Proteinase yscD. Purification and characterization of a new yeast peptidase

A newly recognized peptidase, designated proteinase yscD, was purified from the yeast Saccharomyces cerevisiae. The enzyme cleaves the Pro-Phe bond of the synthetic peptide substrate Bz-Pro-Phe-Arg-4-nitroanilide and the Ala-Ala bond of Ac-Ala-Ala-Pro-Ala-4-nitroanilide, Ac-Ala-Ala-Pro-Phe-4-nitroanilide, and MeO-Suc-Ala-Ala-Pro-Met-4-nitroanilide with high efficiency (Bz-, Ac-, and MeO-Suc are defined as benzoyl, acetyl, and methoxy-succinyl, respectively). [3H]Methylcasein does not serve as a substrate. Optimum pH for cleavage of Bz-Pro-Phe-Arg-4-nitroanilide is in the range of 6.5 to 7; for Ac-Ala-Ala-Pro-Ala-4-nitroanilide the range is between 5.75 and 6. For MeO-Suc-Ala-Ala-Pro-Met-4-nitroanilide the pH optimum was found to be 5.5. The purified enzyme has an apparent Stokes radius of Rs = 37.9 A as judged by gel chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a molecular weight of approximately 83,000 for the enzyme. Mercurials and EDTA were found to be potent inhibitors of proteinase yscD activity.     

Purification and molecular characterization of two inhibitors of yeast proteinase B.

A rapid purification procedure for large scale preparations of yeast proteinase B inhibitors 1 and 2 (IB1 and IB2) is described. By disc gel electrophoresis, amino acid analysis, and end-group determinations, each of the inhibitors is homogeneous. Both inhibitors are polypeptides with molecular weights of 8,500, containing 74 residues. No components other than amino acids could be detected. There is no significant difference in the amino acid compositions of the two inhibitors as analyzed after acid hydrolysis. Both polypeptides are characterized by the total absence of arginine, tryptophan, and sulfur-containing amino acid residues. The proteinase B inhibitors of yeast, therefore, differ fundamentally from proteinase inhibitors of many other organisms, which generally contain a large number of disulfide bridges. Both proteinase B inhibitors have threonine as the NH2-terminal residue and -Val-His-Thr-Asn-COO- as the COOH-terminal sequence. Comparison of peptide maps after tryptic digestion reveals that the two inhibitors differ definitely in only a few tryptic peptides. The inhibitors are rapidly inactivated by digestion with carboxypeptidase A from bovine pancreas at pH 8.5. Inactivation occurs stoichiometrically with the release of threonine, the penultimate residue at the COOH-terminal end of both inhibitors.     

Purification and characterization of proteinase In, a trypsin-like proteinase, in Escherichia coli.

We previously found a trypsin-like proteinase which momentarily appears immediately before DNA synthesis in the cell cycle of Escherichia coli synchronized by phosphate starvation and which is closely related to the initiation of DNA replication (Kato, M., Irisawa, T., Morimoto, Y. and Muramatu, M., unpublished results). The proteinase was named proteinase In. It was purified approximately 2880-fold with a recovery of 15%. The isolated enzyme appeared homogeneous by gel filtration and electrophoresis. Its molecular mass was estimated by analytical gel filtration and SDS/PAGE as approximately 66 kDa. The isoelectric point of proteinase In is 4.9 and its optimal pH is approximately 9. Although protein In hydrolyzes fluorogenic substrate for trypsin, its hydrolytic activity seems markedly affected by amino-acid sequence lying towards the N-terminal from the P1 (lysine, arginine) residue. The proteinase does not hydrolyze N2-benzoyl-D,L-arginine-4-nitronanilide and fluorogenic substrates for chymotrypsin and elastase. The proteinase activity is inhibited by leupeptin, antipain and 4-nitrophenyl 4-guanidinobenzoate, but the effects of tosyl-L-lysine chloromethane, diisopropylfluorophosphate, benzamidine and pentamidine isethionate on the proteinase activity are weak or not inhibitory. Its activity is strongly affected in the presence of NaCl and KCl, and at a concentration of 1.5 M, these increase the activity 14-fold and 13-fold, respectively, above that without salt. Proteinase In was strongly inhibited by various esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, and their inhibitory effects were roughly correlated with those on growth of E. coli. Proteinase activity was found in the cytoplasmic fraction.     

Aminopeptidase Co,a new yeast peptidase

A new aminopeptidase — aminopeptidase Co — has been detected in the yeast $\text{Saccharomyces}\text{cerevisiae}$. The enzyme is only active in the presence of Co²⁺ions. Zn^2+- and Mn²⁺ions are inhibitory. The enzyme activity is also inhibited by chelating agents. Of the p-nitroanilide derivatives tested only those containing basic amino acids are cleaved.     

Purification and characterization of a new 120 kDa alkaline proteinase of Trypanosoma cruzi.

A new alkaline proteinase activity was identified in cell-free extracts of Trypanosoma cruzi epimastigotes on the basis of its ability to hydrolyze the fluorogenic substrate N-Z-Gly-Gly-Arg-AMC. The optimal activity was at pH 8.0. After a three step-chromatography procedure using two anionic columns (DEAE-Sepharose and Mono Q) and a chromatofocusing column (Mono P), the proteolytic activity was associated with a single 120 kDa protein and was called Tc 120 proteinase. The molecular mass of the proteinase was confirmed by direct visualization of the proteolytic activity using a fluorometric assay on SDS-PAGE. The Tc 120 proteinase which also cleaves N-Z-Arg-AMC, N-Z-Phe-Arg-AMC and N-glutaryl-Gly-Arg-AMC substrates, is a cysteine-type proteinase with an unusual low sensitivity to E-64.     

Purification and characterization of a Z-pro-prolinal-insensitive Z-Gly-Pro-7-amino-4-methyl coumarin-hydrolyzing peptidase from bovine serum--a new proline-specific peptidase.

The study of a new proline-specific peptidase from bovine serum is presented. The enzyme readily cleaves the prolyl oligopeptidase (PO) substrate Z-Gly-Pro-MCA, liberating the fluorophore MCA, thus allowing quantification of enzyme activity. Unlike PO, however, this peptidase is completely insensitive to the PO-specific inhibitor Z-Pro-prolinal and has been designated Z-Pro-prolinal-insensitive Z-Gly-Pro-MCA-hydrolyzing peptidase (ZIP). The two peptidases were successfully separated from each other by phenyl Sepharose hydrophobic interaction chromatography and the subsequent purification focused on the isolation of ZIP from bovine serum. In addition to phenyl Sepharose, calcium phosphate cellulose and DEAE anion-exchange chromatography were employed in the purification, with an overall enzyme yield of 33% and a purification factor of 4023. SDS-PAGE and size-exclusion chromatography indicated a dimeric structure with a relative molecular mass of 174 kDa. The enzyme was stable over the pH range 2.5-10.0. Optimal activity was detected in the pH range 7.4-8.0. Isoelectric focusing revealed a pI of 5.68. Inhibition by AEBSF suggests the peptidase may be a serine protease and ZIP possibly contains a cysteine residue near the active site. alpha(2)M failed to inhibit activity, suggesting oligopeptidase specificity. HPLC analysis revealed a broad substrate specificity for proline-containing peptides. Kinetic analysis indicated that ZIP had a high affinity for Z-Gly-Pro-MCA with a K(m) of 54 microM deduced. Bovine serum ZIP exhibits biophysical characteristics both similar to and different from those of PO isolated from a number of sources and may serve an important physiological function in the degradation of bioactive oligopeptides.     

Purification and characterization of a novel proteinase, chymopapain S

Chymopapain (EC 3.4.22.6) possesses an essential and a non-essential thiol group, but enzyme preparations produced hitherto always contained less than two thiol groups. Starting from commercial chymopapain or from papaya latex, we have prepared pure enzyme having two thiol groups, which is demanded by mechanistic investigations. The purification was performed by covalent chromatography on activated thiol-Sepharose column, which specifically binds thiol-containing proteins. Elution was effcted with cysteine in a stepwise manner, first at pH 5 then at pH 8. The pure enzyme had a molecular mass of24 700 as estimated by sodium dodecyl sulfate polyacrylamide gel-electrophoresis. Titration of the pure enzyme with 2,2'-dipyridyl disulfide at pH 9 exhibited a biphasic curve. This indicated that under the conditions employed the nonessential thiol group is more reactive than the essential one, which is a characteristic feature of chymopapain B. On the other hand, the magnitude of the rate constant of the same reaction at pH 4, as well as its pH-dependence, was characteristic of chymopapain A. The enzyme with the hybrid kinetic properties was denoted as chymopapain S. We conclude from the above findings that various forms of chymopapain can be classified by their reaction with 2,2'-dipyridyl disulfide.     

Proteolysis in eukaryotic cells. Proteinase yscE, a new yeast peptidase

A new peptidase, which we call proteinase yscE, was purified from the yeast Saccharomyces cerevisiae. The enzyme cleaves the synthetic substrates Cbz-Gly-Gly-Leu-4-nitroanilide, Cbz-Ala-Ala-Leu-4-nitroanilide, and Suc-Phe-Leu-Phe-4-nitroanilide (Cbz and Suc are defined as benzyloxycarbonyl and succinyl, respectively) at the 4-nitroanilide bond and exhibits a slight activity against [3H]methylcasein. Optimum pH for cleavage of the chromogenic substrates is found to be in the range of 8.2 to 8.6. The purified enzyme has an apparent Stokes radius of Rs = 75.2 A as judged by gel chromatography and is composed of subunits. Mercurials were found to be strong inhibitors of the enzyme activity.     

Solubilization and characterization of yeast signal peptidase

An efficient post-translational assay for solubilized yeast signal peptidase has been developed. The enzyme can be solubilized in nonionic detergent (0.5% Nikkol) without added salt, but salt increased the efficiency of solubilization. Radiosequencing of the cleaved substrate revealed that the enzyme removed the signal peptide. The substrate (prepro-alpha-factor) must be pretreated with sodium dodecyl sulfate to be cleaved. The enzyme displays a broad, alkaline pH optimum, retaining activity at pH 12. Moderately high temperatures (35 degrees C), excess detergent (greater than 0.5% Nikkol), or high salt (greater than 300 mM KOAc) will inactivate the enzyme. Phosphatidylcholine is necessary for optimal activity. The optimal ratio of Nikkol:lipid:sodium dodecyl sulfate is 6.4:2.2:1. The membrane association of yeast signal peptidase is resistant to carbonate extraction, indicating that it is an integral membrane protein.     

Purification and characterization of L-pyrrolidonecarboxylate peptidase from Bacillus amyloiliquefaciens.

Microorganisms capable of producing L-pyrrolidonecarboxylate peptidase [L-pyrrolidonyl peptidase, EC 3.4.11.8] were screened and a strain of Bacillus amyloliquefaciens was chosen as one of the most potent producers of the enzyme. The enzyme was purified from lysozyme-lysate of the bacterial cells by salting out with ammonium sulfate, adsorption on DEAE-cellulose, covalent chromatography on PCMB-Sepharose and by gel filtration on Sephadex G-150. By these procedures, the enzyme was purified about 800-fold with an activity recovery of 9%, and the preparation was electrophoretically homogenous. The enzyme was most active and stable at pH 7-8. The presence of 2-mercaptoethanol and EDTA was effective for stabilizing the enzyme. The molecular weight was estimated to be 72,000 by the gel filtration method and to be 24,000 by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a subunit oligomer, presumably trimer. The enzyme was inactivated by the addition of PCMB, sodium tetrathionate, Hg2+ and Cu2+, but the activity lost was restored by the addition of 2-mercaptoethanol and EDTA. The purified enzyme split amide and ester linkages in L-pyroglutamyl derivatives of L-alanine, beta-naphthylamine, alpha-naphthol, and 4-methylumbelliferone, but was completely inert towards various peptides and esters used as substrates for usual amino- and carboxy-peptidases, and for endopeptidases such as trypsin, subtilisin and alpha-chymotrypsin.     

Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus.

Streptomyces strain K1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70 degrees C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH2-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.     

Purification and characterization of secretory proteinase of Candida albicans.

Proteolytic activity of medically important yeasts was tested in both YCB-BSA agar and medium. All of 134 strains of Candida albicans, 13 of 18 strains of Candida tropicalis and 11 of 18 strains of Candida parapsilosis had this activity, while none of 52 Candida glabrata strains or of 11 Cryptococcus neoformans strains tested had proteolytic activity. Strains of C. albicans fell into five groups based on the level and time-course of in vitro proteinase productivity. Five strains randomly selected from each group were tested for pathogenicity in mice. The strain possessing the strongest pathogenicity was used to purify proteinase. The molecular weight of the proteinase was approximately 44,000 daltons and its isoelectric point was pH 4.2. Optimal pH of the proteinase was 3.2 and the enzyme was stable below pH 7.0 and lost its activity above pH 8.0 at 37 C in a 60-min incubation. The 23 amino acid sequence of the proteinase N-terminus was determined.     

Purification and characterization of barley dipeptidyl peptidase IV

Barley (Hordeum vulgare L.) storage proteins, which have a high content of proline (Pro) and glutamine, are cleaved by cysteine endoproteases to yield peptides with a Pro next to the N-terminal and/or C-terminal amino acid residues. A peptidase cleaving after Xaa-Pro- at the N terminus of peptides was purified from green barley malt. It was identified as a serine-type dipeptidyl peptidase (DPP), based on inhibitor studies, and the nature of the cleavage product. It is a monomeric glycoprotein with an apparent molecular mass of 105 kD (85 kD after deglycosylation), with a pI of 3.55 and a pH optimum at 7.2. Substrate specificity was determined with a series of fluorogenic peptide substrates with the general formula Xaa-Pro-AMC, where Xaa is an unspecified amino acid and AMC is 7-amino-4-methylcoumarin. The best substrates were Xaa = lysine and arginine, while the poorest were Xaa = aspartic acid, phenylalanine, and glutamic acid. The K(m) values ranged from 0.071 to 8.9 microM, compared with values of 9 to 130 microM reported for mammalian DPP IVs. We discuss the possible role of DPP IV in the degradation of small Pro-containing peptides transported from the endosperm to the embryo of the germinating barley grain.     

Purification and characterization of dipeptidyl peptidase I from human spleen.

The lysosomal hydrolase, dipeptidyl peptidase I (DPPI), was purified from human spleen and its enzymatic activity characterized. The enzyme was purified to apparent homogeneity by a combination of differential pH solubility, heat-treatment, affinity chromatography on concanavalin A-agarose and p-hydroxymercuribenzoate-agarose, and gel filtration chromatography on Sephacryl S-300. This procedure resulted in a 1100-fold purification of DPPI protein with a yield of approximately 2% of the total DPPI activity. The enzyme was characterized as a glycoprotein with a pI of 5.4, a molecular mass of 200,000 Da as determined by gel filtration under nondenaturing conditions, and a subunit size of 24,000 Da. Amino acid sequence analysis of peptides isolated from cyanogen bromide and trypsin digests of the 24,000-Da subunit revealed extensive sequence similarity between human and rat DPPI. Purified DPPI exhibited both hydrolytic and transpeptidase (polymerase) activity. DPPI exhibited activity against a variety of dipeptide substrates including peptides with either non-polar or polar residues in the P1 position. In contrast to the reported substrate specificity of bovine and murine DPPI, the human enzyme exhibited a modest preference for peptides with nonpolar residues in the P1 position. DPPI content was found to be highest among cytotoxic lymphocytes and myeloid cells. The high level of DPPI expression in these cell populations correlates with their sensitivity to the toxic effects of leucyl-leucine methyl ester, a substrate for DPPI.     

Purification and characterization of tripeptidyl peptidase I from Dictyostelium discoideum.

A tripeptidyl peptidase I from Dictyostelium discoideum was purified 744-fold to near homogeneity. The enzyme is 214 kDa in size and is composed of two monomers with a M(r) of 107 kDa. It has two pH optima at pH 4.5 and 5.9 and is a serine peptidase with no aminopeptidase or dipeptidyl peptidase activity. The enzyme was relatively specific showing activity on ala-ala-phe-p-nitroaniline but also acted on substrates with proline in the P1 position in contrast to mammalian TPP I. The K(m) values of the enzyme at pH 4.5 for ala-ala-phe-, ala-phe-pro- and ala-ala-pro-p-nitroanilines were 27 microM, 437 microM and 888 microM, respectively. The enzyme is most abundant during the amoeba stage of the life cycle but is present in the early stages of development and may therefore have a dual role in the organism in mobilizing amino acids or in processing specific peptides or proteins.     

Purification and fluorometric assay of proteinase A from yeast

A kinetic assay system which provides reliable measurements of Na-K-ATPase activity on 0.2 to 0.5-mm segments of renal proximal convoluted tubules isolated from collagenase-digested renal cortical slices is described. The use of collagenase digestion provides higher values for Na-K-ATPase, possibly by making the enzyme more accessible to the reaction system. The advantages of a kinetic vs an endpoint assay include the ability to use the same tubule as its own reference for the determination of total, ouabain-sensitive, and ouabain-insensitive ATPase activity. In addition, it allows dose-response studies on the effect of inhibitors on ATPase activity in the same tubule segment.     

Purification and characterization of a cysteine proteinase from silkworm eggs

Eggs of the silkworm, Bombyx mori, contain a high level of a proteinase which is most active in acidic pH region. The proteinase was purified from an extract of eggs by a six-step procedure which included conventional chromatographic fractionations. The molecular mass of the proteinase was estimated to be 350 kDa by gel filtration and 47 kDa by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels, suggesting an octameric structure. The amino acid composition was found to resemble that of mammalian lysosomal cysteine proteinases, in particular cathepsin L. The NH2-terminal 10-residue sequence is Val-Gln-Phe-Phe-Asp-Leu-Val-Lys-Glu-Glu-. The enzyme appears to be a member of the class of cysteine proteinases since it was strongly inhibited by sulfhydryl-reactive compounds and N-[N-(1,3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). The enzyme hydrolyzed various protein substrates, such as hemoglobin, vitellogenin, vitellin, and lipophorin, with maximal activity around pH 3-3.5. The specificity of the cleavage sites in the oxidized B chain of insulin was rather well defined and there was high affinity for hydrophobic residues at the P2 and P3 positions. The cysteine proteinase is thought to be involved in protein degradation during embryonic development of silkworm eggs.     

Purification and characterization of a multicatalytic high-molecular-mass proteinase from rat skeletal muscle.

A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle. The purification procedure consisted of fractionation of the muscle extract by (NH4)2SO4, chromatography on DEAE-Sephacel, fast protein liquid chromatography on Mono Q and gel filtration on Sepharose 6B. The enzyme preparation appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresis. The isoelectric point of the proteinase is at 5.1-5.2. The enzyme has an Mr of about 650 000 and dissociates into eight subunits of Mr 25 000-32 000 when subjected to electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. The proteinase contains hydrolytic activity against N-blocked tripeptide 4-methyl-7-coumarylamide substrates with an arginine or phenylalanine residue adjacent to the leaving group. Maximum activity with the first group of substrates was at pH 10.5, and this activity was inhibited by leupeptin, chymostatin and Ca2+. Maximum activity with the latter group of substrates was at pH 7.5, and was also inhibited by the two microbial inhibitors, but was activated by Ca2+ ions. By using [14C]methylcasein as a substrate, maximum activity was observed at pH9.0, and this proteolytic activity was not affected by leupeptin, was enhanced by chymostatin and inhibited by Ca2+. Similar effects were observed when benzyloxycarbonyl-Leu-Leu-Glu 2-naphthylamide was used as a substrate. These enzymic activities were abolished by p-hydroxymercuribenzenesulphonic acid or mersalyl acid, whereas a small activation was observed with cysteine or dithiothreitol.