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)

Human high molecular weight urokinase, a plasminogen activator, when minimally reduced with 0.01 M 2-mercaptoethanol for 10 h at pH 8.0 and 25 degrees C and then carboxymethylated with sodium iodoacetate, gave two chains, a functionally active heavy chain with about 80% of the original activity and a light chain. These two chains were found to be linked by a single interchain disulfide bond. The functionally active heavy chain can be isolated by an affinity chromatography method with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose. The light chain, which has no enzyme activity, is not adsorbed to the affinity matrix, whereas the active heavy chain was adsorbed and subsequently eluted. The active heavy chain was further purified by gel filtration on Sephadex G-100. This preparation was found to be homogeneous by both analytical and sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The molecular weight of the active heavy chain was determined to be 33,000 by Sephadex G-100 gel filtration and 31,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Its specific activity, with L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide, was determined to be 208,000 IU/mg of protein. Approximately 87% active sites were found by p-nitrophenyl-p'-guanidino-benzoate titration with a molar activity of 7.41 X 10(9) IU/mmol of active site. The active heavy chain when compared to low molecular weight urokinase has a similar molecular weight, specific activity, and amino acid composition. The NH2-terminal residue found in the active heavy chain was lysine which was the same as that found in low molecular weight urokinase, whereas the NH2-terminal residues found in high molecular weight urokinase were serine and lysine. Serine is the NH2-terminal residue of the light chain of high molecular weight urokinase. The steady state kinetic parameters of activation of human Glu-plasminogen by the active heavy chain were also similar to low molecular weight urokinase, as were the amidase parameters of these enzymes. The Michaelis constants of activation (Kplg) were 2.11 and 2.21 microM, respectively; the catalytic rate constants of activation (kplg) were 51.7 and 44.1 min-1, respectively, with second order rate constants, kplg/Kplg of 24.5 and 20.2 microM-1 min-1, respectively.
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PMID:A functionally active heavy chain derived from human high molecular weight urokinase. 634 38

An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60 degrees C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn2+ (to 80%), Mn2+ (to 400%), and Mg2+ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of alpha-hydrogen- and (bulky) alpha,alpha-disubstituted alpha-amino acid amides, alpha-hydroxy acid amides, and alpha-N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, beta-amino and beta-hydroxy acid amides, and dipeptides were not converted. The gene encoding this L-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.
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PMID:L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321. 1633 74

Serine proteases and some cathepsins are present in the stratum corneum. They are known to play a significant role in the pathophysiological mechanism of several dermatological conditions (e.g. atopic dermatitis) and in the induction of itch. Tape stripping of skin is a simple technique used in the investigation of skin barrier function and in the penetration of topically applied drugs. Herein, we show that CE, under stacking conditions, is a well-suited technique to measure the proteolytic activity of enzymes in the stratum corneum. Disks of about 6 mm (id) were cut from adhered tapes and submerged directly in a buffer containing the appropriate peptide substrate. After incubation, the split peptides were separated and detected directly by CE at 214 nm in a borate buffer. The esterase activity on N-benzoyl-tyrosine ethyl ester and the amidase activity on succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and the splitting of hemoglobin were detected by CE. The esterase activity was the highest when compared to the proteolytic activities. Skin scratching increased the enzymatic activity adhered to the tapes. The CE offered over the traditional end-point colorimetric methods the ability to measure the low enzymatic activity and the ability to detect the released peptides directly. This technique is simple, non-invasive, easy to perform and uses non-expensive substrates. It can be useful in quantifying cathepsins and serine proteases in the skin.
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PMID:Capillary electrophoresis of the proteolytic activity of the stratum corneum obtained by tape stripping. 2221 53

Translation is a central cellular process and is optimized for speed and fidelity. The speed of translation of a single codon depends on the concentration of aminoacyl-tRNAs. Here, we used microarray-based approaches to analyze the charging levels of tRNAs in Escherichia coli growing at different growth rates. Strikingly, we observed a non-uniform aminoacylation of tRNAs in complex media. In contrast, in minimal medium, the level of aminoacyl-tRNAs is more uniform and rises to approximately 60%. Particularly, the charging level of tRNA(Ser), tRNA(Cys), tRNA(Thr) and tRNA(His) is below 50% in complex medium and their aminoacylation levels mirror the degree that amino acids inhibit growth when individually added to minimal medium. Serine is among the most toxic amino acids for bacteria and tRNAs(Ser) exhibit the lowest charging levels, below 10%, at high growth rate although intracellular serine concentration is plentiful. As a result some serine codons are among the most slowly translated codons. A large fraction of the serine is most likely degraded by L-serine-deaminase, which competes with the seryl-tRNA-synthetase that charges the tRNAs(Ser) These results indicate that the level of aminoacylation in complex media might be a competition between charging for translation and degradation of amino acids that inhibit growth.
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PMID:Discharging tRNAs: a tug of war between translation and detoxification in Escherichia coli. 2750 88