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 system of intracellular autolytic enzymes of the bacterium Xanthomonas campestris IBPM B-124 was found to include enzymes with muramidase and glucosaminidase activities, while a system of extracellular bacteriolytic enzymes of the same bacterium includes muramidase, muramoylalanine amidase, and endopeptidase. Using a purification technique including fractional precipitation with ammonium sulfate, gel-filtration on Toyopearl HW-55F, and FPLC ion-exchange chromatography on Mono Q, a preparation of intracellular glucosaminidase was purified 435-fold with 16% yield (SDS-PAGE data indicated the presence of minor protein contaminants). Some physicochemical properties of the purified enzyme were determined: molecular mass 26 kD, Km = 5.6 x 10(-4) M with p-nitrophenyl-2-acetamido-2-deoxy-beta-D-glucopyranoside as the substrate, and pH optimum 8.0-8.5. The enzyme is active over a wide range of Tris-HCl buffer concentrations (0.01-0.5 M) and has temperature optimum at 37-40 degrees C. The glucosaminidase activity is sensitive to p-chloromercuribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), and the disodium salt of ethylenediamine tetraacetic acid (EDTA). The properties of this glucosaminidase markedly differ from those of all extracellular bacteriolytic enzymes of Xanthomonas campestris. These findings indicate that the system of autolytic enzymes of this bacterium functions independently and is not connected with the system of extracellular bacteriolytic enzymes.
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PMID:Intracellular glucosaminidase of the bacterium Xanthomonas campestris IBPM B-124: purification and properties. 1104 95

A mutant strain, KLAM59, of Pseudomonas aeruginosa has been isolated that synthesizes a catalytically inactive amidase. The mutation in the amidase gene has been identified (Glu59Val) by direct sequencing of PCR-amplified mutant gene and confirmed by sequencing the cloned PCR-amplified gene. The wild-type and altered amidase genes were cloned into an expression vector and both enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide followed by gel filtration chromatography. The mutant enzyme was catalytically inactive, and it was detected in column fractions by monoclonal antibodies previously raised against the wild-type enzyme using an ELISA sandwich method. The recombinant wild-type and mutant enzymes were purified with a final recovery of enzyme in the range of 70-80%. The wild-type and mutant enzymes behaved differently on the affinity column as shown by their elution profiles. The molecular weights of the purified wild-type and mutant amidases were found to be 210,000 and 78,000 Dalton, respectively, by gel filtration chromatography. On the other hand, the mutant enzyme ran as a single protein band on SDS-PAGE and native PAGE with a M(r) of 38,000 and 78,000 Dalton, respectively. These data suggest that the substitution Glu59Val was responsible for the dimeric structure of the mutant enzyme as opposed to the hexameric form of the wild-type enzyme. Therefore, the Glu59 seems to be a critical residue in the maintenance of the native quaternary structure of amidase.
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PMID:Substitution of Glu-59 by Val in amidase from Pseudomonas aeruginosa results in a catalytically inactive enzyme. 1109 65

An anionic trypsin from pyloric caeca of chum salmon (Oncorhynchus keta) was purified by ammonium sulfate and acetone fractionation followed by affinity chromatography, gel-filtration, and DEAE-anion exchange chromatography. The apparent molecular mass was about 24 kDa as determined by SDS-PAGE. The anionic chum salmon trypsin was moderately active toward esterase substrates such as tosyl-L-arginine methyl ester and tosyl-L-lysine methyl ester. Its amidase activity for benzoyl-L-arginine p-nitroanilide was comparative to those of bovine and Streptomyces griseus trypsins. Kinetic characteristics of anionic chum salmon, bovine, and Streptomyces griseus trypsins toward inverse substrate (p-amidinophenyl ester) were compared. Inverse substrate behaved as a specific substrate for anionic chum salmon trypsin with specific binding, efficient acylation, and relatively slow deacylation.
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PMID:Anionic trypsin from chum salmon: activity with p-amidinophenyl ester and comparison with bovine and Streptomyces griseus trypsins. 1112 64

alpha-Amidase catalyzes the C-terminal amidation of active polypeptides in the Nerve-Endocrine system. It is important for full biological activity of the polypeptides. By using the total RNA of rat atrium as a templet, the cDNA encoding alpha-amidase was amplified by RT-PCR and sequenced. The cDNA was fused with the coding sequence of mel C1 signal peptide to produce fusion gene mel/AE for secretive expression in Streptomyces lividans. The fusion gene mel/AE was inserted into plasmid pIJ680 and the recombinant plasmid pIJ-mel/AE680 was obtained. The results of SDS-PAGE and biological activity showed that the recombinant strain S. lividans TK54[pIJ-mel/AE 680] produced secretly alpha-amidase.
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PMID:[Studies of cloning and expression of rat alpha-amidase gene in Streptomyces lividans]. 1119 60

Sequencing of the Moraxella sp. CK-1 autolysin (cell wall hydrolases) gene showed the presence of an open reading frame which encodes a polypeptide of 273 amino acids with a molecular mass of 33,316 Da. A presumed ribosomal binding site, a possible -10 and -35 region, and rho-dependent terminators were found. The C-terminal region of the mature protein showed considerable homology with the Thermus sp. serine proteinase. Enzyme assay suggests that the recombinant autolysin has amidase or endopeptidase activity. Analysis of the peptidoglycan fragments, following the treatment with the autolysin, indicates that this protein is an N-acetylmuramyl-L-alanine amidase. Insertional inactivation of the autolysin of Moraxella sp. CK-1 chromosome led to a decrease in cell wall hydrolytic activity, clumping of the cells, and color change. No lytic band present in inactivated magA mutant by renaturing SDS-PAGE.
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PMID:Sequence analysis and insertional inactivation of a gene encoding Moraxella sp. CK-1 cell wall hydrolase. 1152 2

Although the four polypeptides of blasticidin S (BS) deaminase (BSD) are packed rather tightly coordinated to the "structural and catalytic" zinc atom of each subunit, the C-terminal region of the enzyme was suggested to be somewhat molten and flexible [M. Kimura, S. Sekido, Y. Isogai, and I. Yamaguchi (2000) J. Biochem. 127, 955-963]. To understand roles of this flexible region, we constructed five C-terminal deletion variants of BSD (each successively deleted from the C-terminal end up to five residues) and analyzed their biochemical properties focusing on the structure and activity of the enzyme. BSD and all of the deletion mutants showed the unique rigid conformation (e.g., characterized by their stabilities in SDS solution) and high levels of resistance against protease digestions. Furthermore, both the wild-type and deletion apoenzymes exhibited similar physical properties in thermodynamic refolding into the stable tetramer conformation. However, these small C-terminal deletions exerted deleterious effects on the catalytic efficiency of the enzyme as indicated by their strongly reduced k(cat)/K(m) value. Judging from the altered kinetic parameters and unaltered structural properties of the deletion variants, these C-terminal residues appear to be directly involved in enzyme-substrate interaction. In this short flexible region, Tyr-126, Trp-128, and Gly-130 were the key residues. Most notably, removal of Gly-130 markedly increased K(m) for BS without affecting its k(cat) value. These results indicate that the flexible C-terminal region is important for catalytic function and that a single Gly residue at the C-terminal end of BSD contributes significantly in facilitating access of a substrate to the active site.
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PMID:The flexible C-terminal region of Aspergillus terreus blasticidin S deaminase: identification of its functional roles with deletion enzymes. 1177 86

We developed a protocol for efficient expression of the functional serine protease, subtilisin E, in Escherichia coli periplasm that permits direct in vivo measurement of the enzyme's catalytic activity. Activity assays and SDS-PAGE/Western blot analysis showed that the levels of expressed subtilisin varied and were correlated with both the culture conditions and the induction procedures. The highest level of subtilisin expression was achieved at 0.10-0.15% (w/v) of arabinose as inducer and a temperature of 20-22 degrees C, and was ca. eightfold higher as compared to the expression level at 30 degrees C. Cultivation of bacterial cells to a steady state of balanced growth before induction was required for uniform subtilisin expression in cell cultures growing in wells of microtiter plates. Amidase and esterase cell-based kinetic assays on microtiter plates were developed based on the direct measurement of subtilisin activity in vivo. Intact E. coli cells displaying wild-type, dimethylformamide-resistant, and temperature-resistant subtilisins were assayed on N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-acetyl-Phe-p-nitrophenyl ester for their amidase and esterase activity, respectively. Additionally, the periplasmic fractions were isolated from the three E. coli strains expressing the respective subtilisins and tested for amidase activity. The amidase activity of the three subtilisins was ca. 15-fold higher than the esterolytic activity when measured in both the intact cells and in the periplasmic fractions. The strategy combining periplasmic expression of subtilisins with two cell-based kinetic assays permits rapid screening of subtilisin mutant libraries for desired activities.
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PMID:A strategy for in vivo screening of subtilisin E reaction specificity in E. coli periplasm. 1200 Nov 68

Human mature sperm cells have a high nuclease and 5-methyldeoxycytidine monophosphate (5-mdCMP) deaminase activity. The deaminase converts the nuclease degradation product 5-mdCMP into dTMP which is further cleaved into thymine and the abasic sugar-phosphate. Both 5-methylcytidine 5' and 3' monophosphates are good substrates for the deaminase. 5-methylcytidine is not a good deaminase substrate and 5-methylcytosine (5mC) is not a substrate. A purified fraction of the deaminase free of nucleases deaminates 5mC present in intact methylated double-stranded DNA. 5-mdCMP deaminase co-purifies on SDS-PAGE with dCMP deaminase and has an apparent molecular weight of 25 kDa. The enzyme requires no divalent cations and has a Km of 1.4 x 10(-7) M for 5-mdCMP and a Vmax of 7 x 10(-11) mol/h/microg protein. The possible biological implications of the deaminase's activities in the present system are discussed.
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PMID:5-Methyldeoxycytidine monophosphate deaminase and 5-methylcytidyl-DNA deaminase activities are present in human mature sperm cells. 1202 31

Turkey seminal plasma contains a serine protease found to be distinct from the spermatozoal acrosin. However, the origin and biological roles of this enzyme are unknown. Our experimental objective was to identify the cellular source of this protease within the male reproductive tract. The enzyme was isolated from seminal plasma using benzamidine-Sepharose 6B chromatography. A synthetic substrate, Nalpha-benzoyl-DL-arginine p-nitroanilide, was used to detect fractions containing the enzyme. The affinity chromatography technique yielded a 150-fold increase in amidase activity. Analysis of the protease by SDS-PAGE revealed two protein bands with relative molecular masses of 37 000 and 61 000. Proteolytic activity was detected within the smaller band as evidenced by casein digestion. Further analysis of the purified protein revealed that the smaller protein band was glycosylated. To determine the cellular source of the protease, a panel of mouse monoclonal antibodies was then developed against the purified protease, and used in immunohistochemistry. Frozen tissue sections from the liver, testis, epididymal region, and deferent duct were fixed in 4% (w/v) paraformaldehyde, permeabilized with 0.2% (v/v) (octylphenoxy)polyethoxyethanol followed by routine immunohistochemistry procedures. Monoclonal antibodies did not bind to tissue sections from either the liver or testis, or to blood plasma proteins. Both the distal portion of the efferent duct and the deferent duct were immunoreactive. We concluded that the protease found in turkey seminal plasma is concentrated to the distal efferent duct and the deferent duct epithelial cells.
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PMID:Localization of a proteolytic enzyme within the efferent and deferent duct epithelial cells of the turkey (Meleagris gallopavo) using immunohistochemistry. 1208 28

AMP-deaminase (EC 3.5.4.6) is a key enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in the liver. Mechanisms regulating activity of the enzyme are not completely elucidated, till now. In this paper experimental data indicating on the potential regulatory significance of changes in oligomeric structure of the enzyme are presented. SDS-PAG electrophoresis of human liver AMP-deaminase revealed the presence of three enzyme fragments. Only largest of them (the protein fragments weighing 68 kDa) reacted immunologically with anti- (human liver) AMP-deaminase antibodies. At physiological pH 7.0, in the absence of regulatory ligands, reaction catalysed by human liver AMP-deaminase was strongly dependent on enzyme concentration used, with half-saturation constant (S0.5) values increasing significantly with the degree of enzyme dilution. Preincubation with activated long-chain fatty acids--substances promoting dissociation of oligomeric enzymes, inhibited the activity of AMP-deaminase studied nearly completely. Gel filtration on Sepharose CL-6B column demonstrated existence of at least three active oligomeric forms of human liver AMP-deaminase. We postulate that oligomeric structure of the enzyme is a factor determining regulatory profile of AMP-deaminase studied.
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PMID:Human liver AMP-deaminase--oligomeric forms of the enzyme. 1248 28


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