Sulfhydryl-dependent Enzymes Research Articles

What caused hemolytic anemia and colicky abdominal pain? Lead!

What caused hemolytic anemia and colicky abdominal pain? Lead!

Butyrylcholinesterase and Erythrocyte Sulfhydryl-dependent Enzyme Hydrolyze Gabexate in Human Blood

Gabexate (GB), a serine protease inhibitor that is widely used for the treatment of acute pancreatitis and disseminated intravascular coagulation, has been reported to be partly hydrolyzed by human serum albumin. However, other enzymes responsible for GB hydrolysis in human blood remain unclear. In this study, we examined in vitro metabolism of GB with human blood samples. The hydrolytic activities of the plasma and erythrocytes at 100µM of GB were 167 ± 26 and 151 ± 9n mol/min/ml blood fraction (mean ± S.D., n = 8), respectively. Kinetic analysis indicated that Vmax and Km values were 1.75µmol/min/ml blood fraction and 529µ Mf or the plasma and 10.6µmol/min/ml blood fraction and 7360µ Mf or the erythrocytes, respectively. The activity of human plasma wa si nhibited by ethopropazine, a butyrylcholinesterase inhibitor (27% inhibition at 100µM). Furthermore, the plasma activity was inhibited by 5,5 � -dithiobis(2-nitrobenzoic acid) (DTNB) (40% inhibition at 5000µM), suggesting the involvement of albumin in the plasma GB hydrolysis. The erythrocyte activity was also decreased by DTNB (56% inhibition at 5000µM), implying that this activity was dependent on the presence of sulfhydryl group(s), while little or no inhibition of the activity was seen with phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, and BW284C51. Butyrylcholinesterase from human serum showed GB hydrolytic activity with Vmax of 363 nmol/min/m gp rotein and Km of 263µM. These results suggest that, in addition to albumin, butyrylcholinesterase and erythrocyte sulfhydryl-dependent enzyme are responsible for GB hydrolysis in human blood.

Spontaneous hydrolysis of 4-trifluoromethylphenol to a quinone methide and subsequent protein alkylation

4-Trifluoromethylphenol (4-TFMP) was cytotoxic to precision-cut rat liver slices as indicated by loss of intracellular potassium. Intracellular glutathione levels decreased and fluoride ion levels increased in a time and concentration-dependent manner. The cytotoxicity of 4-TFMP did not appear to be due to the release of fluoride, however, since equimolar concentrations of sodium fluoride or potassium fluoride were not toxic. The ortho isomer (2-TFMP), which had a threefold slower rate of fluoride release, was much less toxic to liver slices. In incubations without slices, 4-TFMP spontaneously hydrolyzed in aqueous buffer at physiological pH to form 4-hydroxybenzoic acid via a quinone methide intermediate. The quinone methide was trapped by the addition of glutathione. Analysis of the glutathione adduct indicated that all of the fluorine atoms were lost during the hydrolysis, yielding a cresol derivative with the glutathione moiety attached to a benzylic carbonyl group. The glutathione conjugate was the primary product formed at low alkylphenol/glutathione ratios; however, at higher 4-TFMP concentrations additional unidentified products were observed. 4-TFMP also inhibited the in vitro enzyme activity of purlfied glyceraldehyde-3-phosphate dehydrogenase, a sulfhydryl-dependent enzyme, in a time and concentration-dependent manner. Loss of thiol residues closely paralleled the loss in enzyme activity. The coaddition of glutathione prevented 4-TFMP-induced loss of enzyme activity. The cytotoxicity of 4-TFMP therefore appears to be due to spontaneous quinone methide formation and subsequent alkylation of cellular macromolecules.

The effect of cytosol on liver microsomal metabolic activation and demethylation of N-nitrosodimethylamine.

The role of rat liver cytosol in the demethylation and metabolic activation of N-nitrosodimethylamine (NDMA) was examined. Addition of cytosol to liver microsomes from pyridine-pretreated rats enhanced DNA alkylation by NDMA 10- to 14-fold over microsomes alone, while cytosol alone had little DNA alkylating activity. The cytosolic activity responsible for the enhancement of DNA alkylation was heat labile, required NADPH, and was not a general protein effect. Addition of cytosol to purified rabbit liver cytochrome P450 2E1 in a reconstituted system consisting of NADPH-cytochrome P450 reductase, 2E1, and phospholipid produced an 18-fold increase in DNA alkylation over that observed with the reconstituted system alone. The cytosolic activity responsible for the enhancement of DNA alkylation did not work by inhibition of lipid peroxidation, nor did the addition of cytosol affect the level of NADPH present in the reaction mixtures. Attempts to identify the cytosolic component(s) responsible for the DNA alkylation enhancing activity demonstrated no evidence for the involvement of sulfhydryl-dependent enzymes, a flavoprotein, or conjugating enzymes. Studies with semicarbazide and phenylhydrazine suggest that carbonyl groups may be involved in the cytosolic activity. Measurements of NDMA demethylation demonstrated that cytosol addition led to a significant decrease in formaldehyde production, indicating that cytosol was not enhancing the activation of NDMA to a DNA alkylating species by facilitating the cytochrome P450-catalyzed demethylation reaction, and suggested that a cytosolic reaction might be occurring at the expense of formaldehyde formation.

Dibucaine elicits platelet procoagulant activity in factor VIII and factor X activation by a mechanism involving a sulfhydryl-dependent enzyme

Dibucaine, a potent inhibitor of platelet aggregation and platelet release, was found to enhance the ability of fresh gel-filtered or washed human platelets to support factor VIII activation and factor X activation. Dibucaine-treated platelets increased the peak of factor VIII clotting activity by 2-fold compared to activity with untreated platelets. Similarly platelets optimally stimulated by dibucaine (1.0–1.5 mM for 5 min at 37°C) supported as much factor X activation by factors IXa and VIII (measured in a chromogenic assay) as platelets optimally stimulated by ionophore A23187 (15 μM). An assay of platelet calcium-dependent sulfhydryl proteases was devised and used to test the effect of various inhibitors on these platelet proteases. The membrane-permeable sulfhydryl inhibitor Thiolyte MB inhibited platelet calcium-dependent protease activity; whereas, membrane-impermeable Thiolyte MQ did not. Thiolyte MB also blocked the ability of dibucaine-stimulated platelets to support factor X activation. Incubation of fresh, gel-filtered platelets with calpain inhibitor II (N-Ac-L-L-Normethioninal) completely inhibited the calcium-dependent sulfhydryl protease activity of these platelets but did not affect their ability to support factor X activation after subsequent incubation with dibucaine. These data support the interpretation that an intracellular SH-dependent enzyme, which may not be calpain, is involved in the expression of platelet procoagulant activity in dibucaine-treated platelets.

Mechanisms of H2O2-mediated injury to type II cell surfactant metabolism and protection with PEG-catalase

Alterations in type II pneumocyte function, including surfactant biosynthesis, may play a significant role in the development and pathophysiology of oxidant-induced lung injury. The results of this study showed that type II cells exposed to 50-300 microM H2O2 demonstrated a dose-dependent decrease in phosphatidylcholine (PC) synthesis with only minimal changes in cell viability. The activities of the choline-phosphate cytidyltransferase and cholinephosphotransferase, specific enzymes of PC synthesis, were not significantly decreased by the exposure. However, the activity of glycerol-3-phosphate acyltransferase, a sulfhydryl-dependent enzyme involved in an early stage of phospholipid synthesis, was decreased by the exposures in a manner that was similar to that seen for PC synthesis. Further studies showed that incubation of type II cells with polyethylene glycol-conjugated catalase for 1 h resulted in an increase in the cell-associated catalase activity (53 +/- 5 vs. 6.7 +/- 1.5 units/mg protein for controls). Confocal microscopy analysis showed that a significant portion of this activity was located intracellularly. More importantly, these cells were protected from changes in PC synthesis rates when subsequently incubated with 300 microM H2O2. These results indicate that the deleterious effects of H2O2 on type II cell surfactant synthesis may be pharmacologically modified in vitro, a concept that may have utility with regard to the modulation of in vivo lung injuries.

Polymeric thiols as enzyme activators of serum creatine phosphokinase.

Several hydrophilic polymeric thiols were prepared from amino-activated polymeric supports by reaction with N-acetylhomocystein ethiolactone. Supports include agaroses, cellulose, Glycophase controlled-pore glass, and Matrex acrylic beads. Thiol content in these polymers was 3-72 mumol SH/g dry polymer. Several were effective solid-phase activators of the sulfhydryl-dependent enzyme creatine phosphokinase at concentrations comparable to that of monomeric thiol required for enzyme activation. The kinetic activation curves for the polymeric and the monomeric (thioglucose) activators were similar, suggesting unhindered interaction of the enzyme with the polymeric activator.

Phosphorylation characteristics of brain clathrin-coated vesicle endogenous proteins.

Clathrin-coated vesicles purified from bovine brain express protein kinase activity on two principal endogenous vesicle-associated substrates: a 50,000-Mr polypeptide (pp50) and clathrin-associated protein2 (CAP2; the faster-migrating clathrin light chain). Various exogenous substrates, e.g., casein, phosvitin, histone II, and histone III, also are phosphorylated. The pp50 protein kinase activity of clathrin-coated vesicles is not modulated by Ca2+, calmodulin, phosphatidylserine, or cyclic AMP. On the other hand, phosphorylation of the other endogenous substrates requires certain activators, including histone, polylysine, polyarginine, or polyethylenimine. Phosphate incorporation into pp50 was sensitive to divalent cations that inhibit sulfhydryl-dependent enzymes in the following order of potency: Zn2+ greater than Hg2+ greater than Cd2+, Cu2+, and Pb2+. Phosphate incorporation into CAP2 with polylysine present was insensitive to divalent cations. The alkylating agents dithiodinitrobenzene, phenacyl bromide, and N-ethylmaleimide inhibited phosphate incorporation into pp50 up to 90% without affecting incorporation into the other substrates. Vanadium pentoxide inhibited phosphorylation of CAP2 but had a minimal effect on pp50. CAP2 kinase activity was separated from the coated vesicle membrane and from dis-assembled clathrin triskelions, coeluting with the assembly polypeptide complex on a Sepharose 4B column. It retained phosphorylation properties similar to those of intact vesicles. These data imply that clathrin-coated vesicle kinases are elements of the coat proteins and may be involved in the assembly/disassembly of clathrin triskelions or interactions of coated vesicles with other cellular components.

Internalization and degradation of glucagon by human mononuclear cells.

The processing of glucagon by circulating human mononuclear cells was examined. Glucagon bound to the membrane with a turnover time of 4.4 minutes per site after 15 minutes of incubation and 8 minutes per site after 90 minutes. The amount of intact intracellular hormone increased by 3-fold by 90 minutes suggesting a slowing of intracellular processing with prolonged incubation. Excess unlabelled insulin also slowed the processing of glucagon at the degradative step with no effect on binding or internalization of glucagon. Subcellular fractionation of the cells showed that most hormone accumulated in the 500xg pellet and in the 100,000xg supernatant. N-ethylmaleimide blocked intracellular glucagon degradation suggesting a role for intracellular sulfhydryl-dependent enzymes. Kinetic analysis of the dissociation of glucagon revealed a second order process with K values of 2.2 X 10(-2) fm-1 min-1 and 1.4 X 10(-2) fm-1 min-1 for dissociation from membranes and from membranes + intracellular sites, respectively. T 1/2 values were 6 min. for membrane dissociation and 9 min for membranes + cells. These findings suggest that glucagon interaction with mononuclear cells has characteristics similar to other receptor bound ligands including internalization processing and metabolism.

Biochemical and electrophysiologic effects of thallium poisoning on the rat corpus striatum

Rats were given thallium acetate (5 mg of thallium/kg) ip daily for 7 days. The corpus striatum was removed, and several enzymes and protein estimations were made. Succinic dehydrogenase, monoamine oxidase, acid phosphatase, guanine deaminase and protease (cathepsin) activities in the corpus striatum were depleted by thallium, whereas adenosine triphosphatase and adenine deaminase activity were unaffected. Protein content of the corpus straitum was significantly increased, which could be due to a depletion of cathepsin and acid phosphatase; sulfhydryl-dependent enzymes were particularly depleted. Single neuron activity of the caudate nucleus, recorded after iv injection of thallium acetate (10 mg/kg body weigh), showed an increased firing rate of the caudate neurons, which could possibly be due to an imbalance of neurotransmitters.

Isolation of -L-glutaminyl 4-hydroxybenzene and -L-glutaminyl 3,4-benzoquinone: a natural sulfhydryl reagent, from sporulating gill tissue of the mushroom Agaricus bisporus.

Early in the development of spores, there appears in gill tissue of the mushroom Agaricus bisporus a red pigment that inhibits mitochondrial respiration. The inhibitor and its immediate precursor were isolated from the mushroom and identified as gamma-L-glutaminyl 3,4-benzoquinone (I) and gamma-L-glutaminyl 4-hydroxybenzene (II), respectively, neither of which had previously been described. II was synthesized chemically and the synthetic material was identical with isolated II in all regards. An enzyme that oxidizes II to I was isolated concurrently. I reacts unusually rapidly, completely, and at low concentration with the sulfhydryl groups of various mitochondrial enzymes, accounting for its originally observed properties. It may also prove of value as a general inhibitor of sulfhydryl-dependent enzymes.

Peptidases in the skin

Rat and human skin homogenate were fractionated using gel filtration of Sephadex G-100, and several enzymes hydrolysing dipeptide naphthylamides were partially separated. The presence of the following enzymes was demonstrated: 1. an enzyme hydrolysing glycyl-proline naphthylamide optimally at pH 7.6–7.8, corresponding to a similar enzyme found in the rat liver, 2. an enzyme hydrolysing lysyl-alanine naphthylamide optimally at pH 5.3–5.5, corresponding to the so-called dipeptidyl arylamidase II of bovine pituitary, 3. an sulfhydryl-dependent enzyme hydrolysing arginyl-arginine naphthylamide optimally at pH 8.0, corresponding to the so-called dipeptidyl arylamidase III of bovine pituitary, 4. a sulfhydryl-dependent enzyme hydrolysing alanyl-alanine naphthylamide optimally at pH 5.0, corresponding to cathepsin C. This enzyme was found to be present in the human skin only. In addition, aminopeptidase B, earlier demonstrated in the rat skin, was found to be present in the human skin.

Peptidases in the skin

Rat and human skin homogenate were fractionated using gel filtration of Sephadex G-100, and several enzymes hydrolysing dipeptide naphthylamides were partially separated. The presence of the following enzymes was demonstrated: 1. an enzyme hydrolysing glycyl-proline naphthylamide optimally at pH 7.6–7.8, corresponding to a similar enzyme found in the rat liver, 2. an enzyme hydrolysing lysyl-alanine naphthylamide optimally at pH 5.3–5.5, corresponding to the so-called dipeptidyl arylamidase II of bovine pituitary, 3. an sulfhydryl-dependent enzyme hydrolysing arginyl-arginine naphthylamide optimally at pH 8.0, corresponding to the so-called dipeptidyl arylamidase III of bovine pituitary, 4. a sulfhydryl-dependent enzyme hydrolysing alanyl-alanine naphthylamide optimally at pH 5.0, corresponding to cathepsin C. This enzyme was found to be present in the human skin only. In addition, aminopeptidase B, earlier demonstrated in the rat skin, was found to be present in the human skin.

Enzymes in hogs kidney hydrolyzing amino acid naphthylamides.

Hydrolysis of β-naphthylamides of a number of amino acids and dipeptides and of a number of di- and tripeptides by hog kidney homogenate and by fractions obtained by various fractionation procedures has been studied. The substrates were found to be split by a soluble, apparently sulfhydryl-dependent enzyme, and by a particle-bound, metal-activated enzyme. The former constituted only a small part of the total activity. The latter was subfractionated by starch gel electrophoresis into two fractions with identical characteristics. The soluble and particle-bound enzymes differed also in their substrate specificity. The latter enzyme was solubilized, partially purified, characterized by some modifier compounds and compared with enzyme preparations obtained by various fractionation procedures presented by other investigators. Thus enzyme showed ion-determined substrate specificity, i.e., hydrolysis of some of the amino acid naphthylamides was found to be activated by Co++ while the hydrolysis of others was inhibited by the same metal ion.

STUDIES ON THE PATHOGENESIS OF FEVER. 13. THE EFFECT OF PHAGOCYTOSIS ON THE RELEASE OF ENDOGENOUS PYROGEN BY POLYMORPHONUCLEAR LEUKOCYTES.

1. Phagocytosis promotes the release of endogenous pyrogen from polymorphonuclear leucocytes. 2. The release of pyrogen, though initiated by the phagocytic event, is not synchronous with it. 3. The postphagocytic release mechanism is not inhibited by sodium fluoride and, therefore, appears not to require continued production of energy by the cell. 4. The release process, on the other hand, is inhibited by arsenite, suggesting the participation of one or more sulfhydryl-dependent enzymes in the over-all reaction. 5. Particle for particle, the ingestion of heat-killed rough pneumococci causes the release of approximately 100 times as much pyrogen as the ingestion of polystyrene beads of the same size. 6. The pyrogen release mechanism of polymorphonuclear leucocytes separated directly from blood, unlike that of granulocytes in acute inflammatory exudates, is not readily activated by incubation of the cells in K-free saline. Despite this difference, both blood and exudate leucocytes following phagocytosis release large amounts of pyrogen, even in the presence of K(+). The fact that the postphagocytic reaction is uninhibited by the concentrations of K(+) which are present in plasma and extracellular fluids, suggests that this mechanism of pyrogen release may well operate in vivo. 7. As might be expected from the foregoing observations, the intravenous injection of a sufficiently large number of heat-killed pneumococci causes fever in the intact host. Intravenously injected polystyrene beads, on the other hand, are significantly less pyrogenic. Evidence is presented to support the conclusion that the fever in both instances is caused by pyrogen released from the circulating leucocytes which have phagocyted the injected particles. 8. The possible relationships of these findings to the pathogenesis of fevers caused by acute bacterial infections are discussed.