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)

Protease and basic amidase activity was found in the seminal plasma of the domestic turkey. Amidase activity, measured through use of N-alpha-benzoyl-DL-arginine-p-nitroanilide-HCL (BAPNA), was 23-28 times greater for turkey than for guinea fowl or chicken. Within the reproductive tract, seminal plasma from the vas deferens had much greater activity than testicular or epididymal fluids. Turkey seminal plasma enzyme (TSPE) purified by chromatography or isoelectric focusing showed three protein bands by PAGE, each resolving on SDS-PAGE into two subunits with molecular weights of approximately 28,000-32,000 and 38,000-44,000. One of the three proteins also contained a larger subunit (M(r) 76,000-81,000) thought to be transferrin. Turkey acrosin consisted of three subunits with molecular weights below 20,500. Acrosin, but not TSPE, was visualized in native gels with N-alpha-benzoyl-DL-arginine-beta-naphthylamide (BANA)/Fast Garnet stain. Michaelis constants (BAPNA) for TSPE, acrosin, and trypsin were 2.41 +/- 0.12 x 10(-4) M (n = 5), 4.96-6.03 x 10(-4) M (n = 2), and 6.76 +/- 0.95 x 10(-4) M (n = 6), respectively. TSPE, like acrosin and trypsin, was inhibited by benzamidine but not iodoacetamide. While all natural trypsin inhibitors tested inhibited acrosin, TSPE was not inhibited by ovomucoid from chicken or turkey egg white.
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PMID:Proteolytic enzymes in seminal plasma of domestic turkey (Meleagris gallopavo). 767 33

Candida albicans and other pathogenic Candida species can use N-acetylglucosamine as a sole carbon source for growth. GlcNAc induces the enzymes of GlcNAc catabolic pathway; besides, under certain conditions, GlcNAc also induces a change from the yeast to germ tube morphology. Glucosamine-6-phosphate deaminase (EC 5.3.1.10) is the terminal enzyme of the GlcNAc catabolic pathway. We have purified the deaminase from C. albicans and studied its characteristics. The size of the deaminase estimated from SDS-polyacrylamide gel electrophoresis is 28 kDa. N-Acetylglucosamine 6-phosphate, an allosteric activator of the Escherichia coli deaminase, has no effect on the activity of the C. albicans enzyme. The deaminase is induced over 100-fold by GlcNAc and its level is about 0.3-0.5% of the proteins in crude extract. Three cDNA clones were obtained from a lambda gt11 expression library by immunoscreening with deaminase antiserum. C. albicans genomic DNA blot hybridization revealed that the NAG1 gene, encoding the glucosamine-6-phosphate deaminase, is present in a single copy. Hybrid-selected translation and immunoprecipitation experiments revealed that the purified deaminase and the protein encoded by the clones were similar in size and in their antigenicity. DNA sequencing revealed that the largest cDNA clone contained the complete open reading frame, which can code for a 27.5-kDa protein. The NH2-terminal sequence (35 residues) determined from the purified deaminase was identical to the sequence of the deduced protein. The Nag1 protein has about 47% identity with the sequence of the E. coli glucosamine-6-phosphate deaminase. Furthermore, RNA blot hybridization showed that GlcNAc induces the expression of NAG1 gene.
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PMID:Molecular cloning and analysis of the NAG1 cDNA coding for glucosamine-6-phosphate deaminase from Candida albicans. 768 45

Among 29 strains of zygomycetes screened for serine carboxypeptidases, Absidia zychae NRIC 1199 showed the highest enzyme production. Two serine carboxypeptidases, CPZ-1 and CPZ-2, were purified to a homogeneous state from an extract of koji culture of A. zychae NRIC 1199. Purified CPZ-1 and CPZ-2 showed similar properties except the isoelectric point (pI); The pI of CPZ-1 and CPZ-2 were 4.50 and 4.65, respectively. The molecular weights of the CPZ-1 and CPZ-2 were 48,000 by SDS-PAGE and gel filtration. Among the proteinase inhibitors tested, phenylmethylsulfonyl fluoride and monoiodoacetic acid strongly inhibited the enzyme activity. The optimum pHs of CPZ-1 and CPZ-2 were 4.2 towards Z-Glu-Tyr. It is shown that the substrate specificities of CPZ-1 and CPZ-2 were dependent on the presence of bulky amino acid residues in the penultimate position (P1) for the small Z-peptides. However, in spite of the presence of Gly, Asp, Arg, or Pro in the P1 position, oligopeptides were hydrolyzed rapidly. CPZ-1 and CPZ-2 had not only carboxypeptidase but also carboxyamidase and amidase activities, and acted preferentially as a carboxyamidase for C-terminal amidated peptides. The hydrophobicity of P2 and P3 positions and the bulkiness of P1 and P'1 positions of the substrate may be important for carboxyamidase and amidase activities.
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PMID:Purification and characterization of serine carboxypeptidases from Absidia zychae. 776 58

A novel carboxypeptidase (CPD-S3) from Penicillium janthinellum IBT 3991 has been isolated in a two-step purification procedure by cation exchange and affinity chromatography. The enzyme is a serine carboxypeptidase with a denatured molecular mass determined by SDS of 62 kDa of which 32% is carbohydrate. The isoelectric point is 5.1. CPD-S3 exhibits a high stability towards organic solvents and elevated temperatures. Besides the carboxypeptidase activity, CPD-S3 exhibits esterase, amidase, and carboxamidohydrolase activities. CPD-S3 favors substrates of L-configuration with basic amino acid residues in either P1 or P1', and particularly dibasic substrates and medium-sized straight-chain alkyl esters for hydrolysis. In aminolysis of esters, amino acid amides and hydrazines coupled in good yield, but methyl esters poorly, and unlike other carboxypeptidases, free amino acids could not be coupled or transpeptidation effected to form amides. In ester semisynthesis, peptides with neutral, but not basic, residues in P1 could be esterified. The scope of applicability for enzymatic peptide synthesis is limited.
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PMID:Novel serine penicillocarboxypeptidase CPD-S3 from Penicillium janthinellum IBT 3991: purification, characterization, and uses in peptide synthesis and modification. 776 95

The peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) earlier described [Lhernould S., Karamanos Y., Bourgerie S., Strecker G., Julien R., Morvan H. (1992) Glycoconjugate J 9:191-97] was partially purified from cultured Silene alba cells using affinity chromatography. The enzyme is active between pH 3.0 and 6.5, and is stable in the presence of moderate concentrations of several other protein unfolding chemicals, but is readily inactivated by SDS. Although the enzyme cleaves the carbohydrate from a variety of animal and plant glycopeptides, it does not hydrolyse the carbohydrate from most of the corresponding unfolded glycoproteins in otherwise comparable conditions. The substrate specificity of this plant PNGase supports the hypothesis that this enzyme could be at the origin of the production of 'unconjugated N-glycans' in a suspension medium of cultured Silene alba cells.
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PMID:Characterization of the peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) from Silene alba cells. 779 18

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38,994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-L-alanine amidase, which has a M(r) value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.
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PMID:Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwlL, of Bacillus licheniformis. 790 27

The CWLA lytic amidase of Bacillus subtilis 168 was purified and antisera raised against the purified protein. No expression of cwlA could be demonstrated under any conditions by the use of the antisera and cwlA::lacZ fusion analysis. Two lytic enzymes of apparent molecular masses 34 and 30 kDa (as measured by renaturing SDS-PAGE) were found to be mitomycin C-inducible, the larger of which corresponds to a protein immunologically related to CWLA. Both of these inducible lysins were found to be encoded by prophage PBSX. Prophage SP beta was shown by renaturing SDS-PAGE to produce a 43 kDa lytic enzyme unrelated immunologically to CWLA. The smaller of the two PBSX enzymes was purified and found to be an N-acetylmuramyl-L-alanine amidase of 32 kDa (as measured by SDS-PAGE and Coomassie blue staining) which cross-reacts only weakly with the anti-CWLA sera. The potential origin of cwlA and its possible relationship to the other phage lytic enzymes are discussed.
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PMID:Analysis of Bacillus subtilis 168 prophage-associated lytic enzymes; identification and characterization of CWLA-related prophage proteins. 790 56

A mouse monoclonal antibody against boar acrosin and antiserum prepared to highly purified acrosin in female rabbits were used to detect the antigen in various fluids and tissues of boars using an indirect immunofluorescence technique. A strong reaction was found in fluid and epithelial tissue of the seminal vesicles as well as in the germinal cells in the testis. No immunoreactivity was detected in tissues of the epididymides and other organs of the boar. The antigens present in seminal vesicle fluid of boars were partially purified by column chromatography. It was demonstrated that two antigens differing in molecular mass were present and both possessed protease and amidase activity. The higher molecular mass antigen eluted from a gel filtration column in a volume identical to that of proacrosin. The same result was obtained in polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). The low molecular mass antigen was eluted from Sephadex G-75 column together with natural protease inhibitors corresponding in molecular mass to less than 20 kDa. The mobility of the antigen in SDS-PAGE was greater than that of chymotrypsin. It is assumed that the protease from seminal vesicle epithelial resembled acrosin in structure and function. Acrosin may therefore not be specific for spermatozoa.
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PMID:Serine protease activity in boar seminal vesicles and its immunological similarity to sperm acrosin. 802 64

Deficiency of human aspartylglucosaminidase (AGA, glycosylasparaginase, EC 3.5.1.26), a lysosomal amidase, results in the lysosomal storage disease aspartylglucosaminuria (AGU). This disorder is most prevalent in the genetically isolated Finnish population. To facilitate the detailed analysis of this important enzyme, which functions in the final degradation step of glycoproteins, we developed a novel purification method which makes possible a simple five-step 5000-fold purification to apparent homogeneity of human aspartylglucosaminidase from leukocytes. This purification procedure takes advantage of the remarkable SDS resistance of aspartylglucosaminidase as SDS-sensitive proteins aggregate preferentially at low (NH4)2SO4 concentrations in the presence of SDS. This new method should be applicable to the isolation of other SDS-resistant enzymes, e.g., superoxide dismutase. The homogeneous enzyme preparation exhibited a previously unreported fully denatured 19-kDa form of the alpha-subunit of aspartylglucosaminidase on SDS-polyacrylamide gel electrophoresis as a consequence of complete coating by SDS.
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PMID:Large-scale purification of human aspartylglucosaminidase: utilization of exceptional sodium dodecyl sulfate resistance. 805 56

Major histocompatibility (MHC) class II antigens are heterodimeric cell surface glycoproteins consisting of an alpha and a beta chain. Although one-dimensional SDS-polyacrylamide gel electrophoresis analysis of purified MHC class II antigens shows a single diffuse band for each chain, multiple spots of identical molecular size were observed for each chain when analyzed by two-dimensional electrophoresis. The basis of this heterogeneity has not been clearly defined and has been predicted partially to be due to glycosylation and/or phosphorylation of the mature protein. To investigate the role of the three N-linked oligosaccharides of the alpha and beta chains in determining the isoelectric point of each chain, affinity-purified MHC class II antigens from human and rat sources were deglycosylated using asparagine amidase. The complete enzymatic removal of all three N-linked oligosaccharides was confirmed by SDS-polyacrylamide gel electrophoresis as well as by four different lectin-linked Western blot analyses. Two-dimensional gel analysis of the deglycosylated molecules shows no significant difference from the fully glycosylated chains. We have expressed truncated forms of the HLA DR2 chains which lack the transmembrane and cytoplasmically exposed regions in Escherichia coli. Two-dimensional electrophoresis of these single chains also reveal multiple banding patterns. The two-dimensional banding patterns described are unaffected by exposure to acidic or basic conditions, increased gel running time in the first dimension, treatment of the proteins with alkaline phosphatase to remove any potential phosphorylation, or preincubation in the presence of iodoacetamide. Multiple forms of recombinant alpha and beta chains were also observed in Tris-glycine-urea gels which merged into a single band in the presence of SDS. In addition, partially fractionated bands from preparative isoelectric focusing gels, when refocused, showed an identical number of multiple spots spanning the same range of isoelectric points. These results together suggest that each polypeptide chain of MHC class II antigens may exist in multiconformational forms, and the observed charge heterogeneity is independent of glycosylation and phosphorylation of the proteins.
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PMID:Intramolecular charge heterogeneity in purified major histocompatibility class II alpha and beta polypeptide chains. 814 5


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