillamine was the most reactive. N-acetylcysteine weakly reactive and no reaction was seen with captopril. For hydrogen peroxide, the calculated rate constants for the reaction with the thiolate anion all fell within the range 18-26 M(-1) s(-1). With superoxide, our results are consistent with each thiol reacting via a short chain that consumes oxygen and regenerates superoxide. Only with some of the thiols, was the consumed oxygen recovered as hydrogen peroxide. Reported values for the rate constant for the reaction of thiols with superoxide vary over four orders of magnitude, with the highest being > 10(5) M(-1) s(-1). Due to the complexity of the chain reaction, no study so far has been able to obtain accurate values and we consider the best estimates to be in the 30 to 1000 M(-1) s(-1) range.     

Organization of thiol groups of electric-eel electric-organ sodium-plus-potassium ion-stimulated adenosine triphosphatase studied with bifunctional reagents.

The reactions of three bifunctional thiol-blocking reagents of differing cross-linking spans and two monofunctional thiol-blocking reagents with the Na+ + K+-stimulated ATPase of the electric-eel electric organ were examined. 1,5-Difluoro-2,4-dinitrobenzene with a cross-linking span of 0.3--0.5 nm (3--5 A) and high solubility in non-polar solvent was the most efficient inhibitor of enzyme activity; thus essential thiol groups exist in a non-polar environment and are approx. 0.3--0.5 nm (3--5 A) from their nearest thiol-group neighbours. Ligands promoting phosphorylation of the Na+ + K+-stimulated ATPase decreased the number of thiol groups bridged by 1,5-difluoro-2,4-dinitrobenzene and by 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone [0.7--1.0 nm (7--10 A) span]. Phosphorylation is associated with a conformational change in the enzyme.