Penetration of brain and brain tumor. VII. Tumor-binding sulfhydryl boron compounds.
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Animals↗
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A new form of inactive renin in rat brain. A latent renin.
A new inactive (latent) form of renin was found in rat brain extract. It is activated by sulfhydryl compounds such as dithiothreitol but not by proteases such as trypsin. The activated form of latent renin in crude brain extract was again inactivated by the disulfide compound sodium tetrathionate. Latent renin was separated, at least partially, from active renin by affinity chromatography on Affi-Gel Blue. In contrast to a marked (10-fold) increase of latent renin by dithiothreitol, the enzyme activity of active renin was increased by less than 50% by this sulfhydryl compound. Thus, the major part of the activating effect of dithiothreitol does not seem to be due to its effect on renin substrate. Latent renin showed affinity for pepstatin-Sepharose gel. These properties indicate that latent renin is different from inactive renin of the zymogen type, which is activated by protease or acid treatment but not by sulfhydryl compounds and does not show affinity to pepstatin. Latent renin has a molecular weight of 45,000 and is reduced to 34,000 upon activation by dithiothreitol. This observation suggests that latent renin may be a renin-inhibitor complex.
Animals↗
Formation of sulfhydryl groups in the culture medium by human diploid fibroblasts.
When human diploid fibroblasts IMR-90 are cultured in routinely used medium (Eagle's basal medium supplemented with 10% fetal calf serum), sulfhydryl compounds appear in the medium. The major component of these sulfhydryl compounds is cysteine, and it is shown that a part of medium cystine is converted into cysteine by the cells. It is also shown that the sulfhydryl groups of serum albumin, which are masked and barely detectable before the culture, are restored. Probably cysteine formed by the cells reacts with serum albumin to give rise to the protein sulfhydryl groups via sulfhydryl-disulfide exchange reactions. Total sulfhydryl concentrations in the medium are maintained in a considerable level throughout the culture, and a possible physiological function of these sulfhydryl groups is discussed.
Cell Line↗
Four sulfhydryl-modifying compounds cause different structural damage but similar functional damage in murine lymphocytes.
Four thiol-modifying compounds were used to inhibit murine lymphocyte mitogenesis. The compounds were a copper sulfate/O-phenanthroline complex (CuP) to oxidize surface thiols, N-ethyl maleimide (NEM) to alkylate surface and intracellular thiols, D,L-buthionine-S,R-sulfoximine (BSO) to prevent synthesis of glutathione, and hydrogen peroxide, which reacts with various cellular constituents, including sulfhydryls. Splenic lymphocytes were incubated with one of the four compounds, washed, and then stimulated with the B cell mitogen, LPS, or the T cell mitogen, Con A. In spite of their differing chemical reactivities and differing effects on cell viability, lipids, and total, protein, and non-protein thiols, the four sulfhydryl-modifying compounds had very similar effects on the kinetics and inhibition of lymphocyte growth. All compounds had complex effects on mitogenesis, causing enhanced, delayed, or inhibited tritiated thymidine incorporation. Although the total thiol contents of untreated T cells and B cells were found to be equivalent, the LPS response consistently was inhibited by lower concentrations than the Con A response, suggesting that B cells were more sensitive than T cells to thiol modification. To compare compounds the efficiency of inhibition was determined by functionally relating reductions in mitogenesis with reductions in thiol content of the cells. The compounds differed in inhibitory efficiency; thus, damage to some thiols must be more important than damage to others. CuP ablated mitogenesis with the least change in thiol content. Therefore, surface sulfhydryls appear critical in lymphocyte mitogenesis. With all compounds inhibition of mitogenesis occurred over a very narrow range of thiol content, suggesting that the thiols important in inhibition were few in number relative to the total thiol content of the cell.
Animals↗
Probing the major skeletal muscle chloride channel with Zn2+ and other sulfhydryl-reactive compounds.
The sensitivity of the human skeletal muscle Cl- channel, hClC-1, towards various sulfhydryl-reactive agents was tested with the channel expressed in Xenopus oocytes and in human embryonic kidney cells. External but not internal Zn2+, at 1 mM, substantially reduced the current without affecting activation parameters. External Cd2+ and Hg2+ as well as organic mercurial compounds reduced the Cl- currents to a similar degree. With the mutant channel hClC-1 D136G, presumed to have a defective voltage sensor, external Zn2+ also reduced the current without effect on the altered gating. These findings suggest that hClC-1 contains cysteine residues near the extracellular face that may directly influence ion conduction. Since Zn2+ can also bind to histidine side chains, we tested the effect of compounds with either more cysteine- or more histidine-specificity. The results confirm the involvement of cysteine(s) in the observed effects but do not exclude the involvement of histidine(s).
Chloride Channels↗
Neutrophil membrane sulfhydryl groups are involved in stimulated neutrophil adherence to endothelium.
We investigated the role of membrane sulfhydryl groups in adherence of stimulated polymorphonuclear neutrophils to cultured endothelial cells. Treatment of neutrophils with p-chloromercuriphenyl sulfonate (PCMPS), a slowly penetrating sulfhydryl reagent, inhibited phorbol myristate acetate (PMA)- or calcium ionophore A23187-stimulated adherence to cultured human or bovine endothelial cells. At concentrations which completely blocked PMA-stimulated adherence, PCMPS did not cause release of lactic dehydrogenase, inhibit PMA-mediated degranulation or hydrogen peroxide production, or prevent the PMA-induced increased surface expression of CD11b/CD 18 (Mac-1). Coincubation with a competing reduced sulfhydryl compound protected neutrophils from inhibition of PMA-stimulated adherence by PCMPS, whereas coincubation with an oxidized sulfhydryl compound did not. Monobromotrimethylammoniobimane, a nonpenetrating sulfhydryl reagent that is structurally unrelated to PCMPS, also inhibited stimulated neutrophil adherence to endothelium cultures. We conclude that stimulated neutrophil adherence to endothelium involves neutrophil membrane protein sulfhydryl groups.
4-Chloromercuribenzenesulfonate↗
Aminosulfhydryl and aminodisulfide compounds enhance binding of the glucocorticoid receptor complex to deoxyribonucleic acid-coated cellulose and to chromatin.
In the presence of amine-containing sulfhydryl compounds, binding of heat-transformed cytosolic rat liver glucocorticoid receptor complex (GRC) to double-stranded calf thymus DNA-coated cellulose and to rat liver chromatin was enhanced up to 10-fold. These observations were made under conditions when a maximum of 8% of the total GRC bound to DNA in the absence of test compound. Compounds which did not contain both a sulfhydryl and amine group were inactive. Phosphorothioate derivatives of the active sulfhydryl compounds were also inactive. However, pretreatment of the phosphorothioate compounds with alkaline phosphatase restored activity. Upon centrifugation at 8800g, amine-containing disulfide compounds at millimolar concentrations caused considerable sedimentation of the GRC in the absence of DNA-coated cellulose or chromatin and no apparent increase in GRC binding to DNA or chromatin. Amine-containing disulfide compounds at micromolar concentrations did not cause heavy sedimentation of the GRC and enhanced binding of the GRC to DNA-coated cellulose up to 9.5-fold. Thus, diaminosulfhydryl compounds and the disulfide 1,18-diamino-6,13-diaza-9,10-dithiaoctadecane (WR 149,024) possess both the ability to restore and preserve the steroid binding capacity of the glucocorticoid receptor and to enhance binding of the GRC to DNA and chromatin.
Amines↗
Effect of sulfhydryl-containing compounds on the antitumor effects of adriamycin.
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Animals↗
Comparative behavioral toxicity of four sulfhydryl radioprotective compounds in mice: WR-2721, cysteamine, diethyldithiocarbamate, and N-acetylcysteine.
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Acetylcysteine↗
Protective effects of selected sulfhydryl containing compounds against global ischemia in isolated perfused rat hearts.
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Animals↗
Acceleration of nitric oxide (NO) release from FK409, a spontaneous NO releaser, in the presence of sulfhydryl-bearing compounds.
PURPOSE: Recently, we have reported that FK409 spontaneously releases nitric oxide (NO) in solution. In the present study, the influence of L-cysteine (Cys) and glutathione (GSH), which are typical sulfhydryl group-bearing compounds, on NO release from FK409 and biological action of FK409 was examined. METHODS: We evaluated the effects of Cys and GSH on NO release from FK409 by nitrite analysis or detection with a chemiiluminesence analyzer. In a biological study, the influence of Cys on inhibition of rat platelet aggregation of FK409 was investigated. In addition, the above mentioned characteristics of FK409 were compared with those of isosorbide dinitrate (ISDN). RESULTS: FK409 decomposed spontaneously with generation of nitrite in solution. Both Cys and GSH accelerated decomposition of FK409 and nitrite generation from FK409 in a concentration-dependent manner. When the NO levels in the headspace of FK409 solutions (0.5 mM) reached equilibrium with and without 25 mM Cys, the constant rate for NO release from FK409 in the presence of Cys was 13 times larger than that in the absence of Cys. In biological study, FK409 (100 microM) showed 56 and 90% inhibition of rat platelet aggregation in the absence and presence of 10 mM Cys, respectively, whereas ISDN (100 microM) showed 10 and 23% inhibition, respectively. CONCLUSIONS: Decomposition of FK409 with generation of NO is spontaneous, and is accelerated in the presence of sulfhydryl group-bearing compounds, thereby potentiating the biological action of FK409.
Animals↗
Regulation of cytoplasmic tubulin carboxypeptidase activity in vitro by cations and sulfhydryl-modifying compounds.
alpha-tubulin subunits within microtubules (MTs) can be post-translationally detyrosinated by a tubulin-specific carboxypeptidase (TCP) activity to form biochemically distinct MTs. Attempts to characterize and purify TCP have suffered from the inability to detect low levels of activity and to distinguish TCP from other, competing enzyme activities. We recently developed an assay for TCP [Webster et al. (1992) Biochemistry 31:5849] that uses taxol-stabilized MTs as the substrate. In this study, we exploited the increased sensitivity and specificity of this new assay to explore the effects of various agents that might act to either stimulate or inhibit this enzyme in vitro. We tested a variety of both monovalent and divalent cations for their ability to affect TCP, and tested whether the cations were affecting the enzyme, the substrate, or both. We found that TCP displayed salt-sensitive binding to MTs, characteristic of other, more well characterized MT-associated proteins. While both calcium and magnesium stimulated TCP activity over a narrow concentration range (2-10 mM), they inhibited activity at higher concentrations. Other divalent cations tested, including zinc, copper, and cobalt, inhibited TCP at virtually all concentrations tested, but to different levels (zinc > copper > cobalt). Most of the zinc-induced TCP inhibition was attributed to the interference with the normal binding of TCP to MTs. In addition, we examined the involvement of free sulfhydryl groups (which are important for the activities of many types of enzymes) in TCP activity by the addition of sulfhydryl-modifying compounds during the assay, and found that their addition reduced TCP activity mainly (but not solely) by their action on the extract that contained the TCP. Finally, we tested the ability of DL-benzylsuccinic acid, a potent inhibitor of carboxypeptidase A, to inhibit TCP. While carboxypeptidase A has been found, in other studies, to be inhibited by micromolar concentrations, TCP was affected only at concentrations above 20 mM, adding another proof that carboxypeptidase A and TCP are distinct enzyme activities.
Animals↗
Interactions of nitroglycerin and sulfhydryl-donating compounds in coronary microvessels.
Previous studies have shown the effect of nitroglycerin on coronary microvessels < 100 microns in diameter is markedly enhanced by L-cysteine. These studies were performed to examine the mechanisms responsible for this effect. Under control conditions, nitroglycerin caused potent dilations of large (> 200 microns diam) coronary microvessels while having minimal effects on small (< 100 microns diam) coronary microvessels [peak relaxations 85 +/- 4 vs. 23 +/- 3% (mean +/- SE) of endothelin-1-constricted vessels, respectively]. L-Cysteine (100 microM) and N-acetylcysteine (100 microM) markedly enhanced nitroglycerin-induced relaxations of small coronary microvessels (peak relaxation 84 +/- 6 and 87 +/- 12%, respectively) while having no effect on relaxations of vessels > 100 microns. In contrast, neither L-methionine (100 microM) nor glutathione (100 microM) enhanced nitroglycerin's vasodilation of small coronary microvessels. The effects of L-cysteine and N-acetylcysteine on the augmentation of nitroglycerin vasodilatation in smaller coronary microvessels was abolished in the presence of buthionine sulfoximine (100 microM), a potent inhibitor of intracellular glutathione synthesis. Buthionine sulfoximine had no effect on the vasodilatation produced by nitroprusside. These data demonstrate that, in smaller coronary microvessels, L-cysteine and N-acetylcysteine enhance nitroglycerin-induced vasodilatation by increasing intracellular glutathione concentrations. Intracellular glutathione, formed from either L-cysteine or N-acetylcysteine, may participate in the formation of an intermediate of nitroglycerin biotransformation or may maintain a redox potential within coronary microvessels that favors enzymatic bioconversion of nitroglycerin.
Animals↗
Effects of sulfhydryl-containing compounds on nitroglycerin-induced coronary dilatation in isolated working rat hearts.
The effects of various compounds on the time course of the coronary dilating response to nitrovasodilators were studied in working rat hearts. Continuous administration of 100 microM glyceryl trinitrate (GTN) to the perfusate induced a rapid increase in coronary flow. The flow decreased to about 40% of the initial flow rise within 20 min and remained constant during the following 40 min. The flow increase induced by 100 microM SIN 1 (3-morpholino-sydnonimine), however, remained constant throughout the 60-min perfusion period. The decay of the GTN action was reduced in the presence of the thiol-containing agents L-homocysteine, L- and D-cysteine and to a lesser extent, reduced glutathione, while the initial response to GTN was only slightly enhanced. However, N-acetyl-L-cysteine caused a significant increase of the maximum response to GTN whereas the subsequent loss of GTN action was not greatly altered, suggesting that this agent exerts a potentiating action without altering tolerance. Dithiothreitol elicited a slight increase in the maximum effect of GTN, followed by a complete attenuation of the effect. These data suggest that SH group donors could exert different effects on the action of GTN while GTN tolerance is only influenced by L- and D-cysteine, L-homocysteine and reduced glutathione.
Amino Acids↗
High-performance liquid chromatographic determination of penicillamine in whole blood, plasma, and urine.
A high-performance liquid chromatographic method for the determination of penicillamine in plasma, whole blood, and urine samples is described. The method uses a commercially available electrochemical detector at a potential of +0.1 V versus the Ag/AgCl reference electrode. This method is selective and sensitive for sulfhydryl compounds. The chromatography separates penicillamine from other endogenous sulfhydryl compounds with a limit of detection for penicillamine in biological samples of ca. 10(07) M.
Buffers↗
Inhibition by converting enzyme inhibitors of pig kidney aminopeptidase P.
Several inhibitors of angiotensin converting enzyme were also found to inhibit aminopeptidase P, whereas inhibitors of other mammalian aminopeptidases were ineffective. Aminopeptidase P purified from pig kidney cortex was found to contain one atom of zinc per polypeptide chain, confirming its metalloenzyme nature. The concentrations of converting enzyme inhibitors required to cause 50% inhibition (I50) of aminopeptidase P were in the low micromolar range. The most potent converting enzyme inhibitors toward aminopeptidase P were the carboxylalkyl compounds, cilazaprilat, enalaprilat, and ramiprilat (I50 values of 3-12 microM). The sulfhydryl compounds captopril (I50 110 microM) and YS980 (I50 20 microM) were slightly less potent at inhibiting aminopeptidase P. In contrast, the carboxylalkyl compounds benazeprilat, lisinopril, and pentoprilat; the sulfhydryl compound rentiapril; and the phosphoryl compounds ceranopril and fosinoprilat had no inhibitory effect against aminopeptidase P. This compares with I50 values in the 1-6 nM range for these inhibitors with angiotensin converting enzyme. Inhibition of aminopeptidase P may account for some of the effects or side effects noted with the clinical use of converting enzyme inhibitors. These results may provide the basis for the design of more selective inhibitors of angiotensin converting enzyme or mixed inhibitors of aminopeptidase P and angiotensin converting enzyme, or both.
Aminopeptidases↗
Effect of polyamines on acidified ethanol-induced gastric lesions in rats.
The participation of polyamines and nonprotein sulfhydryls in the gastric cytoprotective mechanisms was studied using gastric mucosal lesions produced by acidified ethanol in rats as an experimental model. Treatment with prostaglandin E2 (PGE2), but not cimetidine, prevented the formation of gastric mucosal lesions. Oral administration of cadaverine, spermidine and spermine prevented the lesion formation by acidified ethanol in a dose-dependent manner. Indomethacin or acetazolamide had no influence on the cytoprotective effect of spermine, whereas sulfhydryl blockers such as iodoacetamide and N-ethylmaleimide partially blocked it. Sulfhydryl compounds such as cysteine, reduced glutathione (GSH), and cysteamine prevented the lesion formation induced by acidified ethanol. The concentration of nonprotein sulfhydryls in the gastric mucosa was significantly decreased at 1 hr after administration of acidified ethanol, and this decrease was partially prevented by spermine or PGE2. These results suggest that the cytoprotective effect of spermine may not be mediated by endogenous prostaglandins or alkaline secretion in the gastric mucosa, but may be partially related to endogenous sulfhydryl compounds.
Animals↗
Purification and characterization of rat brain prostaglandin D synthetase.
Prostaglandin D synthetase was purified 2,600-fold from rat brain to apparent homogeneity, as judged by polyacrylamide gel electrophoresis and ultracentrifugation. The purified enzyme was a monomeric protein with a molecular weight of 27,000 +/- 1,000. The pI value, sedimentation coefficient, and partial specific volume were 4.6, 4.1 s, and 0.73 ml/g, respectively. The enzyme was stable between pH 4 and 11 at the temperature lower than 25 degrees C and resistant to a heat treatment under alkaline conditions (pH 8-11). About 50% of the activity was detected after a heat treatment at 100 degrees C for 5 min at pH 10. However, the enzyme was readily inactivated by the isomerase reaction of prostaglandin H2 to prostaglandin D2. The enzyme required sulfhydryl compounds such as dithiothreitol, glutathione, beta-mercaptoethanol, cysteine, and cysteamine for the reaction, but stoichiometric oxidation of these sulfhydryl compounds was not observed. The optimum pH, Km value for prostaglandin H2, and the turnover number were 9.5, 14 microM, and 170 min-1, respectively. The antibody was raised against the purified enzyme in a rabbit, which showed only one positive band in immunoblotting after gel electrophoresis of crude extracts of the brain at the same position as that of the purified enzyme. More than 90% of the prostaglandin D synthetase activity in the brain was absorbed by an excess amount of the antibody, indicating that our preparation is a major component of the enzyme responsible for the biosynthesis of prostaglandin D2 in the brain.
Animals↗