Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: KEGG:D03575 (CoCl2)
1,247 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Carboxypeptidase B of the human pancreas was purified by chromatography on DEAE-cellulose and CM-cellulose columns. Two forms of the enzyme, named carboxypeptidase B1 and B2, were separated. They have similar mol.wts. (34250 +/- 590) as established by polyacrylamide-gel disc electrophoresis and by gel filtration. Carboxypeptidase B2 migrates further towards the anode in disc electrophoresis. When the amino acid content of the enzymes was analysed, carboxypeptidase B2 had four more glycine and three more aspartic acid residues than had form B1. The amino acid sequence of the human carboxypeptidase B1 differs from that of the bovine enzyme only in two places in the N-terminal 20-amino-acid sequence. The N-terminal amino acid in carboxypeptidase B1 and B2 is alanine. The peptide 'map' of the tryptic digest of carboxypeptidase B1 contained more peptides than did that of form B2. The Km, the Vmax. and the pH optimum of the cleavage of the peptide substrate hippurylarginine and the ester substrate hippurylargininic acid were similar for both enzymes. CoCl2 accelerated the peptidase activity, and cadmium acetate enhanced the esterase activity, of human carboxypeptidases B1 and B2. Urea and sodium dodecyl sulphate inhibited the enzymes.
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PMID:Purification of carboxypeptidase B from human pancreas. 1 98

The microsomal fraction of rabbit liver contains an endopeptidase that cleaves synthetic peptides that mimic the amino acid sequences of the processing sites of many proproteins, including the vitamin K-dependent proteins. The endopeptidase (M(r) 69,000) was extracted from liver microsomes with 1% Lubrol and purified about 2,700-fold. The substrate employed for isolation and characterization of the enzyme was the decapeptide acetyl-Ala-Arg-Val-Arg-Arg-Ala-Asn-Ser-Phe-Leu (prothrombin peptide), in which hydrolysis occurred on the carboxyl side of the paired Arg-Arg residues. The purified enzyme, whose activity was enhanced 1.8-fold by 0.1 mM CoCl2, has a Km = 80 microM and Vmax = 21,000 nmol.min-1.mg-1 and a pH optimum of 8.7. Proteolytic cleavage of decapeptide substrates was dependent on an arginine residue at positions P1 and P4. The enzyme was completely inhibited by EDTA and 1,10-phenanthroline as well as by p-chloromercuriphenylsulfonic acid and Hg2+. Inhibitors of serine proteases and cysteine proteases had no effect. Based on the substrate preference, the endopeptidase appears to be a good candidate for the enzyme responsible for the precursor processing of the vitamin K-dependent proteins and a number of other proproteins that are synthesized via the secretory pathway in liver and other tissues.
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PMID:A microsomal endopeptidase from liver with substrate specificity for processing proproteins such as the vitamin K-dependent proteins of plasma. 131 98

An alanine aminopeptidase, which has characteristics different from those of known alanine cleaving aminopeptidases, was partially purified from human serum by Sephadex G-200 gel chromatography and DEAE Sepharose column chromatography. The enzyme exhibited a molecular weight of 58,000 by gel chromatography. The pI of the enzyme was 5.0, and it was inactivated at 60 degrees C in 20 min. The enzyme readily hydrolyzed L-alanine beta-naphthylamide, but hardly hydrolyzed the other tested beta-naphthylamides. The Km value for L-alanine beta-naphthylamide was 0.29 mM, the pH optimum 7.5. The activity of the enzyme was enhanced by chloride ions and by sulfhydryl ethylenediaminetetraacetic compounds, and was inhibited by sulfhydryl blocking ethylenediaminetetraacetic agents, and bestatin. Furthermore, 1.10 phenanthroline and ethylenediaminetetraacetic acid were inhibitory, and the activity was restored by CoCl2 and ZnCl2. The enzyme is a chloride-enhanced thiol dependent metalloaminopeptidase.
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PMID:Low molecular weight alanine aminopeptidase of human serum: separation and some characteristics. 198 Jan 89

Carboxypeptidase H, EC 3.4.17.10, also known as enkephalin convertase, carboxypeptidase E, and crino carboxypeptidase B, is an important enzyme involved in the biosynthesis of bioactive peptides. To assay the enzyme, tissues are homogenized in at least 20 vol (ml/g) of 0.025 M Tris-HCl buffer, pH 8, with 5 mg/ml of bovine serum albumin. After centrifugation, the supernatant is brought to pH 5.6 and centrifuged again. Following a 20-min preincubation in 2 mM CoCl2, the supernatant is incubated with 0.1 mM (final concentration) of the radioactive substrate [3H]benzoyl-Phe-Ala-Arg. The 100-microliters assay is stopped by the addition of 680 microliters of acetonitrile/0.25 M HCl (0.7/1). The 1.5-ml tube is transferred into a scintillation vial and is flushed with 4 ml of Econofluor, a water-immiscible scintillation fluid. The product, [3H]benzoyl-Phe-Ala, recovered in the organic phase, is counted directly with no interference from the substrate remaining in the aqueous phase. The blank is below 1%. Expressed in nanomoles per minute per milligram of tissue, the activity of the soluble enzyme in rat is 0.34 for striatum, 21.0 for pancreatic islet, 16.6 for anterior pituitary, 46.0 for intermediate pituitary, and 10.9 for neural pituitary. In every case 25 microM guanidinoethylmercaptosuccinic acid, an active site-directed inhibitor of carboxypeptidase H, completely inhibits the activity.
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PMID:Radiometric assay for carboxypeptidase H (EC 3.4.17.10) and other carboxypeptidase B-like enzymes. 265 84

A membrane-bound neutral carboxypeptidase B-like enzyme was solubilized from human placental microvilli with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and purified to homogeneity by ion-exchange chromatography and affinity chromatography on arginine-Sepharose. It gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 62,000 with or without reduction. The enzyme is a glycoprotein as shown by its high affinity for concanavalin A-Sepharose and reduction in mass to 47,600 daltons after chemical deglycosylation. It has a neutral pH optimum, is activated by CoCl2, and inhibited by o-phenanthroline, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, or cadmium acetate, indicating it is a metallopeptidase. The enzyme cleaves arginine or lysine from the COOH terminus of synthetic peptides (e.g. Bz-Gly-Arg, Bz-Gly-Lys, Bz-Ala-Lys, dansyl-Ala-Arg, where Bz is benzoyl and dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) as well as from several biologically active substrates: dynorphin A(1-13), Met5-Arg6-enkephalin (Km = 46 microM, kcat = 934 min-1), bradykinin (Km = 16 microM, kcat = 147 min-1), Met5-Lys6-enkephalin (Km = 375 microM, kcat = 663 min-1), and Leu5-Arg6-enkephalin (Km = 63 microM, kcat = 106 min-1). Although the enzyme shares some properties with other carboxypeptidase B-like enzymes, it is structurally, catalytically, and immunologically distinct from pancreatic carboxypeptidase A or B, human plasma carboxypeptidase N, and carboxypeptidase H ("enkephalin convertase"). To denote that the enzyme is membrane-bound, and to distinguish it from other known carboxypeptidases, we propose the name "carboxypeptidase M." Because of its localization on the plasma membrane and optimal activity at neutral pH, carboxypeptidase M could inactivate or modulate the activity of peptide hormones either before or after their interaction with plasma membrane receptors.
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PMID:Human carboxypeptidase M. Purification and characterization of a membrane-bound carboxypeptidase that cleaves peptide hormones. 291 4

Multiple forms of dextranase were detected in both intra- and extracellular fractions of Bacteroides oralis Ig4a. The molecular weights of these enzymes varied from 52,000 to 260,000 by sodium dodecyl sulfate-polyacrylamide-blue dextran gel electrophoresis. The intracellular dextranases were fractionated by chromatography and gel filtration steps, and the dextranases IV and V were obtained. The former was only partially pure. The molecular weights of the dextranases IV and V were estimated to be 120,000 and 105,000, respectively, by SDS-PAGE. The dextranase V was further characterized and it was revealed that the pH- and temperature optima were 5.0, and 55 degrees C, respectively. The Km value was 6.7 x 10(-2) mM for dextran T-70. The enzyme did not exhibit any metal ion requirements, but was inhibited by CoCl2 and HgCl2; lysine and alanine contents were especially high; it hydrolyzed the alpha-1,6-glucan by an exo-type mechanism, and was inactive toward glucans containing alpha-1,3-, alpha-1,4-, and beta-1,4-linkages.
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PMID:Purification and characterization of intracellular dextranase of Bacteroides oralis Ig4a. 327 Sep 38

Two aminopeptidases which hydrolyze Metenkephalin at the Tyr-Gly bond have been solubilized from rat brain membranes and resolved by ion-exchange chromatography. These aminopeptidase are designated MI and MII based on the order in which they are eluted during ion-exchange chromatography. The two aminopeptidases can be distinguished kinetically; aminopeptidase MI hydrolyzes L-arginine beta-naphthylamide 17 times faster than L-alanine beta-naphythylamide, while only a 1.7-fold difference is exhibited by aminopeptidase MII. Aminopeptidase MII exhibits a higher affinity for amino acid beta-naphthylamides, Met-enkephalin, Leu-enkephalin, and the inhibitor puromycin as compared to aminopeptidase MI. Greater than 90% of aminopeptidase MII activity is lost upon dialysis against ethylene-diaminetetraacetate (EDTA) but can be reconstituted with CoCl2 and MnCl2. In contrast, aminopeptidase MI loses only 30% of its activity when dialyzed against EDTA. In addition to cleaving the Tyr-Gly bond of Met-enkephalin, aminopeptidase MII also cleaves the Tyr-Gly bond of alpha- and gamma-endorphin. Hydrolysis of Met-enkephalin by intact membranes derived from whole rat brain occurs primarily by cleavage at the Tyr-Gly bond, with this activity attributable to aminopeptidase MII.
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PMID:Solubilization and characterization of two rat brain membrane-bound aminopeptidases active on Met-enkephalin. 723 6

The presence of high concentrations of membrane-bound carboxypeptidase M in human, baboon, dog, and rat lung was established by employing a variety of techniques. The activity of the enzyme in the membrane-enriched fractions of human, baboon, dog, and rat lung, measured with fluorescent dansyl substrate (DNS-Ala-Arg), was 198, 261, 484, and 153 nmol/h/mg protein, respectively. This activity in the lung was much higher than that found in the heart, liver, or kidney. The enzyme, optimally active around neutral pH, was completely inhibited by 10 microM 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid and was activated by 1 mM CoCl2 to 170%. Antibody to human carboxypeptidase M immunoprecipitated the solubilized carboxypeptidase from human (98%), baboon (81%), and dog (88%) lung membrane fractions. Carboxypeptidase M is attached to lung membranes by a phosphatidylinositol glycan anchor; thus, it is released with bacterial phospholipase C. Membrane fractions from cultured human pulmonary arterial endothelial cells also contained high carboxypeptidase M activity (254 nmol/h/mg protein). A Northern blot of poly(A)+ RNA from various human tissues showed the presence of a high level of carboxypeptidase M mRNA in human lung and placenta. Finally, immunohistochemistry, employing purified antibody to the enzyme, revealed in fluorescent light microscopy that carboxypeptidase M is present in alveolar type I pneumocytes and in macrophages in apparently lower concentration. In contrast, type II alveolar epithelial cells gave negative results. Because carboxypeptidase M cleaves a variety of active peptides (e.g., bradykinin, anaphylatoxins), it may protect the alveolar surface from the effects of these peptides. In addition, carboxypeptidase M could be a marker enzyme for type I cells.
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PMID:High concentration of carboxypeptidase M in lungs: presence of the enzyme in alveolar type I cells. 833 89

An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn(Penaeus japonicus) was partially purified by Fast Protein Liquid Chromatography with hydrophobic interaction (Phenyl Sepharose Hig hPerformance) chromatography and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl-methylsulfonyl fluoride (PMSF),and showed maximal activity at pH 10, having a molecular weight of about 33kDa estimated by SDS-PAGE and gel filtration chromatography. In addition, the toxin was also completely inhibited by FeCl2 but partially inhibited by CaCl2, CuCl2, CoCl2,MnCl2, and ZnCl2, and not inhibited by ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-amino-ethyl ether)N,N,N',N'-tetraacetic acid (EGTA), iodoacetamide, pepstatin A, sodium dodecylsulfate (SDS), and N-tosyl-l-phenyl-alanine chloromethyl ketone (TPCK). Both the crude extracellular products (ECP) and the partially purified toxin are lethal for kuruma prawn at LD50 values of 0.30 and 0.27 microg protein/g body weight, respectively. The addition of PMSF completely inhibited the lethal toxicity of both the ECP and the partially purified toxin, indicating that this serine protease is a lethal factor produced by the bacterium. The 33-kDa protease is, therefore, suggested to be a new toxic protease produced by V. alginolyticus strain Swy.
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PMID:Alkaline serine protease is an exotoxin of Vibrio alginolyticus in kuruma prawn, Penaeus japonicus. 900 88

A novel metallocarboxypeptidase (PfuCP) has been purified to homogeneity from the hyperthermophilic archaeon, Pyrococcus furiosus, with its intended use in C-terminal ladder sequencing of proteins and peptides at elevated temperatures. PfuCP was purified in its inactive state by the addition of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) to purification buffers, and the activity was restored by the addition of divalent cobalt (K, = 24 +/- 4 microM at 80 degrees C). The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) had no effect on the activity. The molecular mass of monomeric PfuCP is 59 kDa as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 58 kDa by SDS-PAGE analysis. In solution, PfuCP exists as a homodimer of approximately 128 kDa as determined by gel filtration chromatography. The activity of PfuCP exhibits a temperature optimum exceeding 90 degrees C under ambient pressure, and a narrow pH optimum of 6.2-6.6. Addition of Co2+ to the apoPfuCP at room temperature does not alter its far-UV circular dichroism (CD) or its intrinsic fluorescence spectrum. Even when the CoPfuCP is heated to 80 degrees C, its far-UV CD shows a minimal change in the global conformation and the intrinsic fluorescence of aromatic residues shows only a partial quenching. Changes in the intrinsic fluorescence appear essentially reversible with temperature. Finally, the far-UV CD and intrinsic fluorescence data suggest that the overall structure of the holoenzyme is extremely thermostable. However, the activities of both the apo and holo enzyme exhibit a similar second-order decay over time, with 50% activity remaining after approximately 40 min at 80 degrees C. The N-blocked synthetic dipeptide, N-carbobenzoxy-Ala-Arg (ZAR), was used in the purification assay. The kinetic parameters at 80 degrees C with 0.4 mM CoCl2 were: Km, 0.9 +/- 0.1 mM; Vmax, 2,300 +/- 70 U mg(-1); and turn over number, 600 +/- 20 s(-1). Activity against other ZAX substrates (X = V, L, I, M, W, Y, F, N, A, S, H, K) revealed a broad specificity for neutral, aromatic, polar, and basic C-terminal residues. This broad specificity was confirmed by the C-terminal ladder sequencing of several synthetic and natural peptides, including porcine N-acetyl-renin substrate, for which we have observed (by MALDI-TOF MS) stepwise hydrolysis by PfuCP of up to seven residues from the C-terminus: Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser.
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PMID:Purification and characterization of a cobalt-activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus. 1059 52


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