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PubMed1976

Is fatty liver induction a general feature of the administration of foreign sulfhydryl compounds?

The intraperitoneal administration of 2-mercaptoethanol or 2-mercaptoacetate (40 muM/100 g body weight) to the rat induces a fatty liver, a marked and early increase of free fatty acids and a decrease of triacylglycerol and phospholipids in the blood. These changes are accompanied by a decrease of the ketone body level and the beta-hydroxybutyrate/acetoacetate ratio in the liver. Under the same experimental conditions, however, administration of 2-mercaptopropionate fails to induce a fatty liver and does not modify the hepatic ketone body level or the blood triacylglycerol and free fatty acid levels. These results led us to conclude that fatty liver induction is not a general feature of foreign thiols, and suggest that increased peripheral fat mobilization as well as decreased hepatic lipoprotein synthesis and/or release are responsible for the 2-mercaptoethanol- and 2-mercaptoacetate-induced fatty liver.

Animals↗
PubMed1978

Influence of oxygen tension, sulfhydryl compounds, and serum on the motility and virulence of Treponema pallidum (Nichols strain) in a cell-free system.

The motility and virulence of Treponema pallidum (Nichols strain) were monitored during incubation in a modified tissue culture medium to study the effects of oxygen tension and medium composition on survival of the organism. A basal medium of Eagle minimal essential medium with 50% fresh, heat-inactivated normal rabbit serum was used inasmuch as better survival occurred with 50% normal rabbit serum than with lower concentrations. Addition of 0.5 to 2.0 mM dithiothreitol or 2.0 mM dithioerythritol to the basal medium led to significantly longer retention of T. pallidum viability in the presence of 3% oxygen than under aerobic or anaerobic conditions. The results of this investigation lend support to the classification of T. pallidum as a microaerophilic organism and provide direction for the design of potentially successful culture systems, with or without tissue culture cells.

Blood↗
PubMed1976

[Effect of sulfhydryl compounds on the automatism of the pacemakers].

Experiments were performed on the isolated rabbit hearts and also on the hearts with complete atrioventricular block; a study was made of the effect of an excess or deficiency of the sulfhydryl groups on the automatism of the cardiac pace makers. Unithiol and cysteine in concentrations of 1-10(-6)-1-10(-4) g/ml were used as donors of sulfhydryl groups; deficiency of these groups was induced by the alloxan administration in concentrations of 1-10(-5)-5-10(-5) g/ml. Changes in the sulfhydryl group content produced no marked effect on the automatism of the synoatrial node. An excess of sulfhydryl groups promoted poststimulation depression of the automatism of the potential pace makers of the cardiac ventricles and could lead to the origination of Luciani's periods. On the contrary, in case of a deficiency of the sulfhydryl groups there was a sharp elevation of the automatism of the ventricular pace makers, the atrioventricular conduction became disturbed, and the poststimulation depression of the automatism became considerably diminished. Disturbances of the cardiac activity caused by the sulfhydryl group deficiency were completely eliminated by unithiol or cysteine.

Animals↗
PubMed1979

Multiple hemoglobins in Triturus cristatus: their degradation by sulfhydryl compounds.

1. Hemolysates of newt (Triturus cristatus) red cells contain four distinct hemoglobin species which have been observed consistently both in individual animals and pooled samples. 2. Hemoglobin heterogeneity in the species arises from existence of multiple hemoglobulins, with no indication of genetic polymorphism. 3. While the relative proportions of the different hemoglobins may vary in different samples, HbII is usually the most abundant, with HbIII and HbIV constituting most of the remainder of the total hemoglobin complement. HbI never exceeds 3-5% of the total hemoglobin. 4. Neither the electrophoretic migration nor the anion exchange properties of the four hemoglobin species are altered by conversion of oxyhemoglobin to the cyanmet derivative, excluding artifacts due to different oxidation states of iron. 5. The average molecular weight of newt total hemoglobulin is 67,182 with no indication of hemoglobin polymers. 6. The use of sulfhydryl-reducing agents (mercaptoethanol, dithiothreitol) as a precaution against aggregation results in extensive degradation of newt hemoglobin through a process similar to "coupled oxidation" by ascorbate. 7. The degradative effects of sulfhydryl-reducing agents on newt hemoglobin suggest that these reagents be used cautiously in any hemoglobin analysis.

Animals↗
PubMed1976

Processing of mengovirus precursor polypeptides in the presence of zinc ions and sulfhydryl compounds.

The effect of zinc ions on the post-translational cleavage of mengovirus polypeptides has been examined. The cleavage of the "A" precursor, which gives rise to the capsid proteins, was the most sensitive at concentrations of zinc chloride from 0.1 to 1.0 mM. Beta-mercaptoethanol and dithiothreitol antagonized the zinc-promoted inhibtion of clevage. Our results indicate that zinc ions interfere with the proper folding of the nascent polypeptide precursor rather than inhibit the proteases responsible for the cleavages. Thus, proper folding of mengovirus polypeptide "A" appears to be necessary for subsequent processing by proteases.

Dithiothreitol↗
PubMed1977

Stability of commonly used thiols and of human creatine kinase isoenzymes during storage at various temperature in various media.

We assessed the stability--at 20, 4 --20, and --80 degrees C--of 2-mercaptoethanol, dithioerythritol, and dithiothreitol, and of semi-purified creatine kinase isoenzymes of human origin in the presence and absence of the three compounds. The isoenzymes plus the sulfhydryl compounds were also assessed an aqueous buffer, in heat-inactivated pooled sera, or in fresh pooled sera with low endogenous total creatine kinase. We also examined the influence of thawing conditions from--20 or--80 degrees C, of fast freezing, and of exposure to fluorescent light. At 20 and 4 degrees C some--SH groups are oxidized. At--20 and--80 degrees C this loss is diminished. It is a function of both temperature and diluent. For stability, creative kinase isoenzymes stored at any temperature require the presence of a suitable sulfhydryl compound. Periodic addition of fresh sulfhydryl solutions stabilized creatine kinase isoenzymes suspended in the two protein-based diluents by 10-30%; for those in an aqueous buffer such additions decreased the activity to stored creatine kinase isoenzymes. Fast freezing does not increase recovery of creatine kinase isoenzyme activity over slow freezing at--80 or--20 degrees C. Thawing should be done as infrequently as possible and should be done rapidly, at 37 degrees C, for the residual enzyme activity to be maximal.

Creatine Kinase↗
PubMed1977

The role of sulfhydryl groups in the binding of glucocorticoids by cytoplasmic receptors of lung and other mammalian tissues.

We examined the levels and stability of glucocorticoid receptor activity in cytosol preparations of rat lung and other tissues. [3H]Dexamethasome binding capacity at 2 C in lung cytosol decreases with a t1/2 of 40 min in the absence of steroid or a sulfhydryl compound. This rapid inactivation of unbound receptor is prevented and reversed by addition of a sulfhydryl compound (t1/2 = 22h); maximal binding occurs with 2 mM dithiothreitol (DTT), or with 20-25 mM mercaptoethanol, thioglycerol or glutathione. Binding activity is also stabilized by formation of the receptor-steroid complex; dissociation of the complex occurs at the same rate (t1/2 = 18.6h) in both the presence and absence of DTT. In the presence of DTT, cytosol of adult rat lung specifically binds 0.82 pmol of [3H]dexamethasone per mg protein (average 19,000 sites per cell) with an equilibrium dissociation contant of 2.5 nM at 2 C. At 37 C, dispersed lung cells show specific nuclear binding of hormone. DTT also increased dexamethasone binding activity in cytosol prepared from lung of adult hamster, adult mouse, fetal monkey and 11 of 15 tissues of adult rat. There was a minimal effect of DTT in reactions using cytosol of fetal rat lung, fetal and newborn human lung, rabbit lung, and liver, kidney, heart and testis of adult rat. Liver contains a heat-stable factor which mimics that stabilizing effect of DTT. The variable requirement for DTT may reflect in part tissue concentrations of endogenous sulfhydryl compounds. Our findings indicate that glucocorticoid binding activity of lung and many other tissues decays rapidly in vitro due to oxidation of receptor sulfhydryl groups. Maintenance of these groups in the reduced form by endogenous tissue factors, addition of sulfhydryl compounds, or binding of glucocorticoid stabilizes receptor and allows its detection in lung and other tissues previously found to contain little if any activity.

Adrenalectomy↗
PubMed1978

Evidence for a role of sulfhydryl groups in catalytic activity and subunit interaction of the cyclic GMP-dependent protein kinase from silkworm.

Guanosine 3':5'-monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) has been isolated from silkworm pupal fat body (Bombyx mori) which is devoid of any adenosine 3':5'-monophosphate-dependent protein kinase. The enzyme displayed catalytic properties which were roughly similar to those described for adenosine 3':5'-monophosphate-dependent protein kinase. This similarity has been found in substrate specificity, optimal Mg2+ dependency, polyamines effects and the lack of dependency upon sulfhydryl compound for activation by cyclic GMP. Treatment of the enzyme with sulfhydryl reagents, N-ethylmaleimide or p-chloromercuribenzoic acid, inhibited the catalytic activity as well as the dissociation of the binding and catalytic activities as shown by means of sucrose-density gradient ultracentrifugation. In the presence of cyclic GMP or histone, the disulfide-linked structure did not dissociate into separate subunits nor did it migrate as the holoenzyme but sedimented as an intermediate form carrying both binding and catalytic activities.

Animals↗
PubMed1978

Effects of beta-mercaptoethanol and chelating agents on the stability and activation of creatine kinase in serum.

We investigated the effects of sulfhydryl compounds on the stability of creatine kinase (CK) in unfrozen human serum and found that both beta-mercaptoethanol and N-acetylcysteine led to accelerated loss of the endogenous serum enzyme activity. This is in contrast to the results of others who have either studied the stability of exogenous enzyme added to human serum or studied endogenous enzyme in frozen serum. The addition of cation chelators to serum markedly improved the stability of the endogenous CK activity. The enhanced stability was independent of chelation of calcium, iron, manganese, copper, or zinc. In addition, cation chelators caused a 16% increase in the CK activity of fresh samples. This latter effect was independent of the activation of CK by BME and could be accounted for by chelation of calcium ions during the assay. The data suggest that addition of cation chelators prior to storage may be useful in enhancing the stability of CK in serum whereas sulfhydryl compounds should be added prior to assay rather than prior to storage.

Calcium↗
PubMed1979

Studies on regulatory functions of malic enzymes. IV. Effects of sulfhydryl group modification on the catalytic function of NAD-linked malic enzyme from Escherichia coli.

Reactions catalyzed by NAD-linked malic enzyme from Escherichia coli were investigated. In addition to L-malate oxidative decarboxylase activity (Activity 1) and oxaloacetate decarboxylase activity (Activity 2), the enzyme exhibited oxaloacetate reductase activity (Activity 3) and pyruvate reductase activity (Activity 4). Optimum pH's for Activities 3 and 4 were 4.0 and 5.0, and their specific activities were 1.7 and 0.07, respectively. Upon reaction with N-ethylmaleimide (NEM), Activity 1 decreased following pseudo-first order kinetics. Activity 2 decreased in parallel with Activity 1, while Activities 3 and 4 were about ten-fold enhanced by NEM modification. Modification of one or two sulfhydryl groups per enzyme subunit caused an alteration of the activities. Tartronate, a substrate analog, NAD+, and Mn2+ protected the enzyme against the modification. The Km values for the substrates and coenzymes were not significantly affected by NEM modification. Similarly, other sulfhydryl reagents such as p-hydroxymercuribenzoate (PMB), 5,5'-dithiobis(2-nitrobenzoate) (DTNB), and iodoacetate inhibited the decarboxylase activities and activated the reductase activities to various extents. Modification of the enzyme with PMB or DTNB was reversed by the addition of a sulfhydryl compound such as dithiothreitol or 2-mercaptoethanol. Based on the above results, the mechanism of the alteration of enzyme activities by sulfhydryl group modification is discussed.

Dithionitrobenzoic Acid↗
PubMed1976

Effect of catechol-thiol conjugates on tyrosinase-dependent tyrosine hydroxylation.

1. The tyrosinase reaction in the presence of a thiol compound was studied using mushroom tyrosinase (EC 1.10.3.1) with regard to catechol-thiol conjugates. 2. Although tyrosine hydroxylation of tyrosinase was extremely decreased in the presence of a thiol compound, the inhibitory effect was removed by the addition of a pyrocatechol-cysteine conjugate, S-(2,3-dihydroxyphenyl) cysteine, which was not oxidized by the enzyme. 3. The pyrocatechol-cysteine conjugate was also able to shorten the lag period of tyrosinase-dependent tyrosine hydroxylation. 4. The sigmoidal reaction curve of tyrosine hydroxylation observed in the presence of sulfhydryl compounds was found to be caused by the catechol-thiol conjugates, the final products of the enzyme reaction, which counteract the inhibitory effect of sulfhydryl compounds. 5. The pyrocatechol-cysteine conjugate, on the other hand, was shown to cause the decrease of the reaction rate of the enzyme during incubation.

Basidiomycota↗
PubMed1975

Purification and characterization of extracellular proteinases of Aspergillus oryzae.

The extracellular proteinases of Aspergillus oryzae EI 212 were separated into two active fractions by (NH4)2SO4 and ethanol fractionation followed by diethylaminoethyl-Sephadex A-50 and hydroxyapatite chromatography. The molecular weight was estimated by gel filtration to be about 70,000 and 35,000 for proteinases I and II, respectively. Optimum pH for casein and hemoglobin hydrolysis was 6.5 at 60 C for proteinase I and 10.0 at 45 C for proteinase II, and for gelatin hydrolysis it was 6.5 at 45 C for both enzymes. The enzymes were stable over the pH range 6 to 8 at 30 C for 60 min. The enzyme activity for both the proteinases was accelerated by Cu2+ and inhibited by Fe2+, Fe3+, Hg2+, and Ag+. Halogenators (e.g., N-chlorosuccinimide) and diisopropyl fluorophosphate inhibited proteinase II. Sulfhydryl reagents such as p-chloromercuribenzoate and iodoacetate inhibited proteinase I. Sulfhydryl compounds accelerated the action of both enzymes.

Ammonium Sulfate↗
PubMed1978

The mercuric and organomercurial detoxifying enzymes from a plasmid-bearing strain of Escherichia coli.

Two separate enzymes, which determine resistance to inorganic mercury and organomercurials, have been purified from the plasmid-bearing Escherichia coli strain J53-1(R831). The mercuric reductase that reduces Hg2+ to volatile Hg0 was purified about 240-fold from the 160,000 X g supernatant of French press disrupted cells. This enzyme contains bound FAD, requires NADPH as an electron donor, and requires the presence of a sulfhydryl compound for activity. The reductase has a Km of 13 micron HgCl2, a pH optimum of 7.5 in 50 mM sodium phosphate buffer, an isoelectric point of 5.3, a Stokes radius of 50 A, and a molecular weight of about 180,000. The subunit molecular weight, determined by gel electrophoresis in the presence of sodium dodecyl sulfate, is about 63,000 +/- 2,000. These results suggest that the native enzyme is composed of three identical subunits. The organomercurial hydrolase, which breaks the mercury-carbon bond in compounds such as methylmercuric chloride, phenylmercuric acetate, and ethylmercuric chloride, was purified about 38-fold over the starting material. This enzyme has a Km of 0.56 micron for ethylmercuric chloride, a Km of 7.7 micron for methylmercuric chloride, and two Km values of 0.24 micron and over 200 micron for phenylmercuric acetate. The hydrolase has an isoelectric point of 5.5, requires the presence of EDTA and a sulfhydryl compound for activity, has a Stokes radius of 24 A, and has a molecular weight of about 43,000 +/- 4,000.

Drug Resistance, Microbial↗
PubMed1976

Comparison of mode of activation of guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases from silkworm.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase (protein kinase G) partially purified from silkworm pupae was selectively activated by cyclic GMP at lower concentrations. Nevertheless, the enzyme seemed to differ from adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) with respect to the mode of response to cyclic nucleotides. The catalytic activity and cyclic GMP-binding activity were not dissociated by cyclic GMP in a manner similar to that described for protein kinase A. The enzyme was not inhibited by regulatory subunit of protein kinase A nor by protein inhibitor. A sulfhydryl compound such as 2-mercaptoethanol or glutathione was essential for the activation by cyclic GMP, and an extraordinary high concentration of either Mg2+ (100 mM) or Mn2+ (25 mM) was needed for maximal stimulation by cyclic GMP. A polyamine such as spermine, spermidine, or putrescine could substitute partly for the cation. Kinetic analysis indicated that Km for ATP was decreased whereas Ka for cyclic GMP was increased significantly at high concentrations of the cation. The effect of the cation to decrease Km for ATP was not evident in the absence of a sulfhydryl compound. These characteristics of protein kinase G described above were not observed for protein kinase A which was obtained from the same organism.

Animals↗
PubMed1976

The respiratory metabolism of Mycobacterium lepraemurium.

The respiratory metabolism of Mycobacterium lepraemurium isolated from Sprague-Dawley rats lepromata using several substrates was investigated. None of the intermediates of the glycolysis cycle as well as of the tricarboxylic acid cycle except succinate was oxidized by purified whole suspensions of M. lepraemurium. Likewise, many sulfur compounds such as cystine, thiourea, thioacetate, thiodiglycol, mercaptoact and some sulfhydryl compounds, e.g., cysteine, dithioerythritol, dithiorthritol, and penicillamine were readily oxidized by murine bacillary suspensions, whereas thioglycolate, thioglucose, and reduced glutathione were oxidized at a slow rate. Succinate was not or was very poorly oxidized by normal cells probably because of impermeability of the cell wall but the addition of succinate to the cell suspensions frozen for 1 min at -40 degrees C considerably enhanced oxygen uptake over the endogenous value. The oxidation of succinate was unaffected by inhibitors rotenone, atabrine, and amytal but was markedly inhibited by thenoyltrifluoroacetone, antimycin A, 2-N-heptyl-4-hydroxyquinoline-N-oxide, and cyanide. The thiol-binding agents, p-hydroxymercuribenzoate and N-ethylmaleimide were also effective inhibitors of succinate oxidation but the process was not affected by uncouplers dinitrophenol, dibromophenol, pentachlorophenol, and carbonyl-cyanide-m-chlorophenylhydrazone. The results indicated that succinate oxidation by M. lepraemurium was mediated by oxidative enzymes involving an electron transport chain with oxygen as the terminal electron acceptor.

Antimycin A↗
PubMed1975

A novel diazonium-sulfhydryl reaction in the inactivation of yeast alcohol dehydrogenase by diazotized 3-aminopyridine adenine dinucleotide.

Diazotized 3-aminopyridine adenine dinucleotide has been found to modify four sulfhydryl groups per molecule of enzyme during the complete inactivation of yeast alcohol dehydrogenase. The reaction of sulfhydryl groups was indicated by titration studies with 5,5-dithiobis(2-nitrobenzoic acid) as well as isolation and quantitation of the cysteinyl derivative released by acid hydrolysis of the modified enzyme. The cysteinyl derivative was identified as S-(3-pyridyl)cysteine. Authentic S-(3-pyridyl)cystein was synthesized and structurally characterized for these studies. Diazonium-sulfhydryl reactions were demonstrated for a number of diazonium derivatives with cysteine, homocysteine, glutathione, and mercaptoethanol at 0-4 degrees and neutral pH. Second order rate constants were determined in reactions of these sulfhydryl compounds with diazotized 1-methyl-3-aminopyridinium chloride, diazotized 3-aminopyridine adenine dinucleotide, and diazotized 3-aminopyridine adenine dinucleotide phosphate.

Alcohol Oxidoreductases↗