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 hydrolase activity that cleaves lysyl-p-nitroanilide (Lys-pNA) has been purified from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 by chromatography on DE52, DEAE Affi-Gel Blue Gel, Hydroxyapatite Bio-Gel HTP and Phenyl Sepharose. The purified aminopeptidase was found to have a native M(r) of 50,000-55,000 by gel filtration chromatography and by FPLC gel filtration on Superose 12 and to be composed of a single polypeptide chain following SDS-PAGE. Enzyme activity was almost completely inhibited by EDTA, amastatin, puromycin and bestatin, while the sulphydryl-reactive agents p-chloromercuribenzoate and iodoacetamide were inhibitory. The enzyme was found to be very unstable during the purification procedures at 4 degrees C and its stability was greatly improved when 10 ml glycerol/l and 2 mM-dithiothreitol were included in the purification buffers. The purified enzyme was found to hydrolyse a wide range of dipeptides, tripeptides and longer peptides provided that proline was not present in the penultimate position from the N-terminus or that a pyroglutamyl residue was not present at the N-terminus. While neither Asp-pNA nor Pro-pNA was hydrolysed by the purified enzyme, the release of N-terminal acidic residues from peptides was observed in addition to the release of N-terminal proline from Pro-Leu-Gly-NH2, Pro-Leu-Gly-Gly and Pro-His-Pro-Phe-His-Leu-Phe-Val-Tyr. This ability of Lys-pNA hydrolase to release N-terminal proline residues was employed in concert with a purified aminopeptidase P preparation to release alternate N-terminal amino acids from Tyr-Pro-Phe-Pro-Gly. The complementary action of these enzymes represents an alternative mechanism to that of post-proline dipeptidyl aminopeptidase for metabolism of proline-containing peptides.
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PMID:Purification and characterization of a lysine-p-nitroanilide hydrolase, a broad specificity aminopeptidase, from the cytoplasm of Lactococcus lactis subsp. cremoris AM2. 1037 45

Lactobacillus casei CRL 705 was screened, among other meat isolates, for its proteinase and aminopeptidase activities toward synthetic substrates and, according to that, selected for specific assays on muscle proteins. The hydrolytic effects of whole cells, cell free extracts (CFE), and the combination of both on muscle sarcoplasmic and myofibrillar protein extracts was evaluated by SDS-PAGE and reverse phase HPLC analyses. The proteinase activity of whole cells caused the degradation of a great number of sarcoplasmic protein bands. A partial hydrolysis was also associated with CFE that when combined with whole cells showed an important additional degradation. Peptide profiles from sarcoplasmic protein extracts were greatly modified regardless of the addition of whole cells or CFE, although their combination intensified these changes. The generation of free amino acids was remarkable when whole cells and CFE were incorporated together to sarcoplasmic protein extracts.
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PMID:Hydrolytic action of Lactobacillus casei CRL 705 on pork muscle sarcoplasmic and myofibrillar proteins. 1055 69

The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ss-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ss-naphthylamide was eluted at 750 microS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10%) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/K M ratio for L-Leu-ss-naphthylamide. The enzyme activity was increased 40% by 0.15 M NaCl, inhibited 94% by 2.0 mM Zn2+, inhibited 91% by sodium p-hydroxymercuribenzoate and inhibited 45% by 0.7 mM o-phenanthroline and 30 microM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 microM), p-nitroaniline (0.25 mM) and ss-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 microM) inhibited 18% the enzyme activity. The aminopeptidase activity in the seeds decayed 50% after two months when stored at 4 degrees C and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.
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PMID:Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase. 1058 29

A leucine aminopeptidase gene of Aquifex aeolicus, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli, and its expression product was purified and characterized. The expressed protein was purified to homogeneity by using heat to denature contaminating proteins followed by ion-exchange chromatography to purify the heat-stable product. The purified enzyme gave a single band on SDS-PAGE with a molecular weight of 54 kDa. Kinetic studies on the purified enzyme confirmed that it was a leucine aminopeptidase. The optimum temperature for its activity was around 80 degrees C and the optimum pH was in the range from 8.0 to 8.5. It was stable at high temperatures and 27% of its activity was retained after heating at 115 degrees C for 30 min. The purified enzyme had a pH stability range between 4.0 and 11.0. This aminopeptidase was highly resistant to organic solvents such as methanol, ethanol, tetrahydrofuran, dimethyl sulfoxide, acetone, acetonitrile, dimethyl formamide, 1-propanol, 2-propanol, and dioxane.
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PMID:Characterization of a solvent resistant and thermostable aminopeptidase from the hyperthermophillic bacterium, Aquifex aeolicus. 1086 5

A cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human liver cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS-PAGE in the presence of beta-ME. These findings suggest that the enzyme exists as a monomeric form in human liver cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys-Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K(cat)/K(m) values of AAP-S at the optimal pH was Arg->Arg-Arg->Met->Leu->Lys->Phe->Lys-Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, DFP, PCMBS, Zn(2+), Cd(2+), Co(2+), Cu((2+)), Hg(2+) and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human liver cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of liver cells and renal tubules, and was ubiquitously localized in various human tissues.
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PMID:Puromycin-sensitive alanyl aminopeptidase from human liver cytosol: purification and characterization. 1097 16

An aminopeptidase was purified 178-fold from an extract of Grifola frondosa by ammonium sulfate precipitation and a series of column chromatographies on phenyl-Toyopearl, Sephadex G-25, and Mono-Q. The molecular mass of the enzyme was estimated to be 27 kDa and 30 kDa by gel filtration and SDS-PAGE, respectively. The enzyme had an optimum pH of 8.5 and was stable between pH 6.0 and pH 10.5, and it also had a high level of heat stability. The enzyme was inactivated by EDTA and o-phenanthroline, and it was also strongly inhibited by bestatin, but no inhibitory effect of DFP was observed. The enzyme preferentially hydrolyzed peptides containing hydrophobic residues in the N-terminal position.
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PMID:Purification and characterization of an aminopeptidase from the edible basidiomycete Grifola frondosa. 1130 80

Cell populations derived from viable Haemonchus contortus L(3) larvae were propagated in vitro in a tissue culture environment for a prolonged period (>48 months). Microscopic evaluation of H. contortus-derived cell populations revealed gross morphological characteristics highly analogous to those described for cell types originating from species of plant nematodes propagated in vitro in a tissue culture environment for a briefer period of time (<6 months). The characterisation of extracts harvested from tissue culture populations of H. contortus-derived cells by SDS-PAGE analysis detected molecular fractions of approximately 29, 45, 55, and 200-kDa that closely correlated with reports for preparations obtained from intact/viable H. contortus larvae. Complementary investigations detected the dual biochemical expression of phosphohydrolase and aminopeptidase-M activities based on the hydrolysis of the synthetic enzyme-specific substrates, para-nitrophenylphosphate and leucine-para-nitroanaline, respectively. The identification of phosphohydrolase and aminopeptidase-M-like biochemical activity in fractions harvested from H. contortus-derived cell populations and propagated in vitro in tissue culture served as evidence validating their parasitic-origin. Further validation of H. contortus-derived cell populations propagated in tissue culture entailed the formulation of Triton X-100 extracts containing potential immunoprotective antigens with SEAM adjuvant and its administration by intramuscular injection (100 microg total protein) to healthy sheep (n=8) on day 0 (left rear-limb) and day +14 (right rear-limb). Animals on day 28 subsequently received a single oral challenge of 10,000 infective L(3)-stage H. contortus larvae. Applying ELISA methodologies, increases in antigen-specific IgM and IgG were detected in ovine serum samples. Interpretation of experimental findings revealed that sheep with the greatest antigen-specific humoral immune responses (IgG titre 1/3125) also demonstrated a degree of reduced abomasal H. contortuslarvae burdens (60% reduction). Polyclonal antibody from immunoprotected sheep was subsequently found to recognise both the: (i), digestive tract; and (ii), antigen extracts associated with intact/viable H. contortus larvae. These experimental findings reveal the potential feasibility of propagating parasite-derived cell populations in an in vitro tissue culture environment in a manner that retains their ability to express immunoprotective antigenic fractions.
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PMID:Characterisation of Haemonchus contortus-derived cell populations propagated in vitro in a tissue culture environment and their potential to induce protective immunity in sheep. 1130 14

A proline-specific dipeptidyl aminopeptidase, dipeptidyl peptidase IV (EC 3.4.14.5), was purified from a cell sonicate soluble fraction of Prevotella loescheii ATCC 15930 by sequential column chromatography. The molecular mass of the native enzyme was estimated as 160 kDa by high-pressure liquid gel filtration column chromatography and unheated sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The subunit molecular mass was 80 kDa when the enzyme was heated to 100 degrees C in the presence of 2-mercaptoethanol before SDS-PAGE, suggesting that the native enzyme consists of two identical subunits and is folded in 2% SDS. The optimum pH, with glycyl-prolyl-4-methyl-coumaryl-7-amide as the substrate, was 8.0; the isoelectric point was 5.2. Purified enzyme showed a strong preference for dipeptide substrates containing proline and, less efficiently, alanine in the P1 position. The enzyme was markedly inhibited by Cd(2+), Zn(2+), Hg(2+), Co(2+), and serine proteinase inhibitor di-isopropylfluorophosphate.
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PMID:Isolation and characterisation of dipeptidyl peptidase IV from Prevotella loescheii ATCC 15930. 1138 67

Juvenile hormone esterase (JHE) from the serum of the cricket, Gryllus assimilis, was purified to homogeneity in a four-step procedure involving polyethylene glycol precipitation, hydrophobic interaction FPLC, and ion exchange FPLC. This procedure could be completed in 4 days and resulted in a greater than 900-fold purification with greater than 30% recovery. The purified enzyme exhibited a single band on a silver-stained SDS PAGE gel and had an apparent subunit molecular mass of 52 kDa. The native subunit molecular mass, determined by gel permeation FPLC, was 98 kDa, indicating that JHE from Gryllus assimilis is a dimer of two identical or similar subunits. The turnover number of the purified enzyme (1.41 s(-1)), K(M(JH-III)) (84 +/- 12 nM) of nearly-purified enzyme, and k(cat)/K(M) (1.67 x 10(7) s(-1) M(-1)) were similar to values reported for other well-established lepidopteran and dipteran JHEs. JHE from Gryllus assimilis was strongly inhibited by the JHE transition-state analogue OTFP (octylthio-1,1,1-trifluoro-2-propanone; I(50) = 10(-7) M) and by DFP (diisopropyl fluorophosphate; I(50) = 10(-7) M). The shapes of the inhibition profiles suggest the existence of multiple binding sites for these inhibitors or multiple JHEs that differ in inhibition. Isoelectric focusing separated the purified protein into 4 isoforms with pIs ranging from 4.7-4.9. N-terminal amino acid sequences (11-20 amino acids) of the isoforms differed from each other in 1-4 positions, suggesting that the isoforms are products of the same or similar genes. Homogeneously purified JHE hydrolyzed alpha-napthyl esters, did not exhibit any detectable acetylcholinesterase, acid phosphatase, or aminopeptidase activity, and exhibited only very weak alkaline phosphatase activity. JHE exhibited a low (11 microM) K(M) for long-chain alpha-naphthyl esters, indicating that JHE may have physiological roles other than the hydrolysis of JH-III. Purification of JHE represents a key step in our attempts to identify the molecular causes of genetically-based variation in JHE activity in G. assimilis. This represents the first homogeneous purification of JHE from a hemimetabolous insect.
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PMID:Purification and characterization of hemolymph juvenile hormone esterase from the cricket, Gryllus assimilis. 1175 93

An extracellular alkaline metalloprotease (MprI) from Alteromonas sp. strain O-7 was purified and characterized. The molecular mass of the purified enzyme was estimated to be 56 kDa by SDS-PAGE. The optimum pH and temperature were pH 10.0 and 60 degrees C, respectively. The gene (mprI) encoding MprI was cloned and its nucleotide sequence was analyzed. The deduced amino acid sequence of MprI showed significant similarity to metalloproteases classified into the thermolysin family. Furthermore, sequence analysis showed that another metalloprotease (MprII)-encoding gene was located downstream from mprI. The deduced amino acid sequence of MprII showed high similarity to metalloproteases of the aminopeptidase family. Similar repeated C-terminal extensions were found in both MprI and MprII.
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PMID:Isolation and characterization of the genes encoding two metalloproteases (MprI and MprII) from a marine bacterium, Alteromonas sp. strain O-7. 1199 19


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