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J Westley

Publications and source records attributed to J Westley.

At least 37 records · Page 2Linked to original sources
PubMed1990

Competitive partial inhibitors of serum albumin-catalyzed sulfur cyanolysis.

Efforts to locate the active site for sulfur cyanolysis catalyzed by bovine serum albumin have led to systematic tests of several compounds that inhibit the catalyzed reaction. Hexanoate and 5-dimethylaminonaphthalene-1-sulfonate bind at the same site and are partial inhibitors competitive with cyanide, uncompetitive with respect to sulfur. Various dansyl amino acids and 1-anilino-8-naphthalene sulfonate display the same inhibitory behavior but bis (1-anilino-8-naphthalene sulfonate) is a total inhibitor competitive with cyanide. These findings are interpreted to indicate that the cyanolysis active site is near, but not at, one of the short-chain fatty acid binding sites on albumin subdomain 2-AB or 3-AB. Both ionic repulsion and steric considerations are implicated in the mechanisms of inhibition.

Anilino Naphthalenesulfonates↗
PubMed1990

15-Acetylthioxy-furodysinin lactone, isolated from a marine sponge Dysidea, sp. is a potent agonist to human leukotriene B4 receptor.

A sesquiterpene thioacetate, 15-acetylthioxy-furodysinin (SK&F 105900) has been isolated from the sponge Dysidea SP. This compound can bind specifically to the human peripheral blood polymorphonuclear leukocyte (PMN) and to the differentiated human monocytic leukemic U-937 cell membrane leukotriene B4 (LTB4) receptors with high-affinity. This compound can also promote a concentration-dependent chemotaxis in PMNs and an intracellular calcium mobilization in U-937 cells that can be blocked by the LTB4 receptor antagonist, LY-223982. Furthermore, the calcium mobilization induced by SK&F 105900 can specifically cross-desensitize with the LTB4-induced calcium mobilization. These observations indicate that SK&F 105900 is a novel and specific high-affinity agonist that can bind to the LTB4 receptors and activate the receptor-mediated signal transduction processes in human PMN and U-937 cells.

Animals↗
PubMed1989

Voltammetric determination of cyanide and thiocyanate in small biological samples.

Practical methods for the quantitative measurement of cyanide and thiocyanate content in small biological samples are presented. The basis of the procedures is electrochemical determination of CN- with a silver rotating disk electrode at 90 mV, relative to the Ag/AgCl reference electrode, or with a dropping mercury electrode in differential pulse mode at -240 mV. Thiocyanate is oxidized to CN- with permanganate under controlled conditions prior to its analysis. Endogenous cyanide and cyanide from SCN- oxidation are recovered, separately, by volatilization and trapping. Emphasis is placed on careful handling of specimens prior to analysis. Precautions include a provision for sequestering cyanide in storage, the use of an antioxidant, and an avoidance of the spontaneous sulfide formation from proteins that occurs in even mildly alkaline solutions.

Cyanides↗
PubMed1989

The sulfur-cyanolysis sites of serum albumin: metabolite competition studies.

In the presence of a source of sulfane sulfur, a cyanolysis reaction catalyzed by serum albumin may contribute to cyanide detoxication. The active site for this catalysis by serum albumin has been investigated in competition studies with ligands that have known albumin binding sites. Despite complications caused by the occurrence of multiple primary and secondary sites for many ligands, the results show that the primary sites for bilirubin, steroids, indoles, aspirin, and palmitate are distinct from that for sulfur. Laurate is a tight-binding partial inhibitor of the cyanolysis reaction, competitive with cyanide rather than with sulfur. In view of the formal mechanism previously established for the catalyzed reaction, this result indicates that the sulfur-cyanolysis site is probably near the site occupied by laurate.

Aspirin↗
PubMed1989

Appendix to the sulfur-cyanolysis sites of serum albumin: metabolite competition studies. Tight-binding inhibitions that yield atypical Henderson plots.

Ordinary tight-binding inhibition in steady-state enzyme systems is conveniently evaluated by means of the Henderson plot. This is a linear plotting form that has an ordinate intercept equal to the total enzyme concentration. However, there are two experimental situations that yield deviations from the common Henderson plot form. These are inhibitor binding in a separate, noninhibitory mode that depletes the concentration of free inhibitor, and partial inhibition, i.e., the retention of partial activity by the enzyme-inhibitor complex. Noninhibitory depletion results in Henderson plots with elevated ordinate intercepts. Competitive partial inhibition yields a characteristic pattern of parabolic Henderson plots.

Binding, Competitive↗
PubMed1988

Mammalian cyanide detoxification with sulphane sulphur.

It has been understood for nearly a century that most of the cyanide entering mammals by either ingestion or inhalation of sublethal quantities is detoxified by combination with cyanide-reactive sulphur. The detoxifying reaction is primarily enzymic, and the enzymes that catalyse it, as well as those that catalyse reactions which generate the cyanide-reactive sulphane sulphur, have been isolated and studied extensively. However, analytical methods suitable for quantitative characterization of the physiological pool of sulphur available for this detoxification have only recently come under development. A new method is based on the evaluation of cyanide depletion under controlled, mild reaction conditions in the presence of a sulphurtransferase and cofactor. The use of differential pulse polarography with nested internal standards makes this a practical procedure for samples containing as little as a few nanomoles of cyanide-reactive material. As measured by this method, the sulphane sulphur concentration in mammalian liver tissue is in the fractional millimolar range, with even less in the bloodstream. Nevertheless, the specific binding of sulphane sulphur by serum albumin, and the rapid cyanolysis of the albumin-bound sulphur, may yet indicate a significant role for the sulphurtransferase capability of serum albumin in cyanide detoxification.

Animals↗
PubMed1987

Isolation and characterization of a prokaryotic sulfurtransferase.

A sulfurtransferase has been purified to apparent homogeneity from the prokaryote Acinetobacter calcoaceticus lwoffi by conventional protein fractionation techniques. Steady-state kinetic studies of the enzyme revealed that its formal mechanism varies with the acceptor substrate employed. With inorganic thiosulfate as the sulfane sulfur-donor substrate and cyanide anion as the acceptor, the enzyme was shown to catalyze the reaction by a double displacement mechanism like that of mammalian rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1). In contrast, with a thiol as the acceptor substrate at relatively high concentrations, the reaction proceeds by a single displacement mechanism, reminiscent of catalysis by another sulfur-transferase, thiosulfate reductase, glutathione-dependent (EC 2.8.1.3). When dithiothreitol is the acceptor substrate, the enzyme cycles through both the single and double displacement pathways, with the flux through each depending differentially on the concentration of dithiothreitol employed. In view of both the relaxed acceptor substrate specificity and the corresponding variability of formal mechanism, the more general name of sulfane sulfurtransferase is proposed for this bacterial enzyme.

Acinetobacter↗
PubMed1987

Methods for in situ visualization and assay of sulfurtransferases.

The dansyl derivative 5-dimethylamino-1-naphthalene thiosulfonate (DANTS) can serve as a sulfane sulfur-donor substrate for several of the sulfurtransferases, the reaction being dependent on the acceptor substrates supplied. Enzymatic cleavage of the sulfur-sulfur bond of DANTS releases the intrinsic fluorescence of the molecule, with an emission maximum of 500-510 nm (excitation at 325 nm). This process permits selective visualization of active sulfurtransferase enzymes separated in nondenaturing polyacrylamide gels, even from impure preparations. This technique was used to locate rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), thiosulfate reductase (EC unassigned), and a recently isolated prokaryotic enzyme that has been called sulfane sulfurtransferase. In addition, a refinement of the thiosulfate reductase assay technique is reported.

Dansyl Compounds↗
PubMed1986

Serum albumin and cyanide detoxication. Kinetic characterization of a reactive albumin-sulfur complex.

Kinetic studies of the serum albumin-catalyzed reaction of cyanide with colloidal elemental sulfur have been carried out to elucidate the formal mechanism of this reaction. The results indicate that one of the several sulfur-binding sites on the protein molecule is involved in a single displacement catalytic cycle in the presence of cyanide. At high velocities the rate of thiocyanate production is limited by an albumin-independent second order process which produces the form of sulfur that serves as substrate. The catalyzed reaction is also subject to substrate inhibition at high colloidal sulfur concentrations.

Cyanides↗
PubMed1985

Effect of hyaluronidase treatment of intact cells on hyaluronate synthetase activity.

Hyaluronidase treatment of mouse oligodendroglioma cells in monolayer culture resulted in a 4-5-fold stimulation of hyaluronate synthetase, assayed in washed membrane preparations [Philipson, L., & Schwartz, N. B. (1984) J. Biol. Chem. 259, 5017-5023]. We now report studies on the mechanism of the hyaluronidase-induced increase in the specific activity of the membrane-bound synthetase complex. The stimulation was dependent on the concentration of hyaluronidase but not on the particular bond cleaved or the nature of the product generated. Analysis of chain growth during cell-free synthesis by the disaccharide ratio method suggested that substantial internal labeling of hyaluronate chains had occurred. With both treated and untreated membranes, greater than 90% of incorporated (and recovered) radioactivity appeared in unsaturated disaccharides. Further analysis showed that hyaluronidase treatment increased both the rate of elongation and the rate of release of elongated chains from the enzyme complex. Hyaluronidase treatment also caused a change in the apparent steady-state kinetic patterns of double-reciprocal plots from intersecting lines for membranes from control cells to a family of parallel lines. Both the overall stimulation of synthesis and the change in apparent kinetic pattern were reversed by brief incubation of washed cells in the absence of hyaluronidase. These results have led to the development of an explicit kinetic model for hyaluronate synthesis which suggests an explanation for the switch in apparent kinetic patterns based on changing concentrations of a postulated key intermediate.

Animals↗
PubMed1985

Chromogenic substrates for sulfurtransferases.

The azo dye 4-(dimethylamino)-4'-azobenzene (DAB) thiosulfonate anion can serve as a sulfur-donor substrate for rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) and for thiosulfate reductase (EC unassigned) with cyanide anion and GSH, respectively, as acceptor substrates. In either case, the dye product is DAB sulfinate, which differs substantially in light absorption at 500 nm. Moreover, DAB sulfinate can serve as a sulfur-acceptor substrate for rhodanese with either inorganic thiosulfate or a colorless thiosulfonate anion as donor, and this reaction provides a second chromogenic assay procedure.

Azo Compounds↗
PubMed1985

Evidence for dynamically determined conformational states in rhodanese catalysis.

Physical and kinetic studies have been used to explore hysteretic effects that are observed in rhodanese catalysis at pH 5 and also at neutral pH when the ionic strength of the medium is high. Experiments that involve observation of changes in intrinsic protein fluorescence of the enzyme and kinetic investigation of its interactions with product thiocyanate anion at pH 5 have implicated enzyme isomerization as the cause of hysteresis. Taken all together, the data indicate that the conformations of enzyme forms in the catalytic cycle are dynamically determined, depending on the relative rates of conformational relaxation and catalysis as influenced by the concentrations of substrates and products.

Fluorescence↗
PubMed1984

Insulin-receptor interactions. Presence of a positive cooperative effect.

Competitive binding curves for 125I-insulin on a cultured rat hepatoma, H4-II-E-C'3, indicated that low competitive insulin concentrations increased label binding. This upward deflection of the binding curve or "hook effect" occurred at physiological concentrations of insulin. Lectins capable of mimicking insulin also stimulated insulin binding on whole cells, membranes, and solubilized receptors, apparently by increasing the affinity of the receptor for the hormone. An anti-receptor antibody and derived FAB fragments also elicited a similar increase in insulin binding affinity. At insulin levels below 10 ng/ml, the binding curves exhibited sigmoidicity consistent with positive cooperativity. This property appears to be intrinsic to the receptor and may be due to its oligomeric structure.

Animals↗
PubMed1984

Thin-layer chromatography of thiosulfonate anions.

Organic thiosulfonates of the form RS(O2)S- can be separated from the characteristic contaminants in synthetic preparations by thin-layer chromatography on silica gel. Thiosulfonates and sulfinates can both be visualized, and differentiated, by treatment of plates with a FeCl3 reagent in acetone. Data are presented for seven aromatic and six aliphatic thiosulfonate anions and the corresponding sulfonates and sulfinates. With exception of the series related to cysteic acid, Rf values are always in the order RS(O)2S- greater than RSO3- greater than RSO2-.

Anions↗