Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A neutral protease with kininogenase activity was isolated from human polymorphonuclear (PMN) leukocytes by cation exchange chromatography and gel filtration. The protease appears heterogeneous by cation exchange chromatography, isoelectric focusing and cationic disc gel electrophoresis, but homogeneous by gel filtration, sucrose density gradient ultracentrifugation, SDS-disc gel electrophoresis and immunoelectrophoresis. By carrying out the electrophoresis of the protease in acrylamide gels of varying concentrations, it was shown that they represent charge isomers. The protease was stable at pH 4-10, but labile to heat, being almost completely inactivated when incubated for 30 min at 70 degrees C. It exhibited proteolytic activity between pH 5 and 9, being maximal at 7.5-8.5. The molecular weight of the PMN protease was estimated to be about 20,000 daltons by gel filtration in aqueous buffer and about 26,000-28,000 daltons by SDS-disc gel electrophoresis and gel filtration in Sepharose 6B in the presence of the dissociating agent guanidine HCl. Its sedimentation coefficient was about 2.7S. Corresponding to the charge heterogeneity, by isoelectric focusing, the kinin-generating and esterolytic activities of the PMN granule lysate focused between pH 6.0 and 11.5, whereas the isolated PMN protease focused between 10.0 and 11.8. With respect to kinin generation, caseinolysis, and alanine esterase activity, the protease was inhibited by DFP and certain chloromethyl ketone inhibitors, as well as the plasma protease inhibitory a1-antitrypsin, a2-macroglobulin and antithrombin III. Both bradykinin and a methionyl-lysyl-bradykinin-like peptide were generated from highly purified kininogens by a lysosomal lysate containing the PMN protease. However, this assay was done with a crude enzyme preparation which contains an aminopeptidase capable of converting lysyl-bradykinin or methionyl-lysyl-bradykinin to bradykinin. When injected intradermally, the protease induced hyperemia, hemorrhage, and moderate enhancement of vascular permeability, but the mixture of the protease and kininogen induced a marked enhancement of vascular permeability.
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PMID:Neutral proteases of human PMN leukocytes with kininogenase activity. 5 89

The isolation and characterization of a microsomal arylaminopeptidase from rat kidney is reported. By treatment of a microsomal arylaminopeptidase-phosphatase-complex with trypsin and subsequent gel filtration of the solubilized proteins on Sepharose 6B a electrophoretic homogeneous arylaminopeptidase was obtained (yield, 3%; enrichment, 900 times). The following properties of the purified enzyme were determined: 1. Molecular weight: 182000 (gel filtration on Sepharose 6B) to 192000 (SDS-polyacrylamide gel electrophoresis). 2. Subunit structure: In the presence of 6 M guanidine - HC1 + 1% BETA-mercaptoethanol the enzyme dissociates into subunits (MW 46700, ESTIMATED BY SDS gel electrophoresis method). 3. Isoelectric point: 4,71 (agarose gel electrophoresis method). 4. UV characteristics: E 280nm/E260NM=1.3. 5. Substrate specifity: optimal substrates L-alanyl derivatives (anilide, beta-naphthyl amide, p-nitroanilide, 4-(phenylazo)-phenylamide and hydrazide). Among these compounds the anilide derivative was hydrolyzed most rapidly. Furthermore, di- and tripeptides, especially L-methionyl-L-leucine, were also split. No hydrolysis was observed with hemoglobin (pH 4.5 and 7.5) and amino acid- or peptide-ester substrates. 6. Optimal pH: 7.5 +/- 0,1; optimal temperature: 45 to 50 degrees C. 7. The enzyme has no transamidation activity with L-alanyl amide both as aminoacyl donator and -acceptor. 8. Influence of effectors: Heavy metal ions (Ni2+, Cd2+, Cu2+, Zn2+), chelating agents (EDTA, o-phenanthroline) and puromycin inhibit the enzyme significantly. SH-group reagents are without any influence. 9. L-alanyl-L-alanyl-4 (phenylazo)-phenylamide, a dipeptide aryl aminopeptidase substrate, is hydrolyzed by the purified enzyme preparation according to a consecutive or step by step mechanism.
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PMID:[Isolation and characterization of a microsomal arylaminopeptidase from rat kidney]. 97 46

An aminopeptidase isolated from the cytoplasmic fraction of a cell extract of Streptococcus mitis ATCC 903 was purified 330-fold by ion-exchange chromatography, gel filtration, and hydroxyapatite chromatography. The partially purified enzyme had a broad substrate specificity. Twelve aminoacyl-beta-naphthylamide substrates were hydrolyzed and also several di-, tri-, tetra-, and pentapeptides and bradykinin. The enzyme hydrolyzed arginine-beta-naphthylamide at the highest rate. Optimal conditions for activity were at pH 7.0-7.2 and at 37-40 degrees C. The molecular weight of the enzyme was estimated to be 93,000. The enzyme was activated by Co2+ ions. Hg2+ inhibited the activity completely. SDS, EDTA, urea, and pCMB also inhibited activity. Inhibition by EDTA could be completely reversed by dialysis and addition of Co2+ ions. Reducing agents, sodium fluoride, and PMSF had no effect on the activity of the enzyme. The isoelectric point of the enzyme was at pH 4.3. High substrate concentrations inhibited activity. Substrate inhibition increased in the presence of high concentrations of Co2+ ions.
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PMID:Purification and characterization of an aminopeptidase from Streptococcus mitis ATCC 903. 136 97

Cytosolic aminopeptidase P was obtained in highly purified form from human leukocytes by a four-step procedure. Buffy coats were the starting material. A M(r) of 140,000 was obtained by size-exclusion HPLC for the native enzyme. As shown by SDS/PAGE under reducing and denaturing conditions, the enzyme consisted of likely identical subunits with M(r) of 71,000. Purified aminopeptidase P cleaved off, specifically and efficiently, the N-terminal residues from peptides with N-terminal Xaa-Pro sequences. The penultimate proline was not replaceable by hydroxyproline, alanine and glycine in di-, tri- and tetrapeptides. Polyproline was not hydrolyzed. Dipeptides were cleaved (Arg-Pro, Phe-Pro > Trp-Pro > Pro-Pro) although slower than longer peptides. Cleavage was observed of several biologically active peptides; C-terminal fragment (residues 201-206) of C-reactive protein, oxytocin fragment Tyr-Pro-Leu-Gly, morphiceptin, peptide Gly-Pro-Arg-Pro (inhibitor of fibrin polymerization) and kentsin. In addition, cleavage of a protein, interleukin-6, was also demonstrated. Aminopeptidase P was maximally activated by Mn2+, and to a lesser extent by Co2+. The activity was optimal at pH 8. Ni2+, Zn2+ and especially Cd2+ caused marked inhibition. EDTA, 1,10-phenantroline and dithiothreitol were also inhibitory. Carbobenzoxy-phenylalanine, as well as several N-carbobenzoxy-proline-containing peptides, caused partial inhibition. The observed resistance of Gly-Pro, Pro-Gly, Pro-Phe and Pro-Ile to hydrolysis by the purified enzyme strongly indicates absence of known proline-specific dipeptidases in the aminopeptidase-P preparation.
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PMID:Aminopeptidase P from human leukocytes. 144 89

NADP-malate dehydrogenase (L-malate: NADP oxidoreductase, EC 1.1.1.82) from leaves of Pisum sativum has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis. In the crude leaf extract and in the absence of protease inhibitors in the isolation medium, the N-terminus of NADP-MDH was found to be highly susceptible to proteolysis. Evidence of proteolysis during purification includes observations of reduced subunit size on SDS-PAGE and reduced specific activity. Experiments were carried out to investigate the function of the N-terminal amino acid sequence extension of NADP-MDH. Limited proteolysis of highly active (600 units/mg protein) NADP-MDH using aminopeptidase K yielded catalytically active monomers of 34.7 kDa. The results support the conclusions that the N-terminal region is located at the surface of the protein, and that for maintenance of the native NADP-MDH dimer an N-terminal amino acid sequence is important.
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PMID:Limited proteolysis of NADP-malate dehydrogenase from pea chloroplast by aminopeptidase K yields monomers. Evidence of proteolytic degradation of NADP-malate dehydrogenase during purification from pea. 147 42

Aminopeptidase H was purified from fresh chicken breast muscle by ammonium sulfate fractionation and successive chromatographies on DEAE-cellulose, Ultrogel AcA 34, activated thiol-Sepharose 4B, phenyl-Sepharose CL-4B and DEAE-cellulose again. The purified enzyme migrated as a single band on SDS/PAGE. Aminopeptidase H exhibits activity against both L-leucine beta-naphthylamide and alpha-N-benzoyl-DL-arginine beta-naphthylamide. The molecular mass of this enzyme was found to be 52 kDa on SDS/PAGE and 400 kDa on Sepharose 6B column chromatography. The optimum pH for the hydrolysis of both substrates was 8.0 and this activity was remarkably enhanced by reducing agents. The enzyme was strongly inhibited by monoiodoacetate and leupeptin, but not affected by EDTA, phenylmethylsulfonyl fluoride, pepstatin, bestatin or puromycin. Aminopeptidase H has been shown to hydrolyze di-, tri- and tetrapeptides in the manner of an aminopeptidase, as well as the beta-naphthylamide derivatives of amino acids. However, the enzyme has not been shown to hydrolyze proteins such as hemoglobin, bovine serum albumin, myofibrillar proteins or sarcoplasmic proteins.
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PMID:Purification and properties of aminopeptidase H from chicken skeletal muscle. 151 89

Five-hundred-and-six fresh isolates of rumen bacteria were tested for their ability to hydrolyse the synthetic substrate for dipeptidyl aminopeptidase type I, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA), using a gel overlay technique. Twelve positive isolates were small Gram-negative rods which resembled Bacteroides ruminicola in their biochemical and morphological properties. SDS-PAGE of whole cell extracts indicated that two were similar to B. ruminicola strain B14, six resembled B. ruminicola strain M384, and four were similar to B. ruminicola GA33. All hydrolysed GlyArg-MNA, Ala2 and Ala5, and showed no activity against Leu-MNA. Ala3 and Ala2, but no Ala4, was produced from Ala5. The different groups had different, distinctive activity profiles. The two remaining positive isolates were Lactobacillus spp. with an exceptionally high Leu-MNA activity. It was concluded that, although different strains may only be distantly related, B. ruminicola forms the most important group of bacteria in the rumen to possess a dipeptidyl aminopeptidase type I activity.
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PMID:Selective isolation of bacteria with dipeptidyl aminopeptidase type I activity from the sheep rumen. 152 49

The isolation of cytosol aminopeptidase (CAP) from intestinal mucosa has involved difficult procedures because of low yield, instability of the enzyme and many other factors related to purification. The authors resolved these difficulties in the purification of the enzyme from intestinal mucosa of rats using hydrophobic chromatography on phenyl-TOYOPEARL 650S and ion exchange HPLC on Mono Q 5/5. In this procedure, a 60-fold purification was achieved and the purified enzyme revealed a single band with 56kDa molecular weight by SDS polyacrylamide gel electrophoresis. The purified enzyme preferentially hydrolyzed the Leucyl-containing peptides of Leu-Gly and Leu-Gly-Gly. However, it showed no activity on synthetic substrates of Leu-beta-naphthylamide, Gly-p-nitroanilide, Phe-p-nitroanilide or Met-beta-naphthylamide. The Km and Vmax for Leu-Gly were 0.91 mmol/l and 16.4 mol/min/mg protein, while for Leu-Gly-Gly they were 0.77 mmol/l and 20.6 mol/min/mg protein. The purified enzyme was heat-labile and quickly became less active in highly concentrated ammonium sulfate. The optimum pH was 6.5-10. The enzyme activity was stimulated by Mn2+ and Mg2+, while it was inhibited by EDTA, 1,10-phenanthroline, 2-mercaptoethanol and p-hydroxymercuribenzoate. The results suggested the participation of metal ions and the SH group in the enzyme activity. Furthermore the cytosol aminopeptidase was distinct from the brush-border membrane aminopeptidase in molecular weight, immunoreaction to cytosol aminopeptidase antiserum and the specificities to the substrates.
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PMID:[Purification and characterization of cytosol aminopeptidase from rat intestinal mucosa]. 188 Jan 98

The aminopeptidase was isolated from cell-free extracts of Xanthomonas rubrilineans by protein precipitation by isopropyl ester with subsequent purification by affinity chromatography on CABS-Sepharose, bacitracin-Sepharose, gel filtration through Sephadex G-200 and ultrafiltration, the total yield being 32% with 2200-fold purification. The enzyme was homogeneous during SDS-PAAG electrophoresis. Apart from the broad spectrum of the peptidase activity, aminopeptidase possesses a hydrolase activity towards beta-lactam antibiotics and an esterase activity towards L- and D-amino acids. Besides, this enzyme catalyzes the acetyl transfer reaction during cephalexin synthesis from the D-phenylglycine ester and 7-aminodesacetoxycephalosporanic acid. The maximal enzyme activity during L-Ala-pNA and cephalexin hydrolysis is manifested at pH 6.5. The enzyme is stable at pH 4.0-8.0 and is inhibited by o-phenanthroline, p-chloromercuribenzoate, hydrogen acetate and N-bromosuccinimide. The molecular mass of the enzyme is 270-280 kDa. The enzyme is a tetramer; the molecular mass of each of its four subunits is 70 +/- 2 kDa. The isoelectric point for the enzyme is 6.8. The amino acid composition of the enzyme appears as follows: Asp63, Thr33, Ser32, Glu72, Gly55, 1/2Cys3-4, Val45, Ile24, Leu53, Tyr23, Phe24, Lys23, His16, Arg36, Pro60, Met25, Ala55.
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PMID:[Intracellular aminopeptidase from Xanthomonas rubrilineans, hydrolyzing alpha-amino acid esters and cefalexin]. 209 52

An intracellular aminopeptidase (EC 3.4.11.-) was purified from the extreme thermophilic archaebacterium, Sulfolobus solfataricus. The molecular weight of the native enzyme was about 320,000, as calculated by gel-filtration studies, and a subunit Mr of 80,000 was estimated by SDS-polyacrylamide gel electrophoresis. The temperature optimum of the enzyme was at 75 degrees C and the pH optimum was found to be 6.5. The aminopeptidase was highly active against the chromogenic substrates L-Leu-p-NA and L-Ala-p-NA. The enzyme was inhibited by EDTA, but the activity could be partially restored by removal of the EDTA and incubation with Co2+ or Mn2+. Bestatin, a typical inhibitor of aminopeptidase, fully inhibited the enzyme activity, but inhibitors of serine proteinases had no effect. Beside a high thermostability, the enzyme showed a remarkable stability against 6 M urea, organic solvents and acetonitrile.
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PMID:Isolation and characterization of an intracellular aminopeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus. 210 44


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