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

Aminopeptidase yscXVI was purified from the yeast Saccharomyces cerevisiae. By SDS-PAGE the enzyme has a molecular weight of 45,000 Da, and in chromatofocusing, elution was observed at pH 6.2. The synthetic substrate cystinyl-4-nitroanilide (Km 22.5 microM, Vmax 12.9 mU/mg) is cleaved most efficiently in the pH range 7-8. Besides cleaving this standard substrate, aminopeptidase yscXVI acts on several other 4-nitroanilide substrates with unsubstituted N-terminal L-amino acids. Highest hydrolysis rate was measured with Lys-4-nitroanilide and Leu-4-nitroanilide. The activity of aminopeptidase yscXVI is abolished by chelating agents and restored by Zn2+, Mn2+ and Co2+ ions. Bestatin and amastatin are both strong inhibitors of the enzyme, with Ki values of 0.53 microM and 0.93 microM, respectively. Aminopeptidase yscXVI is detectable in the logarithmic growth phase, stationary phase, and in starved cultures of yeast.
 ...
PMID:Purification and characterization of the cystinyl bond cleaving yeast aminopeptidase yscXVI. 848 90

The production and properties of an aminopeptidase from Capnocytophaga gingivalis were studied. C. gingivalis was grown in continuous culture over a range of dilution rates and the cell-bound and extracellular levels of aminopeptidase and trypsin-like protease (TLPase) measured. At high growth rates (0.6 mu rel) TLPase specific activity was low and found exclusively as cell-bound activity; at low growth rates (0.0375 mu rel), specific activity was high and 26% was found as extracellular activity. In contrast, aminopeptidase specific activity was highest at 0.3 mu rel and the ratio of cell-bound to extracellular activity was relatively constant at all growth rates. Only about 5% of the total activity was extracellular. The aminopeptidase, which has a wide specificity towards artificial substrates, was purified to homogeneity, as judged by SDS-PAGE, from the supernatant fluid of cells grown in continuous culture in a tryptone/glucose/thiamine medium. The enzyme has a molecular mass of 61 kDa, a pl of 6.3, a pH optimum close to 7.5 and showed a requirement for magnesium or calcium ions. The N-terminal sequence of the first 10 amino acids (Asp-Val-Asn-Met-Leu-Trp-Tyr-Val-x-Arg...) showed no similarity to any published sequence. This enzyme in its cell-bound or extracellular form may be important in the nutrition and pathogenesis of C. gingivalis in the human oral cavity.
 ...
PMID:Capnocytophaga gingivalis aminopeptidase: a potential virulence factor. 857 2

A 1.4-kb gene encoding the "small" sialidase isoenzyme of Clostridium perfringens A99, including its own promoter, was previously cloned in and expressed by Escherichia coli JM 101. Since all attempts to purify this enzyme to homogeneity were unsuccessful, a new strategy was developed. The structural gene was amplified by means of a PCR technique and inserted into the plasmid vector pQE-10, transferring a six-histidine affinity tag (His6) to the N-terminus of the protein. In order to minimize proteolytic degradation of the sialidase protein, the gene was subcloned into the Escherichia coli strain BL21(DE3)pLys S with reduced protease activity. The sialidase production was increased about 2.5-fold when compared with that of the original clone. The enzyme, released by lysozyme treatment of the bacterial cells, was purified by metal chelate chromatography on Ni-nitrilo-triacetic acid agarose to apparent homogeneity in SDS-PAGE. The 42-kDa protein was enriched 62-fold with a yield of 82% and a specific activity of 280 U mg-1. A total amount of 1 mg sialidase was obtained from 1 liter of bacterial culture. For future studies, including crystallization experiments, the histidine affinity tag was removed from the sialidase enzyme by aminopeptidase K. The sialidase was then separated from aminopeptidase K by ion-exchange chromatography, resulting in an overall yield of 83% and a specific activity of 305 U mg-1 using 4-methylumbelliferyl- alpha-D-N-acetylneuraminic acid under standard conditions. The two forms (with or without the histidine tag) of sialidase exhibited similar kinetic properties when compared to the wild-type enzyme.
 ...
PMID:Expression and purification of a recombinant "small" sialidase from Clostridium perfringens A99. 877 61

Pyroglutamyl aminopeptidase type-1 (PAP-I) is reported to be a soluble, broad specificity aminopeptidase, capable of removing the pyroglutamic acid (pGlu) residue from the amino terminus of pGlu-peptides (e.g. TRH, LHRH, neurotensin and bombesin). The central aim of this study was to undertake, for the first time, the complete purification and characterisation of a PAP activity observed within the cytosolic fraction of bovine whole brain and to compare the properties of the enzyme with previous findings. A series of chromatographic steps (DEAE-Sepharose, Sephacryl S-200 and Activated Thiol Sepharose 4B) generated a soluble PAP activity purified to near homogeneity with a total active yield of 6.6% The enzyme displayed a native molecular mass of approximately 23,700 Da, which compares well with that value obtained under denaturing conditions via SDS-PAGE (24,000 Da), suggesting that the enzyme exists as a monomer. The expression of PAP activity displayed an absolute requirement for the presence of a disulphide bond-reducing agent such as DTT, whilst optimum activity was observed at pH 8.5. strong inhibition of PAP activity was observed with a number of different agents, including transition metal ions, sulphydryl-blocking agents and 2-pyrrolidone (a pGlu analog). A broad pyroglutamyl substrate specificity, which excludes substrates commencing with the pGlu-Pro bond, was also demonstrated for the bovine brain enzyme. Based on a comparison of these findings with those reported for PAP-I in other mammalian tissues, the soluble PAP activity observed in bovine whole brain can tentatively be classified as a pyroglutamyl aminopeptidase type-1 (EC 3.4.19.3).
 ...
PMID:Bovine brain pyroglutamyl aminopeptidase (type-1): purification and characterisation of a neuropeptide-inactivating peptidase. 881 36

Bleomycin (BLM) hydrolase, which hydrolyzes the carboxyamide bond in the beta-amino-alanine moiety, was purified from newborn rat skin. The enzyme was purified 2,500-fold over the crude extract to apparent homogeneity in five steps in the presence of 2-mercaptoethanol: 45-55% ammonium sulfate fractionation, followed by chromatographies on Sephacryl S-200, DEAE-cellulofine, Phe-Superose, and Mono Q ion-exchange. The native enzyme had a molecular mass of 280 kDa according to gel filtration. The subunit molecular mass was estimated as 48 kDa by SDS-PAGE, indicating that the enzyme was comprised of six identical subunits. The amino acid sequence of its NH2-terminus was determined to be acetyl-Met-Asn-Asn-Ala-Gly-Leu-Asn-Ser-Glu-Lys-, which was not found in the amino acid sequence database. The optimum pH of the enzyme was 7.5 with pepleomycin (PLM). The Km and Vmax values were 2.1 mM and 6.8 mu mol center dot mg-1 center dot h-1 for PLM, and 1.8 mM and 7.2 mu mol center dot mg-1 center dot h-1 for BLM-A2, respectively. The enzyme activity was inhibited by iodoacetic acid, N-ethylmaleinimide (NEM), and p-chloromercuribenzoic acid (pCMB) as well as divalent cations such as Cu2+, Cd2+, Hg2+, and Zn2+. It was effectively inhibited by a cysteine protease inhibitor E-64. However, cystatins A and C did not inhibit the activity. BLM hydrolase exhibited broad aminopeptidase substrate specificity towards aminoacyl-beta-naphthylamides such as basic, neutral, and hydrophobic amino acid residues, as well as acidic residues. These results indicated that BLM hydrolase represents a new family of cysteine proteases. Western blotting and immunohistochemical analyses showed that BLM hydrolase is ubiquitous in various rat tissues but at low levels in lung and adult skin tissues, suggesting that this enzyme plays an important role in the metabolism of antibiotics.
 ...
PMID:Purification and characterization of bleomycin hydrolase, which represents a new family of cysteine proteases, from rat skin. 890 72

An aminopeptidase with a very broad substrate specificity was purified to homogeneity from Lactobacillus helveticus SBT 2171 by FPLC. The enzyme was purified 144-fold from a cell-free extract with a yield of 16%. The purified enzyme appeared as a single band on an SDS-PAGE gel. It had a molecular mass of 95 kDa and an isoelectric point of 4.9. The enzyme hydrolysed a large range of naphthylamide- and nitroanilide-substituted amino acids, as well as several di-, tri- and oligopeptides. It also exhibited significant proline-iminopeptidase-like activity, since it hydrolysed several proline-containing peptides. Prolyl-p-nitroanilide was hydrolysed with a low affinity (Michaelis-Menten constant 0.6 mM) and a Vmax of 2.5 mumol min-1 (mg protein)-1 while lysyl-p-nitroanilide was hydrolysed with a high affinity [Km 0.003 mM; Vmax 37.5 mumol min-1 (mg protein)-1]. The aminopeptidase activity, which was optimal between pH 6.0 and 8.0 and at 50 degrees C, was very stable at 30 degrees C for more than 7 d. The activity lost by treatment with the thiol-blocking reagents could be restored with beta-mercaptoethanol, while Co2+ and Mn2+ restored the activity of the EDTA-treated enzyme. Immunological experiments with antibodies raised against the aminopeptidases from Lactococcus lactis and Lb. helveticus clearly showed that both aminopeptidases are at least immunologically different from each other.
 ...
PMID:A new, broad-substrate-specificity aminopeptidase from the dairy organism Lactobacillus helveticus SBT 2171. 893 7

A relatively simple and rapid method is described for the isolation of basal cell membranes (BCM) from the human placenta at term which showed considerable improvement in the yield, purity and membrane characteristics as compared to the earlier described methods. The method is based on thorough washings of the syncytium in balanced salt solution, selective grinding, hypotonic lysis, sonication, incubation with EDTA and then more conventional differential centrifugation and ultracentrifugation. The isolated material showed smooth surfaced vesicular structure of various sizes as revealed by both positive and negative staining and transmission electron microscopic analysis. The membrane was highly enriched in Na+/K+, Ca2+ and Mg2+ dependent ATPase activities while the cross contamination with brush border surfaces was low as revealed by the marker enzyme assays specific for the brush border membrane (BBM) such as the disaccharide hydrolases, aminopeptidase and alkaline phosphatase. The membrane showed a relatively low lipid/protein ratio and the lipid composition represented by a variety of phospholipids (phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl inositol and phosphatidyl serine), neutral lipids (cholesterol, triacyl glycerol, free fatty acids) and glycosphingolipids (ganglioside, cerebroside and sulfatide). It also contained plasmalogens. On SDS-PAGE analysis and Coomassie blue staining reaction, the isolated membrane showed 14 major bands with as many minor ones with a molecular weight ranging between 30-110 kDa.
 ...
PMID:Isolation and characterization of the basal cell membranes of the human term placenta. 893 20

A new type of major aminopeptidase was purified from bovine brain by ammonium sulfate fractionation and TMAE-fractogel (anion exchange), arginine-Sepharose 4B, Sephadex G-150, and Sephadex G-100 column chromatography. The purified enzyme showed a maximum activity at pH 7.2, and its molecular size was estimated to be 98,000 by gel filtration and 104,000 by SDS-PAGE with or without 2-mercaptoethanol. Further properties were activation by thiol reagents; inhibition by EDTA, puromycin, bestatin, amastatin, actinonin, leuhistin and probestin; and very low concentrations of Cu2+, Cd2+, Pb2+, Al3+, Fe3+, and Zn2+ inhibited activity. The enzyme hydrolyzed several amino acyl-7-amido-4-methylcoumalin derivatives (amino acid-MCA). The order of MCA-substrate specificity expressed as kcat/Km is Lys-MCA > Arg-MCA > Leu-MCA > Met-MCA > Phe-MCA > Tyr-MCA > Ala-MCA >> Gly-MCA, Pro-MCA, Ser-MCA, Asn-MCA. Immunoreactivity of the antibody against the purified aminopeptidase was observed in human brain and most rat tissues examined including brain, liver, kidney, lung, heart, and skeletal muscle at the same molecular size as in bovine brain aminopeptidase. Most of the Lys-, Leu-, Met-, and Phe-MCA degrading activity in crude bovine and human brain extracts was absorbed by the aminopeptidase IgG, suggesting that this aminopeptidase is a major enzyme, sharing at least Lys-, Leu-, Met-, and Phe-MCA degrading aminopeptidase activities in the brains.
 ...
PMID:A new type of major aminopeptidase in bovine brain. 898 39

Aminopeptidase A (glutamyl aminopeptidase; EC 3.4.11.7) has been cloned from porcine brain and kidney cortex cDNA libraries and the complete primary sequence of the enzyme deduced. This predicts a type II integral membrane protein of 942 amino acids with 14 potential N-linked glycosylation sites and a His-Glu-Xaa-Xaa-His zinc binding motif. Aminopeptidase A was purified from porcine kidney cortex by a combination of anion exchange and hydrophobic interaction chromatographies following its release from the membrane by trypsin. The purified protein migrated as three major polypeptides on SDS-polyacrylamide gel electrophoresis of M(r) 147,000, 107,000, and 45,000. N-Terminal sequencing revealed that both the Mr 147,000 and 107,000 polypeptides had the same N-terminal sequence resulting from cleavage of aminopeptidase A by trypsin at the Lys-42-Asp-43 bond just outside the membrane-spanning hydrophobic region. Immunoelectrophoretic blot analysis following electrophoresis under nonreducing conditions revealed that the trypsin-cleaved form of the enzyme no longer migrated as a disulfide-linked dimer, placing the interchain disulfide link N-terminal to Lys-42. N-Terminal sequencing of the M(r) 45,000 polypeptide in the purified preparation of aminopeptidase A revealed that it resulted from cleavage at the Asn-602-Gly-603 bond by an endogenous protease. This posttranslational proteolytic cleavage occurred in porcine kidney cortex microvillar membranes but not in porcine intestinal microvillar membranes. Incubation of purified porcine kidney aminopeptidase N (membrane alanyl aminopeptidase; EC 3.4.11.2) with trypsin resulted in a similar fragmentation pattern to that observed in aminopeptidase A, suggesting that these and other members of the type II membrane-spanning zinc aminopeptidase family may have two distinct domains: an N-terminal domain, containing the zinc binding site and residues identified as being involved in catalysis, and a C-terminal domain of unknown function, that are separated by a protease-susceptible region.
 ...
PMID:Proteolytic fragmentation reveals the oligomeric and domain structure of porcine aminopeptidase A. 906 31

Chicken aminopeptidase H is a cysteine protease possessing endopeptidase as well as aminopeptidase activity [Rhyu, M. R., Nishimura, T., Kato, Y., Okitani, A. & Kato, H. (1992) Eur. J. Biochem. 208, 53-59]. This enzyme exhibits molecular masses of 400 kDa on gel filtration and 52 kDa on SDS/PAGE, indicating that it consists of eight subunits with the same molecular mass. In the current study, we cloned the cDNA for the catalytic subunit of chicken aminopeptidase H. The open reading frame of the aminopeptidase H gene consists of 1362 base pairs encoding a 52-kDa protein consistent with the molecular mass determined on SDS/PAGE; the deduced amino acid sequence contains all the partial sequences determined for the purified enzyme. The sequence is similar to that of the bleomycin hydrolase of rabbit lung, which has been partially determined. The recombinant 52-kDa protein expressed in COS7 cells exhibited both aminopeptidase and endopeptidase activities, which were inhibited by monoiodoacetic acid. Furthermore, the expression of aminopeptidase H in COS7 cells was also recognized on immunoblotting. This gene is the first one for aminopeptidase H in an animal tissue whose sequence has been completely determined.
 ...
PMID:cDNA cloning and expression of chicken aminopeptidase H, possessing endopeptidase as well as aminopeptidase activity. 915 54


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>