Purification and properties of rat brain dipeptidyl aminopeptidase

Dipeptidyl aminopeptidase, which hydrolyzes the 7-(Gly-Pro)-4-methylcoumarinamide, has been purified from the brains of 3 week-old rats. It was purified about 2,600-fold by column chromatography on CM-cellulose, hydroxyapatite and Gly-Pro AH-Sepharose. This enzyme hydrolyzed Lys-Ala-beta-naphthylamide well with an optimum pH of 5.5. It was inhibited by diisopropyl fluorophosphate, phenyl-methanesulfonyl fluoride, some cations, and puromycin, but was not inhibited by p-chloromercuribenzoate, N-ethylmaleimide, dithiothreitol, EDTA, iodoacetic acid, and bacitracin, indicating that rat brain dipeptidyl aminopeptidase is a serine protease. This enzyme showed a molecular weight of 220,000 by gel filtration and of 51,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The properties of purified rat brain dipeptidyl aminopeptidase were similar to those of bovine pituitary dipeptidyl peptidase II, but the molecular weight and substrate specificity of these enzymes were different.     

The yeast aminopeptidase Y

A metal-dependent aminopeptidase (EC 3.4.11.-), designated APase Y, has been purified to homogeneity by conventional methods. The enzyme is composed of a single polypeptide chain with molecular mass of 102 kilodaltons, estimated by sodium dodecyl sulphate - polyacrylamide gel electrophoresis, with a blocked N-terminal amino acid. It possesses neither endopeptidase nor carboxypeptidase activity and is strongly inhibited by metal-chelating agents, Zn2+, and the protein inhibitor from Neurospora crassa. APase Y is insensitive to Cl anions, S--S reducing reagents, serine protease inhibitors, and the peptidase inhibitor benzamidine. Co2+, Hg2+, and p-chloromercuribenzoate can activate the enzyme up to 22, 20, and 55%, respectively. The holoenzyme is resistant to yeast endopeptidases A, B, and Y, whereas the apoenzyme (obtained after treatment with chelators) is susceptible to the serine endopeptidases B and Y. The enzyme catalyzes hydrolysis of most L peptides possessing free alpha-amino (or imino) group by stepwise removal of N-terminal residue. Peptides with L-leucine at the N terminus are cleaved preferentially. The enzyme is unable to catalyze hydrolysis of X--Pro type peptide bonds, and inefficiently hydrolyzes bonds between Asp--X and Glu--X. L-leucine p-nitroanilide hydrolyzes optimally at pH 8.2 with a Km value of 1 mM. The purified enzyme is stable during storage in 0.05 M phosphate buffer, pH 6.7, containing 40-50% glycerol, at -20 degrees C.     

Aminopeptidase from a thermophilic strain of Bacillus licheniformis

Aminopeptidase is isolated and purified from the culture liquid of the thermophilic strain of Bacillus licheniformis. The aminopeptidase predominantly splits off N-terminal leucin in short peptides and hydrolyzes leucinamide as well. The molecular weight of the enzyme is about 60 kDa. The enzyme is able to form aggregates. Optimum of aminopeptidase activity was demonstrated at pH 8.0-8.3 and temperature of 85 degrees C. The enzyme is inactivated by metal-binding reagents and reducing substances, and is activated by cobalt and PCMB ions. The EDTA-inactivated enzyme activity is reduced by cobalt and zinc ions, however the latter has no activating action. The enzyme under study is characterized by high thermostability: in the presence of the substrate at the temperature of 90 degrees C the reaction linearity is retained for not less than 2 h and without the substrate the half-life of the aminopeptidase at 90 degrees C is 145 min. Extracellular aminopeptidase of the thermophilic strain of B. licheniformis is a new enzyme differing from the aminopeptidases described by the present in high thermostability, induced, evidently, by the presence of one or several disulphide bonds in the enzyme molecule.     

Purification and characterization of chitinase B from moderately thermophilic bacterium Ralstonia sp. A-471

Chitinase B was purified from a culture medium of Ralstonia sp. A-471 by precipitation with (NH4)2SO4 and column chromatography with DEAE-Toyopearl 650 M and Sephacryl S-200. The purified enzyme was homogeneous on SDS-PAGE. The molecular weight was 45,000 by SDS-PAGE. The optimum pH was 5.0 and stable pH was from 5.0 to 10.0. In the early stage of the reaction, chitinase B produced beta-anomer of (GlcNAc)2 from the substrate (GlcNAc)6, whereas (GlcNAc)4 produced almost at equilibrium, indicating that the enzyme predominantly hydrolyzes the second glycosidic linkage from the nonreducing end of (GlcNAc)6.     

Purification and characterization of protease III from Escherichia coli.

An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield. The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4. Protease III is very sensitive to metal-chelating agents and reducing agents. The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+. Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins. When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000. Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26).     

Purification and characterization of a steroid-binding sialoglycoprotein from rat ventral prostate

An androgen-dependent sialoglycoprotein was purified from the secretion of rat ventral prostate by chromatofocusing and DEAE-Sepharose column chromatography. It showed a native molecular weight of 47,000 and consisted of two dissimilar subunits with molecular weights of 20,000 and 18,000. However, each subunit contained a common peptide with molecular weight of 16,000. It also contained 442 +/- 62 micrograms sialic acids per milligram protein and bound pregnenolone with a binding affinity of 1.2 microM-1. Its amino acid composition was similar to those of other known prostatic steroid-binding proteins. Hence, we propose that it is the sialylated form of rat prostatic steroid-binding protein.     

Isolation, partial purification and pharmacodynamics of a slow contractile substance in the venom sac extract of the wasp Vespa cincta Fabr

The venom of V. cincta contains acetylcholine (ACh), histamine and 5-hydroxytryptamine (5-HT). Blockers of these agonists did not block completely the hypotensive and smooth muscle contractile activity of venom. On smooth muscle, there was a residual slow contraction. The active substance which produced this slow contraction was separated by solvent extraction, gel filtration and TLC. The purified material (which has been provisionally designated "Vecikinin") lowered cat, rat and guinea pig blood pressure, increased amplitude of cardiac contraction, and increased capillary permeability. Vecikinin contracted several smooth muscle preparations (rat uterus, rat ascending colon, guinea pig ileum, guinea pig colon and rat ileum), while relaxing rat duodenum. Its contractile activity was not lost on boiling, but acid or alkali-boiling reduced its contractile activity. It was inactivated on incubation with chymotrypsin and carboxypeptidase but not with trypsin, pepsin or leucine aminopeptidase. It is a peptide, appears to be of low molecular weight, and could be distinguished from substance P, angiotensin, bradykinin and hornet or wasp kinin.     

The purification and characterization of a proline dipeptidase from guinea pig brain

A proline dipeptidase (EC 3.4.13.9) from guinea pig brain was purified to over 90% homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, calcium phosphate-cellulose chromatography, chromatofocusing, and gel filtration on Sephadex G-200. A purification factor of 2718-fold was obtained with a yield of 7%. The purified enzyme was found to have an apparent molecular weight of 132,000 and to consist of two dissimilar subunits of molecular weights 64,000 and 68,000. The substrate specificity of the enzyme is not that of a strict proline dipeptidase. Although it preferentially hydrolyzes proline dipeptides (Leu-Pro) it also hydrolyzes prolyl dipeptides (Pro-Leu) and dipeptides not containing proline (Leu-Leu). The purified enzyme preparation exhibited weak aminoacylproline aminopeptidase activity against Arg-Pro-Pro but it did not exhibit any post-proline dipeptidyl aminopeptidase, post-proline cleaving endopeptidase, proline iminopeptidase, prolyl carboxypeptidase or carboxypeptidase P activities when tested with a large variety of peptides and arylamides. With all of the proline and prolyl dipeptides examined the enzyme exhibited biphasic kinetics (two distinct slopes on Lineweaver-Burk plots). However, with Leu-Leu as substrate normal Michaelis-Menten kinetics were obeyed.     

Antisera to synthetic peptide recognize high molecular weight enkephalin-containing proteins

Antisera to a synthetic peptide corresponding to the 95-117 sequence of proenkephalin were used to develop a sensitive radioimmunoassay. Gel-filtration of acid extracts of bovine adrenal medulla and purified chromaffin granules revealed that the antisera recognized high molecular weight material (Mr approximately 5,000-30,000). The material in peak I ( Mr 20 ,000-30,000) and peak II (Mr 10,000-20,000) was further purified by immunoaffinity chromatography. Sequential digestion of each of these fractions with trypsin and carboxypeptidase B generated immunoreactive Met-enkephalin. This study demonstrates that antisera against a synthetic peptide cross-react with high molecular weight enkephalin-containing precursors, validating the use of these antisera in studies of enkephalin biosynthesis.     

Purification and specificity of prolyl dipeptidase from bovine kidney.

Prolyl dipeptidase (iminodipeptidase, L-prolyl-amino acid hydrolase, EC 3.4.13.8) was purified 180-fold from bovine kidney. The enzyme which was obtained in a 10% yield was completely separated from a number of known kidney peptidases including an enzyme of very similar substrate specificity, proline aminopeptidase (L-prolyl-peptide hydrolase, EC 3.4.11.5). The specific activity of the enzyme with L-prolylglycine as substrate is 1600 units of activity per mg protein. Optimum activity of the enzyme is at pH 8.75 and the molecular weight on gel filtration was estimated to be 100 000. The isoelectric point of the enzyme is pH 4.25. Studies of substrate specificity showed that the enzyme preferentially hydrolyzes dipeptides and dipeptidyl amides with L-proline or hydroxy-L-proline at the N-terminus. Longer chain substrates with N-terminal proline were not hydrolyzed.     

Purification and characterization of proline-beta-naphthylamidase, a novel enzyme from pig intestinal mucosa

An enzyme hydrolyzing proline-beta-naphthylamide was purified to apparent homogeneity from porcine intestinal mucosa. The purified enzyme appears to consist of three identical subunit polypeptides with a molecular weight of about 58,000 each, associated noncovalently. The enzyme is a glycoprotein, and the subunit polypeptide contains 3 residues each of mannose and N-acetylglucosamine. A wide variety of peptidase substrates were tested for the enzyme, and the results showed that it hydrolyzes only aminopeptidase substrates, such as proline-beta-naphthylamide, glycine-beta-naphthylamide, leucine-beta-naphthylamide, and alanine-beta-naphthylamide. Among these substrates, proline-beta-naphthylamide is most efficiently hydrolyzed as judged by the kcat/Km value. The optimum pH for this substrate is around 9. The enzyme also hydrolyzes efficiently the ester substrates of these amino acids. No hydrolytic activity was observed for the peptide and protein substrates tested. The proline-beta-naphthylamidase activity was drastically inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, indicating that the enzyme is a serine hydrolase, whereas it was slightly inhibited by aminopeptidase inhibitors, such as amastatin, bestatin, and puromycin. No significant homology was found for the NH2-terminal sequence of 27 amino acid residues with any known protein sequences. From these results we conclude that the enzyme is a protein which has not been described before.     

Identification and characterisation of proteinase inhibitors and their genes from seeds of apple (Malus domestica)

Trypsin and papain proteinase inhibitors have been identified and purified from aqueous extracts of apple seeds (Malus domestica). Superdex G75 gel filtration chromatography identified a higher molecular weight (HMW) papain inhibitory fraction (22-26 kDa) and a lower molecular weight papain and trypsin inhibitory fraction (6-12 kDa). The lower molecular weight fraction was separated into a trypsin inhibitor (designated Trp1) and early (designated Pap1) and late (designated Pap2) eluting papain inhibitors after anion exchange (Hitrap SP) chromatography. For Pap2, two inhibitory peaks (designated Pap2-1 and Pap2-2) were identified after further anion exchange (Resource S) chromatography. Each of these lower molecular weight inhibitors was purified by reverse phase HPLC to homogeneity as determined by SDS-PAGE and by mass spectrometry. The HMW papain inhibitory fraction was purified further by anion-exchange (Hitrap Q followed by Resource Q) column chromatography where a minor inhibitor (HMWPap1) and major inhibitor (HMWPap2) fraction were identified. The relative abundance in seeds of apple and the spectrum of proteinase inhibition has been determined for all of these inhibitors. Reverse-phase HPLC separated HMWPap2 into a minor (HMWPap2-1) and a major (HMWPap2-2) inhibitory fraction, and SDS-PAGE and mass spectrometry confirmed that HMWPap2-2 was purified to homogeneity. Amino acid composition data were obtained from Trp1, Pap1, Pap2-2, and HMWPap2-2, and N-terminal sequence data from Trp1, Pap2-1, Pap2-2, and HMWPap2-2, with two of these sequences (Pap2-2 and HMWPap2-2) perfectly matching predicted protein sequences based on EST sequences from an apple database. The relationship of these inhibitors with those of other species is discussed.     

Separation of the protective enzyme bleomycin hydrolase from rabbit pulmonary aminopeptidases

Bleomycin (BLM) hydrolase inactivates the BLM class of antitumor antibiotics and protects against BLM-induced pulmonary fibrosis. This enzyme is poorly characterized but believed to be an aminopeptidase B. In the present report, both BLM hydrolase and aminopeptidase B from rabbit pulmonary cytosol were retained by arginyl-Sepharose and BLM-Sepharose affinity columns, further suggesting that these two enzymes are similar. When, however, BLM hydrolase was purified over 1800-fold by using our newly developed high-speed liquid chromatography assay for BLM hydrolase coupled with fast protein liquid chromatography, we found that this partially purified BLM hydrolase preparation lacked aminopeptidase B activity. Furthermore, BLM hydrolase was completely separated, by using anion-exchange Mono Q chromatography, from all the aminopeptidases identified in rabbit pulmonary cytosol: one aminopeptidase B, two aminopeptidases N, and one aminopeptidase with both aminopeptidase B and aminopeptidase N activities. Pulmonary BLM hydrolase also had a higher molecular weight than pulmonary aminopeptidase B. In contrast to aminopeptidase B, BLM hydrolase was not activated by NaCl and was much less stable at 4 degrees C. In addition, bestatin was a potent inhibitor of aminopeptidase B but had little effect on BLM hydrolase, while leupeptin was a potent inhibitor of BLM hydrolase but was less effective against aminopeptidase B. Thus, pulmonary BLM hydrolase and aminopeptidase B have affinity for each other's substrate, but they are clearly distinct enzymes on the basis of charge characteristics, molecular weight, stability, and sensitivity to inhibitors and activators.     

Localization and purification of two enzymes from Escherichia coli capable of hydrolyzing a signal peptide

The signal peptide generated during the maturation of prolipoprotein by the purified prolipoprotein signal peptidase can be isolated in substrate amounts (Dev, I. K., and Ray, P. H. (1984) J. Biol. Chem. 259, 11114-11120). This signal peptide is degraded predominantly from the carboxyl terminus by cell-free extracts of Escherichia coli. The signal peptide is degraded (at least 300-fold) more rapidly than other cellular proteins in E. coli. Greater than 90% of the signal peptide hydrolase activity is localized in the cytoplasm. Two enzymes from the cytoplasmic fraction responsible for the degradation of the signal peptide have been identified and purified to near homogeneity. The major activity is associated with a monomeric protein with a molecular weight of 68,000 (S.E. 3,400) as determined by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme appears to be similar to the oligopeptidase (Vimr, E. R., Green, L., and Miller, C. G. (1983) J. Bacteriol. 153, 1259-1265) that hydrolyzes N-acetyl tetra alanine. The second protein represents approximately 5% of the total cytoplasmic activity and has been shown to be a dimer with a monomer molecular weight of 81,000 (S.E. 5,300). This enzyme is similar to protease So (Chung, H. C., and Goldberg, A. L. (1983) J. Bacteriol. 154, 231-238).     

Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosine 3': 5'-monophosphate-dependent protein kinase.

Turkey gizzard smooth muscle light chain kinase was purified by affinity chromatography on calcium dependent regulator weight of 125,000 +/- 5,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When myosin light chain kinase is incubated with the catalytic subunit of cyclic AMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of myosin kinase. Brief tryptic digestion of the 32P-labeled myosin kinase liberates a single radioactive peptide with a molecular weight of approximately 22,000. Phosphorylation of myosin kinase results in a 2-fold decrease in the rate at which the enzyme phosphorylates the 20,000-dalton light chain of smooth muscle myosin. These results suggest that cyclic AMP has a direct effect on actin-myosin interaction in smooth muscle.     

Substrate specificity of collagenolytic proteases from the hepatopancreas of the of the Kamchatka crab]

The substrate specificity of two isozymes of collagenolytic protease of the crab (Paralithodes camtschatica) was studied. It was found that both proteases can effectively hydrolyze type I and III collagens, as well as gelatin, the set of products yielded by enzymatic hydrolysis being different for isozymes A and C. Hydrolysis of some well-known peptides revealed that isozyme A predominantly cleaves the peptide bonds containing arginine and lysine residues, whereas isozyme C predominantly hydrolyzes bonds containing hydrophobic amino acids. The catalytic constants for the hydrolysis of several low molecular weight substrates in the presence of P. camtschatica proteases were determined, which allowed to attribute isozyme A to trypsin-like, and isozyme C to chymotrypsin-like proteinases. The peptide substrates of collagenase, Pz-Pro-Leu-Gly-Pro-D-Arg and Z-Gly-Pro-Ala-Gly-Pro-Ala are not hydrolyzed isozymes of crab collagenolytic protease.     

Isolation and characterization of peptidoglycans in urine from patients with mucopolysaccharidoses

Urinary dermatan sulfate (DS) and heparan sulfate (HS) were purified from mucopolysaccharidosis patients. DS shows average mol. wt 8600-12,000 (approx. one half of tissue DS), iduronic acid content 99.1-99.6% (81.8% in tissue DS), core peptide mostly di- or tri-peptide (--Ser--Gly--or--Ser--Gly--Glu--). Molecular weight of HS ranged from 2500 to 20,000, averaging about 5000. Highly sulfated HS was found in the low molecular weight fraction, and no bound core peptide. By contrast, HS in the high molecular weight fraction bound one sulfate per repeating unit, which include core peptide.     

Dipeptidyl Aminopeptidase III of Guinea-Pig Brain: Specificity for Short Oligopeptide Sequences

Abstract: A dipeptidyl aminopeptidase III-type activity has been purified from the cytoplasm of guinea-pig brain using arginyl-arginyl-7-amido-4 methylcoumarin as substrate. The enzyme was purified 754-fold relative to the crude homogenate and with a 12.7% recovery. The purified enzyme was found to have a relative molecular weight of 85,000 and consists of one polypeptide chain of relative molecular weight 80,000, on the basis of its migration on calibrated sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel. It is highly sensitive to the presence of chelating agents, sulphydryl reactive agents, and the dipeptide Tyr-Tyr. Dithiothreitol (1 m M ) reduced activity by 28%, and 36 and 65% inhibition was noted with phenylmethylsulphonyl fluoride and puromycin (both at 1 m M ), respectively. Little or no inhibition was observed with bestatin, bacitracin, captopril, amastatin, and arphamenine B. The purified enzyme released dipeptide moieties from a wide range of peptides including enkephalin sequences and also angiotensin sequences up to the octapeptide angiotensin II. These sequences inhibited the hydrolysis of arginyl-arginyl-7-amido-4-methylcoumarin by dipeptidyl aminopeptidase III with K _i values in the micromolar range. No hydrolysis was observed with angiotensin I or with peptide sequences containing more than 10 amino acids. No hydrolysis was observed also with peptide sequences containing a Pro residue on either side of the sissile bond. Peptides containing less than four amino acids were not hydrolysed.     

A tumor growth inhibitory factor and a tumor growth promoting factor isolated from unfertilized ova of shad (Alosa sapidissima)

In the present study, a cytostatic tumor growth inhibitory peptide and a tumor growth promoting peptide with molecular weights of 20,000-30,000 Da have been identified in the supernatant fraction of unfertilized ova from Shad. The factors can be separated by gel chromatography, thus indicating that the factors are individual molecules. Both of the factors are nondialyzable, heat stable, and resistant to trypsin digestion and periodate oxidation.     

Purification of two high molecular weight proteases from rabbit reticulocyte lysate

We have purified two high molecular weight proteases approximately 400-fold from rabbit reticulocyte lysate. Both enzymes hydrolyze 125I-alpha-casein and 4-methylcoumaryl-7-amide peptides with tyrosine, phenylalanine, or arginine at the P1 position. Both are inhibited by hemin, thiol reagents, chymostatin, and leupeptin. They differ, however, by other criteria. Degradation of 125I-lysozyme-ubiquitin conjugates and succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide by the larger 26 S protease is stimulated by ATP. Based on sedimentation, gel filtration, and nondenaturing polyacrylamide gel electrophoresis, the ATP-dependent protease has a molecular weight of 1,000,000 +/- 100,000 and is a multisubunit complex. The smaller 20 S protease has a molecular weight of 700,000 +/- 20,000 and is composed of 8-10 separate subunits with Mr values between 21,000 and 32,000. It does not require nucleotides for degradation of protein or peptide substrates. This smaller enzyme is similar, if not identical, to the "multicatalytic proteinase complex" first described by Wilk and Orlowski (Wilk, S., and Orlowski, M. (1983) J. Neurochem. 40, 842-849).