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)

Rhizobium species produce an inducible acyl carrier protein (ACP), encoded by the nodF gene, that somehow functions in an exchange of cell signals between bacteria and specific plant hosts, leading to nodulation of plant roots and symbiotic nitrogen fixation, as well as a constitutive ACP needed for the synthesis of essential cell lipids. The periplasmic cyclic glucans of Rhizobium spp. are also involved in specific rhizobium-plant interaction. These glucans are strongly similar to the periplasmic membrane-derived oligosaccharides (MDO) of Escherichia coli. E. coli ACP is an essential component of a membrane-bound transglucosylase needed for the biosynthesis of MDO, raising the possibility that either or both of the rhizobial ACPs might have a similar function. We have now isolated the constitutive ACP of R. meliloti and determined its primary structure. We have also examined its function, together with those of ACPs from E. coli, Rhodobacter sphaeroides, and spinach, in the MDO transglucosylase system and as substrate for the E. coli ACP acylase enzyme. All four ACPs act as acceptors of acyl residues, but only the E. coli ACP functions in the transglucosylase system.
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PMID:Isolation and characterization of the constitutive acyl carrier protein from Rhizobium meliloti. 214 77

Ampicillin acylase, which is known to have a novel substrate spectrum, was purified to homogeneity from Pseudomonas melanogenum by the crude extract preparation and chromatography with S-Sepharose, hydroxyapatite, CM-cellulose C-52, and CM-Sepharose. The molecular weight of the native enzyme was calculated to be 146,000 by Protein PAK-300 sw HPLC chromatography. SDS-polyacrylamide gel electrophoresis revealed that the enzyme consisted of two identical subunits with a molecular weight of 72,000. The enzyme was a glycoprotein containing 13% of total carbohydrate, and its isoelectric point was 7.2. The enzyme catalyzed both synthesis and hydrolysis of ampicillin and hydrolysis of the ester bond of phenylglycinemethylester hydrochloride substrate. The substrate specificity showed that the enzyme required a free amino group on the alpha-carbon of the acyl group. Chemical modification by diethylpyrocarbonate or N-bromosuccinimide resulted in time-dependent inactivation of the enzyme, and other results suggest the participation of essential histidine residue(s) in the catalytic activity of ampicillin acylase. Substrates of the enzyme, 6-aminopenicillanic acid and ampicillin, exhibited protective effects against N-bromosuccinimide inactivation, suggesting that the modification occurred near or at the active site.
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PMID:Purification and properties of ampicillin acylase from Pseudomonas melanogenum. 216 18

Penicillin acylase from E. coli (EC 3.5.1.11) was found to hydrolyze N-phenylacetylated 1-aminoethylphosphonic acid and its esters. The enzyme preferentially converts the R-form of the substrates: the ratios of the bimolecular rate constants of penicillin acylasecatalyzed hydrolysis of R- and S-forms of 1-(N-phenylacetamino)-ethylphosphonic acid and its dimethyl- and diisopropyl-esters are 58000, 2300, 1800; these derivatives were shown to have the greatest values of the catalytic constants for enzymatic hydrolysis of all known substrates for penicillin acylase: 237, 148 and 134 s-1; the corresponding Km values are 3.7 10(-5), 6.8 10(-4) and 6.2 10(-4) M at pH 7.0. The kinetics of enzymatic hydrolysis of 1-(N-phenylacetamino)-ethylphosphonic acid was investigated up to high degrees of conversion. The inhibition of penicillin acylase by high concentrations of the R-form of the substrate (with substrate inhibition constant of 0.07 M) and competitive inhibition by the reaction product, phenylacetic acid (Ki = 3.5 10(-5) M), was observed.
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PMID:[Kinetic characteristics and enantioselective action of penicillinase in the hydrolysis reaction of N-phenylacetyl derivatives of 1-aminoethylphosphonic acid and its esters]. 220 9

N-Acetyl-L-glutamate has been examined with regard to its ability to activate carbamoyl phosphate synthetase I (EC 6.3.4.16). Substance(s) inhibitory to carbamoyl phosphate synthetase, present even in the partially purified preparation of rat liver extracts, interfered with the measurement of acetylglutamate. In the experiments using chelating agents, metals were apparently involved in this inhibition. When the partially purified preparation of liver extract was placed on a Chelex 100 column, the inhibitor was eliminated and accurate measurements of acetylglutamate content could be made. Evidence supporting the validity of this improved method is given. A significant difference was observed between acetylglutamate levels determined by the present method and by the one using aminoacylase I (N-acylamino acid amidohydrolase, EC 3.5.1.14) to hydrolyze acetylglutamate followed by assay of the glutamate generated. We searched for the presence of glutamate derivatives other than acetylglutamate. When impure tissue preparations containing acetylglutamate were treated with a commercial preparation of aminoacylase, there was an excess amount of glutamate apparently derived from compounds other than acetylglutamate. This can lead to an overestimation of the tissue levels of acetylglutamate.
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PMID:An improved method for determination of N-acetyl-L-glutamate by its function as an activator of carbamoyl phosphate synthetase I. 230 60

The aim of this study was to set up an in vitro system to study nephrotoxicity of xenobiotics which allows exposure at low concentrations for long periods (1-5 days). A very pure preparation of isolated proximal tubular cells (PTC) from rat kidney (Boogaard et al., Toxicol Appl Pharmacol 101: 135-143, 1989) was brought into primary culture. Cells grew to confluence in 3 days and could be maintained up to 8 days in a modification of Dulbecco's modified Eagle's medium Ham F12 nutrient mixture supplemented with fetal calf serum. Fibroblast growth was completely suppressed by replacement of L-valine by D-valine and of L-arginine by L-ornithine. Polarity was retained: in cells grown on filters organic anions were transported at the basolateral membrane while D-glucose transport was located at the apical membrane. Inhibition of the latter was used to assess the functional integrity of the cells after exposure to nephrotoxins. The newly grown cells expressed gamma-glutamyltranspeptidase activity since incubation with the glutathione-conjugate of 1,1-dichloro-2,2-difluoroethylene (DCDFE) induced cytotoxicity. Both beta-lyase and acylase activities were expressed because the cysteine-S-conjugate and the corresponding mercapturate of DCDFE showed cytotoxicity. Cultured cells showed toxicity on prolonged exposure to very low concentrations of gentamicin, cephaloridine, cisplatin and the cysteine-S-conjugate of chlorotrifluoroethylene. The lowest concentrations at which toxicity can be observed are 1-3 orders of magnitude lower in primary cultures than in freshly isolated PTC in suspension. This indicates that this cell model is suitable to investigate mechanisms of nephrotoxicity in vitro, at prolonged exposure to the low concentrations that are relevant in vivo levels.
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PMID:Primary culture of proximal tubular cells from normal rat kidney as an in vitro model to study mechanisms of nephrotoxicity. Toxicity of nephrotoxicants at low concentrations during prolonged exposure. 232 15

1. In this study the processes underlying the renal selectivity of the vasodilator prodrug CGP 22979 (N-acetyl-L-glutamic acid-N-[N2-(5-n-butyl-2-pyridyl) hydrazide]) were studied in rats. 2. The active drug CGP 18137 (2-hydrazino-5-n-butyl pyridine) selectively accumulated in the renal tissue following administration of the prodrug. 3. The kidney concentrations of active drug following prodrug administration were significantly lower than control values when either buthionine sulphoximine, glutathione or probenecid was coadministered (29 +/- 11; 33 +/- 14 and 61 +/- 20% of control values, respectively). Inhibition of gamma-glutamyl transpeptidase by AT-125 did not cause a significant decrease of renal CGP 18137 levels. 4. In order to correlate tissue drug concentrations with pharmacological effect, the renal haemodynamic responses to CGP 22979 were measured and the effect of buthionine sulphoximine, glutathione and AT-125 on these responses evaluated. All three of the compounds attenuated the renal response to the prodrug: an approximately 50% lesser decrease in renal resistance was found. The compounds had no effect on the haemodynamic actions of CGP 18137 itself. 5. In vitro, it was found that kidney cytosol was able to convert the prodrug, whereas microsomes were not, unless acylase was added. 6. The results indicate that, upon prodrug administration, gamma-glutamyl transpeptidase is not involved in the renal accumulation of CGP 18137 but is partly responsible for the renal haemodynamic responses to CGP 22979. Active transport of the prodrug into the tubular cells appears to be the major reason for the renal selectivity. A model is proposed for the renal action of CGP 22979, in which the important parts are the uptake of the prodrug via a transport system followed by an intracellular conversion to the active drug.
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PMID:Renal selective N-acetyl-gamma-glutamyl prodrugs: a study on the mechanism of activation of the renal vasodilator prodrug CGP 22979. 233 67

The solution conformation properties of penicillin G acylase (EC 3.5.1.11) have been characterised by near- and far-ultraviolet circular dichroism, steady-state and time-resolved fluorescence spectroscopy and differential sedimentation velocity. The enzyme (86 kDa) was found to be spherical and stable unfolding over a narrow range of urea concentrations in an apparently cooperative fashion with a mid-point of 4.5 M urea. Separation of its constituent alpha and beta peptides (23.8 kDa and 62.2 kDa, respectively) was accompanied by loss of enzyme activity and unfolding, the kinetics of unfolding being highly dependent upon urea concentration. Urea gradient gel electrophoresis showed that the separated beta peptide aggregates over a wide range of urea concentrations but that the alpha peptide refolds reversibly to a compact state. Physical studies showed that the refolded alpha peptide has a compact but asymmetric structure with more alpha helix than the native enzyme, but is more sensitive to denaturant. The latter is suggested to be due to a hydrophobic patch detected by 8-anilino-1-naphthalene sulfonic acid binding and which is normally covered by the beta peptide in the native enzyme. The results of these investigations indicate that the alpha peptide constitutes a folding domain and suggest that it plays a key role in folding of the precursor for penicillin acylase.
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PMID:The folding and solution conformation of penicillin G acylase. 240 Dec 88

Enzymatic parameters such as pH, temperature and substrate concentration were studied for the hydrolysis of 7-PADCA by penicillin G acylase. Optimum pH and temperature were 8.0 and 50 degrees C, respectively. Km value of soluble and immobilized enzyme for 7-PADCA was 2.3 x 10(-5) M and 7.5 x 10(-5) M, respectively. At 7-PADCA concentration of 5% and an IME: 7-PADCA ratio of 1:2.5, the hydrolysis was complete in 110 min.
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PMID:Enzymatic hydrolysis of 7-phenylacetamidodesacetoxycephalosporanic acid by immobilized penicillin G acylase. 248 71

The penicillin G acylase gene cloned from Arthrobacter viscosus 8895GU was subcloned into vectors, and the recombinant plasmids were transferred into Escherichia coli or Bacillus subtilis. Both E. coli and B. subtilis transformants expressed the A. viscosus penicillin G acylase. The enzyme activity was found in the intracellular portion of the E. coli transformants or in the cultured medium of the B. subtilis transformants. Penicillin G acylase production in the B. subtilis transformants was 7.2 times higher than that in the parent A. viscosus. The A. viscosus penicillin G acylase was induced by phenylacetic acid in A. viscosus, whereas the enzyme was produced constitutively in both the E. coli and B. subtilis transformants carrying the A. viscosus penicillin G acylase gene.
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PMID:Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis. 250 7

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[Whole nucleotide sequence of penicillin G acylase gene and its flanking region from E. coli]. 266 30


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