Thiol-blocking Reagents Research Articles

Partial purification and substrate specificity of a ubiquitin hydrolase from Saccharomyces cerevisiae

A ubiquitin hydrolase that removes ubiquitin from a multi-ubiquitinated protein has been purified 600-fold from Saccharomyces cerevisiae. Four different ubiquitin-protein conjugates were assayed as substrates during the purification procedure. Enzymic activities that removed ubiquitin from ubiquitinated histone H2A, a ubiquitin-ubiquitin dimer and a ubiquitin-ribosomal fusion protein were separated during the purification from an activity that removed a single ubiquitin molecule linked by an isopeptide bond to a ubiquitinated protein. The size of the native enzyme was 160 kDa, based on its sedimentation in a sucrose gradient, and the subunit molecular mass was estimated to be 160 kDa, based on a profile of proteins eluted in different fractions by thiol-affinity chromatography. The partially purified hydrolase was not inhibited by a variety of protease inhibitors, except for thiol-blocking reagents. The natural substrate for this enzyme may be the polyubiquitin chain containing ubiquitin molecules bound to each other in isopeptide bonds, with one of them linked to a lysine residue of a protein targeted for intracellular proteolysis.

Chicken liver Pz-peptidase, a thiol-dependent metallo-endopeptidase

Pz-peptidase was purified from chicken liver as a protein of Mr 80,000 and pI 5.2. The purified enzyme hydrolysed phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg, 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys. 7-methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-(2,4-dinitropheny l)Lys, benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate, Ac-Ala4 (at the Ala-1-Ala-2 bond) and bradykinin (at the Phe-5-Ser-6 bond). No hydrolysis of proteins was detected. Loss of activity in the presence of EDTA or 1,10-phenanthroline was time-dependent. Metal ions found to restore activity after treatment with EDTA were Zn2+, Mn2+, Ca2+, Co2+ and Cd2+, in decreasing order of effectiveness. Ni2+, Fe2+ and higher concentrations of Zn2+ were inhibitory. Inhibition by N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate and related compounds showed Ki values (down to 5 nM) somewhat lower than those for the rat enzyme. Pz-peptidase was activated by low concentrations of 2-mercaptoethanol and dithiothreitol, but inhibited by higher concentrations. p-Chloromercuribenzoate and some other thiol-blocking reagents were inhibitory. Inactivation by diethyl pyrocarbonate that was reversible by hydroxylamine showed the presence of essential histidine residue(s). We conclude that chicken Pz-peptidase is a metallo-endopeptidase with thiol-dependence. Moreover, the properties of chicken Pz-peptidase agree with those described for mammalian soluble metallo-endopeptidase and endo-oligopeptidase A. consistent with the view that these three types of activity are all attributable to the single enzyme for which the name thimet peptidase has been proposed.

Role of cysteine residues in ribonuclease H from Escherichia coli. Site-directed mutagenesis and chemical modification

The role of the three cysteine residues at positions 13, 63 and 133 in Escherichia coli RNAase H, an enzyme that is sensitive to N-ethylmaleimide [Berkower, Leis & Hurwitz (1973) J. Biol. Chem. 248, 5914-5921], was examined by using both site-directed mutagenesis and chemical modification. Novel aspects that were found are as follows. First, none of the cysteine residues is required for activity. Secondly, chemical modification of either Cys-13 or Cys-133 with thiol-blocking reagents inactivates the enzyme, but that of Cys-63 does not. Thus the sensitivity of E. coli RNAase H to N-ethylmaleimide arises not from blocking of the thiol group but from steric hindrance by the modifying group incorporated at either Cys-13 or Cys-133.

Purification and characterization of [formula omitted] amidase from human serum

Purification to homogeneity of the N- acetylmuramoyl- l-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) from human serum has been achieved with a high yield. By molecular sieving chromatography, a molecular weight of 120 000–130 000 has been found for the native enzyme. Polyacrylamide gel electrophoresis under native conditions gave a unique band of M r= 125 000. The same technique performed under denaturing conditions revealed that the protein is a dimer composed of one subunit of M r= 57000 and another of M r= 70000. In isoelectrofocalization assays, the amidase behaved as an acidic protein. Ethylenediaminetetraacetate inhibited the enzyme activity; the Mg 2+ requirement was confirmed. The simultaneous presence of sulfhydryl groups and disulfide bonds in the protien was evidenced by the inhibitions produced by different thiol-blocking reagents and by several thiol-bearing substances. Direct measurements established the presence of two accessible thiol groups and the occurence of nine disulfide bonds per protein molecule. Studies of substrate hydrolyzing capacities showed a marked preference for the muramoyl tripeptide derived from the Echerichia coli or Bacillus cerreus mureins, the disaccharide tetrapeptide and the bis disaccharide tetra-tetrapeptide from E. coli were also good substrates. Activities on small muropeptides of other composition are also reported. Whole (insoluble) peptidoglycans representing the main bacterial chemotypes were submitted to the enzyme action; although with weak specific activities, the human amidase was nevertheless able to release soluble peptides from some of them. A bacteriolytic capacity on some microorganisms cannot be excluded. Results are discussed and the human enzyme is compared to presently known microbiol muramoyl amidases.

Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus.

Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.

Nonequivalence of the Two Subunits of Horse Erythrocyte Glutathione Transferase in Their Reaction with Sulfhydryl Reagents

Glutathione transferase (EC 2.5.1.18) from horse erythrocytes has been purified and some molecular and kinetic properties have been investigated. It appears to be a dimeric protein composed of subunits of about 23 kDa, indistinguishable either in sodium dodecyl sulfate or in urea electrophoresis. Amino acid composition, substrate specificities, sensitivity to inhibitors, CD spectra, and immunological studies provide evidence that the horse enzyme is related to the pi class transferases. This enzyme has only two reactive thiol groups/dimer whose integrity appears to be essential for the activity. A peculiar feature of these protein thiol groups is that they react nonidentically with a number of thiol blocking reagents, i.e. iodacetamide, bromopyruvate, N-ethylmaleimide, and 1-chloro-2,4-dinitrobenzene. Also many disulfides react with one thiol group 5- to 10-fold more rapidly than with the other. The two mixed disulfides so formed also have different rates of reactivation by dithiothreitol. All the structural and kinetic data reported in this paper indicate a nonsymmetrical association of two identical subunits, or alternatively heterodimeric structure with subunits of very similar charge and size.

Purification and Characterization of Sorbitol Dehydrogenase from Bovine Brain

Sorbitol dehydrogenase (EC 1.1.1.14) was isolated from bovine brain and purified 3,000-fold to apparent homogeneity, as judged by polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 36 units/mg of protein; a molecular weight of 39,000 for each of the four identical subunits and 155,000 for the intact enzyme were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography, respectively. The presence of one Zn2+ per subunit was confirmed by atom absorption spectroscopy; inactivation of the enzyme by metal-chelating agents points to the essential role that Zn2+ plays in the catalytically competent enzyme. The enzyme is also inactivated by thiol-blocking reagents; with respect to inactivation by sodium pyrophosphate, sorbitol dehydrogenase is different from closely related alcohol dehydrogenase.

Putative convertase involved in the proteolytic conversion of rat proalbumin to serum albumin.

We have found a proteolytic activity in Golgi membranes which efficiently converts [35S]methionine-labeled proalbumin, isolated from pulse-labeled rat hepatocytes in culture, to serum albumin in an in vitro assay system. The proalbumin-converting activity was dependent on Ca2+ and the maximum activity was observed at pH 5.5-6.0. Since the enzyme activity was found to be resistant not only to both leupeptin and E-64 but also to thiol-blocking reagents, it is unlikely that cathepsin B is involved in the proteolytic conversion of proalbumin occurring in the Golgi complex.

Characterization of collagenolytic activity from strains of Bacteroides gingivalis.

Strains of B. gingivalis were shown to produce collagenolytic activity capable of dissolving reconstituted collagen (type I) fibrils and of cleaving the helical domain of types I. II and III collagens at 22° C. The catalytic activity was dependent on free thiol groups and on metal ions, as indicated by inhibition by thiol blocking reagents and metal chelators. The activity was associated with the bacterial cells and was not secreted to the medium. Under optimal conditions. 100 Units of collagenase per gram cell pellet (wet weight) were released by detergents such as Triton X‐100 and SDS. Zymography of detergent extracts revealed that collagen‐degrading strains, but not an inactive control strain (W), contained a discrete Mr 90 000 gelatin cleaving protease which may be identical to the collagenolytic enzyme. The initial attack on the helical domain of type I collagen occurred near the COOH‐terminus. The a1 and a2 chains were cleaved at the same site, generating a major helical fragment consisting of three shortened (Mr 82 000) a‐chains. Subsequent cleavages of this shortened collagen molecule resulted in generation of multiple fragments from the component a‐chains in the Mr 60 000 to 6000 range. This cleavage pattern was clearly distinct from the characteristic 3/4–1/4 pattern produced by vertebrate collagenases. Type II and III collagens were also cleaved first near the COOH‐terminus, generating fragments of similar size to those produced from type I collagen. In view of its ability to dissolve reconstituted collagen fibrils at 35°C and its ability to attack the helical domain of interstitial collagens in solution at 22°C, we suggest that this enzyme tentatively be classified as a true collagenase.

Purification and characterization of protease Re, a cytoplasmic endoprotease in Escherichia coli.

Protease Re, a new cytoplasmic endoprotease in Escherichia coli, was purified to homogeneity by conventional procedures, using [3H]casein as the substrate. The enzyme consists of a single polypeptide of 82,000 molecular weight. It is maximally active between pH 7 and 8.5 and is independent of ATP. It has a pI of 6.8 and a Km of 10.8 microM for casein. Since diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride inhibited this enzyme, it appears to be a serine protease. Protease Re was sensitive to inhibition by L-1-tosylamido-2-phenylethylchloromethylketone but not to that by 1-chloro-3-tosylamido-7-aminoheptanone, thiol-blocking reagents, chelating agents, or various peptide aldehydes. Re also degraded [125I]globin, [125I]glucagon, and 125I-labeled denatured bovine serum albumin to acid-soluble products (generally oligopeptides of greater than 1,500 daltons), but it showed no activity against serum albumin, growth hormone, insulin, or a variety of fluorometric peptide substrates. It also hydrolyzed oxidatively inactivated glutamine synthetase (generated by ascorbate, oxygen, and iron) four- to fivefold more rapidly than the native protein. Protease Re appears to be identical to the proteolytic enzyme isolated by Roseman and Levine (J. Biol. Chem. 262:2101-2110, 1987) by its ability to degrade selectively oxidatively damaged glutamine synthetase in vivo. Its role in intracellular protein breakdown is uncertain.

Distribution of glutamate racemase in lactic acid bacteria and further characterization of the enzyme from Pediococcus pentosaceus.

We investigated the distribution of glutamate racemase (EC 5.1.1.3) in bacteria, and found that the enzyme occurs exclusively in lactic acid bacteria. Pediococcus pentosaceus IFO 3182 produces the enzyme most abundantly. The enzyme purified from extracts of the Escherichia coli clone cells carrying the plasmid pICR221, which contains the enzyme gene of P. pentosaceus, does not require cofactors for its catalytic activity [N. Nakajima, K. Tanizawa, H. Tanaka and K. Soda, Agric. Biol. Chem., 50, 2823 (1986)]. This cofactor dispensability was re-confirmed with the enzyme purified from cell extracts of P. pentosaceus. On the basis of kinetic parameters obtained by measurements of the initial velocities, the glutamate racemase reaction was categorized into a typical racemization with a calculated equilibrium constant of 1.05. D- and L-Glutamate were the specific substrates for the enzyme, although L-homocysteinesulfinate, a sulfur analog of L-glutamate, slightly served as a substrate. L-α-Aminoadipate behaved as a competitive inhibitor in the racemization of glutamate. The enzyme was inactivated significantly by treatment with various thiol-blocking reagents.

Non-protein-mediated transfer of phosphatidic acid between microsomal and mitochondrial membranes

The transfer of phosphatidic acid between rat liver microsomes loaded with [ 32P]-phosphatidic acid and rat liver mitochondria was studied in the absence of added lipid transfer proteins. It was found that during 1 h at 37 °C in the medium containing 100 m m KCl, 20–30% of phosphatidic acid but only 2.5% of phosphatidylcholine were transferred. This spontaneous transfer of phosphatidic acid remained the same after pretreatment of microsomes and mitochondria with 125 m m KCl or microsomes alone with 1 m m Tris, pH 8.6, procedures reported to remove adsorbed lipid transfer proteins. This transfer was insensitive to thiol-blocking reagents. The initial rate of this non-protein-mediated transfer of phosphatidic acid was virtually independent of the concentration of the acceptor membranes (mitochondria), thus indicating that it occurs by diffusion of the phospholipid through the aqueous phase rather than by membrane collision. About 80% of phosphatidic acid synthesized in the outer mitochondrial membrane was recovered in the inner membrane after a 1-h incubation, pointing to a high rate of the intermembrane transfer of this phospholipid within intact mitochondrion.

Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.

High affinity specific binding of the thyrotrophin releasing hormone metabolite histidylproline to rat brain membranes

Histidylproline a metabolite of Thyrotrophin Releasing Hormone specifically binds to both high and low affinity sites in fresh rat brain membranes. Characterisation of the high affinity site under optimal conditions demonstrated an equilibrium dissociation constant (K D) of approximately 9 nM and maximum binding capacity of approximately 120 fmols/mg protein. Kinetic analysis of [ 3H]-His-Pro binding is limited by low binding density, instability of the high affinity site and rapid degradation of the radioligand. The thiol blocking reagent pHydroxymercuriphenylsulphonic acid (HMPS) inhibited [ 3H]-His-Pro degradation but also reduced binding of the peptide to membranes. The results are discussed with reference to the lack of specific binding sites in brain for the proposed neuropeptide and TRH metabolite cyclo(His-Pro).

Purification and properties of a cysteine proteinase from germinating rice seeds.

A proteinase found in rice seeds during germination was purified by (NH4)2S04 fractionation, CM-Sephadex chromatography, Sephadex G-100 gel filtration, Phenyl-Sepharose CL-4B chromatography, and chromatofocusing. The purified enzyme had a molecular weight of about 23,000 and an isoelectric point at pH 5.15. Its optimum pH for hydrolysis lay at 5.5 and at 5.75 when measured with α-N-benzoyl-dl-arginine-2-naphthylamide and casein, respectively. The proteinase was activated by thiol compounds and it was inhibited by common thiol-blocking reagents, particularly by leupeptin, antipain, and E-64. It was also inhibited, in a non-competitive manner, by a cysteine proteinase inhibitor purified from rice seeds. These results suggests that this enzyme belongs to the cysteine proteinase class (EC 3.4.22).

Ingensin, a fatty acid-activated serine proteinase from rat liver cytosol

The enzyme responsible for the succinylleucyllvalytyrosine methylcoumarylamide- (SLLVT-) degrading activity was purified from the postmitochondrial supernatant of rat liver (Yamamoto, T., Nojima, M., Ishiura, S. and Sugita, H. (1986) Biochim. Biophys. Acta 882, 297–304). The enzyme, named ingensin, was activated by saturated fatty acids, especially myristic acid, as well as by unsaturated linoleic acid and arachidonic acid. Although 2-mercaptoethanol activated ingensin 2-fold and p-chloromercuribenzoate and HgCl 2 completely inhibited its peptide-hydrolyzing activity, the enzyme is activated by the addition of a thiol-blocking reagent, monoiodoacetic acid. Ingensin was also inhibited by a specific serine proteinase inhibitor, diisopropyl fluorophosphate, but not by a specific cysteine proteinase inhibitor, E-64-c. These results suggest that the enzyme is a serine proteinase with an active thiol group(s) near the active site. We have found that the addition of glycerol and nordihydroguaiaretic acid lowered the extent of its activation by fatty acids as well as its intrinsic peptide-hydrolyzing activity.

Purification and characterization of human hepatic cysteine-conjugate beta-lyase.

Cysteine-conjugate beta-lyase (EC 4.4.1.13) was purified about 880-fold from human liver obtained post mortem. The purification procedure included (NH4)2SO4 precipitation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration on Sephadex G-200, and chromatofocusing. The purified enzyme cleaves the C-S bond of several S-aryl-L-cysteines to yield equimolar amounts of thiols, pyruvic acid and ammonia via an alpha beta-elimination reaction. The Mr of the enzyme was estimated to be 88,000 by gel filtration. The enzyme is thermolabile, has a pH optimum of 8.5, and an apparent Km of 0.7 mM towards S-(p-bromophenyl)-L-cysteine. The enzyme requires pyridoxal 5'-phosphate as a cofactor, and hence the enzyme activity was completely abolished by hydroxylamine. No effect of EDTA or thiol-blocking reagents was observed on the activity of the enzyme.

A thiol protease of peritoneal macrophages in the guinea pig.

Proteolytic enzymes of the guinea pig peritoneal exudate macrophages were investigated using synthetic fluorogenic peptide substrates. Among several enzymes, t-butyloxycarbonyl-phenylalanyl-seryl-arginine 4-methylcoumaryl-7-amide cleaving enzymes had the highest activity, and the activity in exudate macrophages was about 3 times stronger than that in resident macrophages. The molecular weight of the enzyme was around 35,000 and optimal pH around 6.5-7.0. It was inhibited by thiol-blocking reagents, suggesting a thiol protease.

Some properties of human and bovine brain cathepsin B

Cathepsin B has been purified 750-fold to apparent homogeneity from human and bovine brain cortex using ammonium sulfate fractionation (30-70%), chromatography on Sephadex G-100, CM-Sephadex C-50, and concanavalin A-Sepharose. Enzyme was assayed fluorometrically at pH 4.0 with pyridoxyl-hemoglobin in the presence of 1 mM DTT and 1 mM EDTA. Properties of the enzyme from the two sources proved to be similar. On disc PAGE the purified preparation produced two bands associated with proteinase activity that are due to existence of two multiple forms of brain cathepsin B with pI 6.1 and 6.8. The enzyme is completely inactivated by thiol-blocking reagents, leupeptin, E-64, and demands thiol compounds for its ultimate activity. Z-Phe-Ala-CHN2 is a potent inhibitor of the enzyme (K2nd = 1280 M-1S-1) in contrast to Z-Phe-Phe-CHN2 (K2nd = 264 M-1S-1). pH optimum in the reaction of hydrolysis of Pxy-Hb is 4.0-6.0, KM(app.) = 10(-5) M. Cathepsin B splits azocasein: pH optimum 5.0-6.0, KM(app.) = 2.2 X 10(-5) M, but inclusion of urea in the incubation medium depresses the azocaseinolytic activity of the enzyme 1.5-fold. It does not split Lys-NNap, Arg-NMec and is not inhibited by bestatin. The specific activity of brain cathepsin B with Z-Arg-Arg-NNapOMe at pH 6.0 is 10-fold higher than with Bz-Arg-NNap, Z-Gly-Gly-Arg-NNap is a poor substrate. With Z-Arg-Arg-NMec and Bz-Phe-Val-Arg-NMec the specific activity is 80 and 35%, respectively of that with Z-Phe-Arg-NMec.

Purification and properties of S-adenosylmethionine: aldoxime O-methyltransferase from Pseudomonas sp. N.C.I.B. 11652.

An enzyme catalysing the O-methylation of isobutyraldoxime by S-adenosyl-L-methionine was isolated from Pseudomonas sp. N.C.I.B. 11652. The enzyme was purified 220-fold by DEAE-cellulose chromatography, (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and chromatography on calcium phosphate gel. Homogeneity of the enzyme preparation was confirmed by isoelectric focusing on polyacrylamide gel and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme showed a narrow pH optimum at 10.25, required thiol-protecting agents for activity and was rapidly denatured at temperatures above 35 degrees C. The Km values for isobutyraldoxime and S-adenosyl-L-methionine were respectively 0.24 mM and 0.15 mM. Studies on substrate specificity indicated that attack was mainly restricted to oximes of C4-C6 aldehydes, with preference being shown for those with branching in the 2- or 3-position. Ketoximes were not substrates for the enzyme. Gel filtration on Sephadex G-100 gave an Mr of 84 000 for the intact enzyme, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated an Mr of 37 500, suggesting the presence of two subunits in the intact enzyme. S-Adenosylhomocysteine was a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 0.027 mM. The enzyme was also susceptible to inhibition by thiol-blocking reagents and heavy-metal ions. Mg2+ was not required for maximum activity.