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)

Two mutations of the porphobilinogen (PBG) deaminase gene resulting in cross-reacting immunological material (CRIM) positive forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA and cloning of the amplified products in a bacterial expression vector. Both mutations resulted from G to A transitions in exon 10 of the gene and produced arginine to glutamine substitutions in the abnormal protein. Expression of mutant cDNA in Escherichia coli reveals that one but not the other of these amino acid changes results in a striking decrease of the optimal pH of the mutated enzyme. One or the other of these two mutations accounted for the defect causing AIP in six unrelated patients among the eight patients evaluated with the CRIM positive subtype of this disorder.
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PMID:Two different point G to A mutations in exon 10 of the porphobilinogen deaminase gene are responsible for acute intermittent porphyria. 224 28

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

Lipoproteins (d = 1.05-1.12 g/ml) were obtained from pooled serum by density gradient ultracentrifugation and used as a source for isolation of apolipoprotein (a) (apo(a]. It was found that both these lipoproteins and purified apo(a) possess negligible amidolytic and proteolytic activity. After preincubation of lipoproteins and apo(a) with collagen-Sepharose, the increase in enzymatic activity was observed. The activation of purified apo(a) also occurred upon its storage in the cold. After two week storage at 7 degrees C, the amidase activity, as measured by splitting of the substrate D-Pro-Phe-Arg-pNA, was increased from 0.009 U/mg to 0.85 U/mg. The amidase activity was completely inhibited by phenylmethylsulfonyl fluoride (10(-3) M) and by soybean trypsin inhibitor (10(-5) M); it was not inhibited by aprotinin (10(-6) M). Activated apo(a) did not split azocasein but converted plasma prekallikrein to kallikrein and degraded apolipoprotein B-100.
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PMID:Enzymatic properties of active form of human apolipoprotein (a). 240 48

Glandular kallikrein shows a special selectivity for D-Val-Leu-Arg-4-methoxy-2-naphthylamide in comparison with other potential oligopeptide substrates and it provides a useful histochemical substrate, although the reaction may not always be specific. However, in cat submandibular saliva, a biochemical assay using the closely related D-Val-Leu-Arg-7-amino-4-trifluoromethylcoumarin (AFC) as substrate, which affords more sensitive detection, showed that soya bean trypsin inhibitor causes no inhibition. This indicates that there are unlikely to be contaminating enzymes competing for the substrate in this body fluid. Support for this observation has been gained by the useful new enzyme overlay membrane technique for fluorescent assessment of reactive bands of enzymes after isoelectric focusing, using membranes of cellulose acetate impregnated with D-Val-Leu-Arg-AFC. Comparison of results after isoelectric focusing of purified cat submandibular kallikrein with samples of cat submandibular saliva confirmed that the substrate is monospecific for kallikrein in saliva of the cat. This knowledge has enabled us to start assessing the dynamics of the secretion of kallikrein by the gland. Testing individual drops of saliva has shown that an amazingly rapid mobilization of kallikrein occurs in high concentrations on sympathetic nerve stimulation. The corresponding oligopeptide-based inhibitor D-Val-Leu-Arg-chloromethyl ketone was found to be strongly inhibitory of the amidase reaction by kallikrein but showed a low specificity for kallikrein. Nevertheless, its effects have been tested in vivo by the intravascular route and it caused an increase in the resting salivary vascular resistance whether administered close-arterially or intravenously. Thus, it would seem that a kallikrein-like protease does influence the background tone in the vessels and the source of this enzyme is thought to be mast cells.
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PMID:Use of different derivatives of D-Val-Leu-Arg for studying kallikrein activities in cat submandibular glands and saliva. 241 50

The composition of the peptidoglycan of Bordetella pertussis and the nature of its turnover products was determined by a new combination of analytical techniques: high performance liquid chromatography of an enzymatic peptidoglycan hydrolysate and fast atom bombardment mass spectrometry and fast atom bombardment collision-activated dissociation tandem mass spectrometry. Sixteen major components of the peptidoglycan were purified, and assignment of complete or partial chemical structures was achieved for nine and seven species, respectively. At this level of resolution, a previously unrecognized heterogeneity of monomeric (five new species; nine total) and dimeric species (five new species; five total) was detected. No species containing diaminopimelyl-diaminopimelic acid cross-links or lysyl-arginine substitutions were found. Previous estimates of total cross-linkage and average chain length were revised downward to 32% and 21 disaccharide residues, respectively. Detection of a chemically novel species, a disaccharide octapeptide monomer, in both the peptidoglycan hydrolysate and culture supernatant fluid, suggests that an N-acetyl-muramyl-L-alanine amidase acts on the intact peptidoglycan of Bordetella and participates in cell wall turnover. Five peptidoglycan turnover products were identified in the supernatant fluid of late logarithmic phase cultures, including the 1,6-anhydro monomeric species known as tracheal cytotoxin. Peptidoglycan turnover was detected at a low rate of approximately 10%/generation, a value sufficient to account for the generation of all tracheal cytotoxin found in culture supernatant fluids.
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PMID:Unusual composition of peptidoglycan in Bordetella pertussis. 254 84

Human pancreatic kallikrein (H. Panc. K.) was purified from human pancreas by serial liquid chromatographies. The final preparation had a specific activity of 9.2 AU/A280 (AU: amidase unit for H-Pro-Phe-Arg-MCA) and its N-terminal sequence coincided with the reported sequence determined from cloned cDNA analysis. In HPLC (gel filtration), one symmetrical peak corresponding to a molecular weight of 48,000 was obtained. In SDS-PAGE without 2-mercaptoethanol, one band corresponding to a molecular weight of 52,000 was obtained. Protease inhibitor specificities of H. Panc. K. were the same as those of human urinary kallikrein (HUK) and hog pancreatic kallikrein (hog Panc. K.), while anti-HUK rabbit antibody inhibited the activities of H. Panc. K. and HUK, but not that of hog Panc. K. From the analysis of affinity for concanavalin A and erythroagglutinating phytohemagglutinin, the carbohydrate parts of H. Panc. K. are relatively rich in bi-(or multi-) antennary complex type sugar chains with bisecting GlcNAc compared with those of human salivary kallikrein and HUK. These findings will be a help to clarify the physiological and pathophysiological roles of H. Panc. K. in the pancreas and pancreatic diseases, especially in acute pancreatitis.
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PMID:[Purification of human pancreatic kallikrein and organ-specificities of human glandular kallikreins]. 260 Nov 18

Human pancreatic kallikrein (H.Panc.K.) was purified from human pancreas by ion exchange chromatography on DEAE-cellulose, affinity chromatographies on p-aminobenzamidine Sepharose 6B and aprotinin aminocellulofine, followed by gel filtration on Sephacryl S-200. The final preparation had a specific activity of 9.2 AU/A280 (AU; amidase unit for H-Pro-Phe-Arg-MCA) and its N-terminal sequence coincided with the reported sequence for H.Panc.K.. In HPLC (gel filtration), one symmetrical peak corresponding to a molecular weight of 48,000 was obtained. In SDS-PAGE without 2-mercaptoethanol (2-ME), one band corresponding to a molecular weight of 52,000 was obtained, but with 2-ME, 2 bands, 52,000 and 30,000, were obtained. Km value for MCA was 4.9 x 10(-2) mM. Proteinase inhibitor specificities of H.Panc.K. were the same as those of human urinary kallikrein (HUK) and hog pancreatic kallikrein (hog Panc.K.), while anti-HUK antibody inhibited the activities of H.Panc.K. and HUK, but not that of hog Panc.K.. From the analysis of affinity for concanavalin A (Con A) and erythroagglutinating phytohemagglutinin (E-PHA), the carbohydrate parts of H.Panc.K. are relatively rich in biantennary complex type sugar chains with bisecting GlcNAc compared with those of human salivary kallikrein (H.Saliv.K.) and HUK.
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PMID:Characterization of human pancreatic kallikrein. 261 57

Purified human serum butyrylcholine esterase (approximately 90-kDa subunit), which also exhibits aryl acylamidase activity, was subjected to limited alpha-chymotrypsin digestion. Three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa were found to be produced, as observed by SDS-gel electrophoresis of the chymotryptic digest. The purified butyrylcholine esterase could fully bind to a Ricinus-communis-agglutinin-Sepharose column but after chymotryptic digestion about 15-20% of the enzyme activity remained unbound and was recovered in the run-through fractions. Sephadex G-75 chromatography of the chymotryptic digest showed an enzymatically active fragment eluted at an approximate molecular mass of 20 kDa, apart from the undigested butyrylcholine esterase eluted at the void volume. The butyrylcholine esterase fragment that did not bind to Ricinus communis agglutinin also was eluted at an approximate molecular mass of 20 kDa from a Sephadex G-75 column. This enzymatically active low-molecular-mass fragment from Sephadex G-75 chromatography showed a single protein band of approximately 20 kDa on SDS-gel electrophoresis. Neutral sugar analysis of the approximately 20 kDa fragment showed the presence of mannose only, whereas the undigested butyrylcholine esterase showed the presence of both mannose and galactose. Amino-terminal-sequence analysis of the approximately 20 kDa fragment showed the sequence Arg-Val-Gly-Ala-Leu, which agrees with amino acid residues 147-151 reported for human serum butyrylcholine esterase [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. Both cholinesterase and aryl acylamidase activities were co-eluted in all chromatographic procedures. The results suggested that limited alpha-chymotrypsin digestion of human serum butyrylcholine esterase resulted in the formation of a approximately 20-kDa enzymatically active fragment with Arg147 as its N-terminal residue and which was devoid of galactose.
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PMID:Isolation of a galactose-free 20-kDa fragment exhibiting butyrylcholine esterase and aryl acylamidase activity from human serum butyrylcholine esterase by limited alpha-chymotrypsin digestion. 264 20

A serine protease shown to be trypsin was purified from the pyloric caeca of Atlantic cod (Gadus morhua), and resolved into three differently charged species by chromatofocusing (pI 6.6, 6.2 and 5.5). All three trypsins had similar molecular mass of 24.2 kDa. N-terminal amino acid sequence analysis of cod trypsin showed considerable similarity with other known trypsins, particularly with dogfish and some mammalian trypsins. The apparent Km values determined at 25 degrees C for the predominant form of Atlantic cod trypsin towards p-tosyl-L-arginine methyl ester and N-benzoyl-L-arginine p-nitroanilide were 29 microM and 77 microM respectively, which are notably lower values than those determined for bovine trypsin (46 microM and 650 microM respectively). The difference was particularly striking when the amidase activity of the enzymes was compared. Furthermore, the kcat values determined for the Atlantic cold trypsins were consistently higher than the values determined for bovine trypsin. The higher catalytic efficiency (kcat/Km) of Atlantic cod trypsin as compared to bovine trypsin may reflect an evolutionary adaptation of the poikilothermic species to low environmental temperatures.
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PMID:Purification and characterization of trypsin from the poikilotherm Gadus morhua. 270 66

Acrosin, a sperm-specific acrosomal proteinase, has an essential role in the fertilization process. Low levels of acrosin appear to be associated with subfertility and infertility, and the acrosin activity of spermatozoa may potentially be a useful indicator of semen quality. The standard acrosin tests employed by research laboratories are too complicated and/or time consuming for clinical use; therefore, a simple assay has been developed to assess total acrosin activity (acrosin and activatable proacrosin). To perform the test, liquefied semen is centrifuged over Ficoll, the washed sperm pellet is suspended in a detergent (Triton X-100)-substrate (N-alpha-benzoyl-DL-arginine p-nitroanilide) buffer, pH. 8.0, and the amidase activity is determined spectrophotometrically after a 3-hour incubation period. Amidase activity can be inhibited with benzamidine, indicating that the activity is primarily or entirely due to acrosin. The absence of detergent in the incubation medium results in greatly reduced activity. The assay is repeatable, linear with increasing sperm concentration, sensitive to a lower limit of 2 x 10(6) spermatozoa, and the results correspond to those obtained with a standard acrosin extraction and assay technique. Storage of ejaculates at 3 to 6 C or at 22 to 24 C for 24 hours does not affect the acrosin activity significantly but much higher temperatures can cause a loss of activity. Freezing ejaculates results in a large decrease in sperm acrosin activity. Leukocytes show minimal activity in the assay. Sperm populations prepared by a swim-up procedure average approximately a 2-fold higher acrosin activity than the original ejaculates. Preliminary experiments indicate that the average sperm acrosin activity of ejaculates whose spermatozoa successfully fertilize human eggs in vitro is significantly higher than those that do not fertilize eggs.
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PMID:A simple, clinical assay to evaluate the acrosin activity of human spermatozoa. 274 33


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