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: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A set of isogenic mutants of Bacillus subtilis 168, insertionally inactivated in the genes encoding a number of lytic enzymes and a sigma factor (sigma D, which controls the expression of a number of autolysins) was constructed. Phenotypic analysis of the mutants determined the individual and combined roles of the autolysins in vegetative growth. The major vegetative autolysins of B. subtilis, LytC (50 kDa amidase) and LytD (90 kDa glucosaminidase), were shown to have roles in cell separation, cell wall turnover, antibiotic-induced lysis and motility. LytC was also shown to have a role in general cell lysis induced by sodium azide. Renaturing SDS-PAGE of cell-wall-binding protein extracts of the mutant strains revealed the presence of a novel autolysin that was previously masked by LytC. This 49 kDa enzyme was shown to be sigma D-controlled and was identified as a candidate cell separation and cell wall turnover enzyme. A multiple mutant strain, lacking LytC, LytD and the 49 kDa enzyme, retained at least ten bands of autolytic activity. These may correspond to individual or proteolytically processed novel autolysins, the functions of which are unknown. The multiple mutant strains facilitate the study of these, and other lytic enzymes, to determine their cellular functions.
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PMID:The role of autolysins during vegetative growth of Bacillus subtilis 168. 953 64

Susceptibilities of several preparations of Staphylococcus aureus cells to various peptidoglycan hydrolases with known bond specificity were analyzed by zymography. The substrates were intact S. aureus cells, cells boiled in the presence of SDS and cells treated with trichloroacetic acid after treatment with boiling SDS solution (TCA-cells). Twofold dilutions of lysostaphin (LS), lysozyme (LZ), S. aureus 51 kDa glucosaminidase (GL) or S. aureus 62 kDa amidase (AM) were electrophoresed, and the minimal enzyme dose showing a visible bacteriolytic band was defined as MBD (minimal bacteriolytic dose). Under the same experimental conditions, this method gave reproducible results. As the substrate for zymogram, TCA-cells were the most sensitive to LS, LZ and AM, whereas the three substrate were equally sensitive to GL. A zymographic analysis of methicillin-resistant S. aureus treated with methicillin together with previous studies suggest that this method can be used for the preliminary characterization of S. aureus cell wall peptidoglycan.
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PMID:Zymographic characterization of Staphylococcus aureus cell wall. 957 Feb 89

Previously, we purified and characterized a pro-phenol-oxidase (pro-PO) of 79 kDa from coleopteran insect, Holotrichia diomphalia larvae [Kwon et al. (1997) Mol. Cells 7, 90-97]. Here, we describe the identification of two pro-PO-activating factors (PPAF), named PPAF-I and PPAF-II, directly involved in the activation of the isolated pro-PO. When pro-PO was incubated with either PPAF-I or PPAF-II, no phenol oxidase activity was observed. However, incubation of pro-PO with both PPAF-I and PPAF-II specifically exhibited phenol oxidase activity. The purified PPAF-I with a molecular mass of 33 kDa on SDS/PAGE had characteristics of a serine protease. It exhibited amidase activity against fluorogenic peptide substrates, tert-butoxycarbonyl-phenylalanyl-seryl-arginyl-4-methylcoumaryl-7-amide being the best among the substrates examined. The activity was completely inhibited by 0.02 mM p-nitrophenyl-p'-guanidinobenzoate HCl and diisopropylflurophosphate. The NH2-terminal sequence of PPAF-I had significant sequence similarity to those of serine proteases. On the other hand, the purified PPAF-II had a molecular mass of 40 kDa on SDS/PAGE and 400 kDa determined by gel filtration, indicating an oligomeric protein. The NH2-terminal sequence of PPAF-II showed no similarity to known proteins. PPAF-II exhibited no amidase activity against the fluorogenic substrates. Reconstitution experiments and immunoblotting analysis using affinity-purified antibody against pro-PO demonstrated that PPAF-I first cleaves the intact pro-PO to an intermediate of 76 kDa with no phenol oxidase activity, and then, PPAF-I converts the intermediate to the active phenol oxidase of 60 kDa in the presence of PPAF-II. These results indicate that the activation of pro-PO system in hemolymph of H. diomphalia larvae is accomplished by at least two activating factors, a serine protease and a protein cofactor.
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PMID:In vitro activation of pro-phenol-oxidase by two kinds of pro-phenol-oxidase-activating factors isolated from hemolymph of coleopteran, Holotrichia diomphalia larvae. 965 93

A glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase was purified 58-fold from Pseudomonas nitroreducens in a two-step procedure involving osmotic shock and carboxymethyl-Sepharose chromatography with a yield of 26%. The molecular mass of the native enzyme was 58 kDa. SDS/PAGE revealed that it consisted of two non-identical subunits with molecular masses of 35 and 21 kDa. The isoelectric point of the purified enzyme was 5.3. The enzyme had an optimal pH of 5.5 and an optimal temperature of 43 degrees C. The purified enzyme exhibited not only GL-7-ACA acylase activity but also gamma-glutamyltranspeptidase activity. The Km values of the enzyme for GL-7-ACA and L-gamma-glutamyl p-nitroanilide were 10.41 mM and 5.92 microM respectively.
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PMID:An acidic glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas nitroreducens. 975 63

Peptide-N4-(N-acetyl-beta-d-glucosaminyl asparagine amidase) from Aspergillus tubigensis (PNGase At) was expressed in baculovirus-infected insect cells. The recombinant PNGase At was secreted and purified to homogeneity with a yield of 9.5 mg per liter of infected cell medium. Recombinant PNGase At migrated upon SDS-PAGE as a single-chain protein with a molecular mass of 78 kDa. This contrasts with the native Aspergillus enzyme which is "nicked" and migrates as two subunits each with a molecular weight about 43 kDa. Quantitation of total sugar by phenol-sulfuric acid suggests that the enzyme expressed in baculovirus-infected insect cells was substituted with 8-10 chains of carbohydrate of which 75% was released by Endoglycosidase F1. ESI-MS analysis of the oligosaccharides released from the recombinant PNGase At revealed similarity in the number of glycosylated residues but a significant difference in their composition, when compared to the carbohydrates of the native PNGase At. Despite differences in the primary structure and in the composition of glycan residues, the recombinant enzyme had the same specific activity as the native enzyme.
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PMID:Overexpression of PNGase at from baculovirus-infected insect cells. 979 Aug 95

Butyrylcholinesterase (BChE) was purified from monkey serum and the catalytic activities were examined. The enzyme has a molecular mass of approximately equal to 74 kDa as seen by SDS-gel electrophoresis. Monkey serum BChE also exhibits an amine sensitive aryl acylamidase (AAA) and a metallocarboxypeptidase activity. The tyramine activation of the aryl acylamidase activity and the metal chelator inhibition of the peptidase activity were characteristics similar to those of the human enzyme. Studies on 65Zn2+ binding and zinc chelate Sepharose chromatography showed that monkey serum BChE and human serum BChE have similar characteristics. Limited alpha chymotrypsin digestion of monkey serum BChE followed by Sephadex gel chromatography cleaved the enzyme into a 36 kDa fragment exhibiting peptidase activity. However the 20 kDa fragment corresponding to cholinesterase and aryl acylamidase activity was not detectable possibly due to the unstable nature of the fragment. Immunological studies showed that a polyclonal antibody against human serum BChE cross reacted with monkey serum BChE. The identical nature of the catalytic activities of human serum BChE and monkey serum BChE supports the postulate that all three catalytic activities co-exist in the same enzyme. This is the first time that purification and characterisation of the monkey serum BChE which has the highest sequence identity and immunological identity with that of human serum BChE, is being reported.
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PMID:Serum butyrylcholinesterase of non-human primate shows amine sensitive aryl acyl amidase and metallopeptidase activities and characteristics similar to those of the human serum enzyme. 980 63

A membrane-associated galactosyltransferase from Trypanosoma brucei was purified 34000-fold by affinity chromatography on UDP-hexanolamine-Sepharosetrade mark. Using SDS/PAGE under reducing conditions, the isolated enzyme ran as a relatively broad band with apparent molecular masses of 53 kDa and 52 kDa, indicative of glycosylation and the existence of two isoforms. N-Glycosylation of the enzyme was subsequently confirmed using Western blotting and either specific binding of concanavalin A or peptide-N4-(N-acetylglucosaminyl)asparagine amidase digestion. The de-N-glycosylated enzyme ran with apparent molecular masses of 51 kDa and 50 kDa, indicative of a single N-glycosylation site. The galactosyltransferase exhibited a pH optimum at 7.2 and had a pronounced requirement for Mn2+ ions (KM=2.5 mM) for its action. The transferase activity was independent of the concentration of Triton X-100. The enzyme was capable of transferring galactose from UDP-galactose to a variety of galactose-based acceptors in alpha-glycosidic linkages. The apparent KM values for UDP-galactose and for the preferred acceptor substrate N-acetyl-lactosamine are 46 microM and 4.5 mM respectively. From these results we would like to suggest that the galactosyltransferase functions in the processing of terminal N-acetyl-lactosamine structures of trypanosomal glycoproteins.
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PMID:Purification and characterization of an alpha-galactosyltransferase from Trypanosoma brucei. 1002 34

1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M(r) 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the Km for S-adenosyl-L-methionine of 1.74 mM and kcat of 0.56 s-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M(r) 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a Km for ACC of 4.8 mM and kcat of 3.52 s-1. The presence of 7 mM Cu2+ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.
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PMID:Synthesis and degradation of 1-aminocyclopropane-1-carboxylic acid by Penicillium citrinum. 1022 40

Administration of 100 and 200 microg/ml of cisplatin [cis -diammine dichloro platinum (II)] for 1 h to growing Dictyostelium discoideum cells severely affects folic acid chemotaxis and phagocytotic function in this organism. Following cisplatin treatment, cells show a much lower uptake of FITC labelled bacteria and a reduced plaque forming ability when plated on Eschericia coli seeded normal agar. Folic acid chemotaxis and folate deaminase activity are greatly inhibited in cisplatin-treated Dictyostelium cells. SDS-PAGE analysis shows a greater association of actin and myosin with the cell cortex of treated cells. These results have been discussed in relation to cisplatin's known ability to raise the levels of cytosolic calcium.
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PMID:Cisplatin inhibits folic acid chemotaxis and phagocytotic functions in Dictyostelium discoideum. 1056 44

We established an efficient overproduction-purification system for blasticidin S deaminase (BSD) using the cDNA cloned from Aspergillus terreus. The estimated molecular mass of the purified enzyme indicated BSD was a tetramer. This tetrameric form was very resistant to denaturation by SDS and showed heat-modifiable behavior on SDS-PAGE; i.e., BSD migrated much slower (as a single band of 36 kDa) in its active conformation than its completely denatured polypeptide (13 kDa) if heat treatment in 2% SDS was not performed before electrophoresis. As predicted from the presence of the catalytic zinc-coordinating sequence motif conserved in the cytosine nucleoside/nucleotide deaminase family, BSD also contained one zinc per deaminase subunit. However, the predicted catalytic function appeared not to be the only role of this zinc in the enzyme. First, titration of the zinc-chelating -SH groups with p-hydroxymercuriphenylsulfonate led to dissociation of the BSD tetramer into unstable monomers or dimers. Second, depletion of zinc on reconstitution of chemically denatured BSD (with either guanidine-HCl or acidic pH) resulted in improper folding of the polypeptide. These results suggest that zinc also plays a structural role in maintenance of the protein structure. When we introduced mutations at Glu-56 (the proposed active site) and Cys-91 (a proposed catalytic zinc-binding Cys) in BSD, none of the resulting mutants (E56D, E56Q, C91A, C91S, and C91H) showed any detectable activity, as judged with the spectrophotometric assay. Replacements of Cys-91 resulted in gross perturbation of the enzyme structure although the catalytically essential Glu-56 was not necessarily required for proper folding of the enzyme. These results further support our proposal that the catalytic zinc coordinated by the conserved sequence motif is also structural in BSD.
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PMID:Expression, purification, and characterization of blasticidin S deaminase (BSD) from Aspergillus terreus: the role of catalytic zinc in enzyme structure. 1083 62


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