scispace - formally typeset
Search or ask a question

Showing papers on "Enzyme assay published in 1993"


Journal ArticleDOI
TL;DR: It is proposed that the carbonyl groups of the HNE-derived Michael addition products may undergo secondary reactions with the amino acid groups of lysine residues to yield inter- and intrasubunit cross-links.

509 citations


Journal ArticleDOI
TL;DR: The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.
Abstract: 1. A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-gamma, 10 u ml-1) plus lipopolysaccharide (LPS, 10 ng ml-1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2. The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3. The decline in the NO synthase activity was significantly slowed down in cells cultured in a low L-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4. The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5. SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6. The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.

410 citations


Journal ArticleDOI
TL;DR: A mechanism by which HNE may, in part, mediate free radical damage is described and a method for the detection of the lysine-HNE adduct is introduced.

280 citations


Journal ArticleDOI
TL;DR: Clinical Pharmacology and Therapeutics (1993) 53, 503–514; doi:10.1038/clpt.1993.63
Abstract: Clinical Pharmacology and Therapeutics (1993) 53, 503–514; doi:10.1038/clpt.1993.63

274 citations


Journal ArticleDOI
TL;DR: Chitin deacetylase, the enzyme that catalyzes the hydrolysis of acetamido groups of N-acetylglucosamine in chitin, has been purified to homogeneity from mycelial extracts of the fungus Mucor rouxii and further characterized.
Abstract: Chitin deacetylase, the enzyme that catalyzes the hydrolysis of acetamido groups of N-acetylglucosamine in chitin, has been purified to homogeneity from mycelial extracts of the fungus Mucor rouxii and further characterized. The enzyme exhibits a low pI (approximately 3). Its apparent molecular mass was determined to be approximately 75 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and approximately 80 kDa by size-exclusion chromatography, suggesting that the enzyme exists as a monomer. Carbohydrate analysis of purified chitin deacetylase revealed that the enzyme is a high-mannose glycoprotein and that its carbohydrate content is approximately 30% by weight. Chitin deacetylase is active on several chitinous substrates and chitin derivatives. The enzyme requires at least four N-acetylglucosamine residues (chitotetraose) for catalysis, and it is inhibited by carboxylic acids, particularly acetic acid. When glycol chitin (a water-soluble chitin derivative) was used as substrate, the optimum temperature for enzyme activity was determined to be approximately 50 degrees C and the optimum pH was approximately 4.5.

232 citations


Journal ArticleDOI
TL;DR: It is suggested that a defect in this key Krebs cycle enzyme could contribute to an impairment of cerebral energy metabolism and the brain dysfunction in AD.
Abstract: We measured the activity of the a-ketoglutarate dehydrogenase complex (α-KGDHC), a rate-limiting Krebs cycle enzyme, in postmortem brain samples from 38 controls and 30 neuropathologically confirmed Alzheimer's disease (AD) cases, in both the presence and absence of thiamine pyrophosphate (TPP), the enzyme's cofactor. Statistically significant correlations between brain pH and lactate levels and α-KGDHC activity in the controls were observed, suggesting an influence of agonal status on the activity of α-KGDHC. As compared with the controls, mean α-KGDHC activity, with added TPP, was significantly (p < 0.005) reduced in AD brain in frontal (-56%), temporal (-60%), and parietal (-68%) cortices, with the reductions (-25 to -53%) in the occipital cortex, hippocampus, amygdala, and caudate failing to reach statistical significance. In the absence of exogenously administered TPP, mean a-KGDHC activity was reduced to a slightly greater extent in all seven AD brain areas (-39 to -83%), with the reductions now reaching statistical significance in the four cerebral cortical areas and hippocampus. A statistically significant negative correlation was observed between α-KGDHC activity and neurofibrillary tangle count in AD parietal cortex, the brain area exhibiting the most marked reduction in enzyme activity; this suggests that the enzyme activity reduction in AD brain may be related to the disease process and severity. In each brain area examined, TPP produced a greater stimulatory effect on α-KGDHC activity in the AD group (23–280% mean stimulation) as compared with the controls (-4 to ±50%); this TPP effect could be explained by reduced endogenous TPP levels in AD brain. Reduced brain α-KGDHC activity could be consequent to loss of neurons preferentially enriched in α-KGDHC, a premortem reduction in TPP levels (which may have affected enzyme stability), elevated brain levels of the α-KGDHC inhibitor ammonia, or an actual failure in the expression of the gene encoding the enzyme. We suggest that a defect in this key Krebs cycle enzyme could contribute to an impairment of cerebral energy metabolism and the brain dysfunction in AD.

170 citations


Journal ArticleDOI
TL;DR: The phenotypic features of strain GJ1B, an unidentified marine bacterium that degrades agar, were investigated and its agarolytic system was characterized using 13C-NMR spectroscopy to analyse the agarose degradation products.
Abstract: The phenotypic features of strain GJ1B, an unidentified marine bacterium that degrades agar [Young, K. S., Bhattacharjee, S. S. & Yaphe, W. (1978) Carbohydr. Res. 66, 207–212], were investigated and its agarolytic system was characterized using 13C-NMR spectroscopy to analyse the agarose degradation products. The bacterium was assigned to the genus Alteromonas and the new combination A. agarlyticus (Cataldi) is proposed. An α-agarase, i.e specific for the α(13) linkages present in agarose, was purified to homogeneity from the culture supernatant by affinity chromatography on cross-linked agarose (Sepharose CL-6B) and by anion-exchange chromatograpy (Mono Q column). The major end product of agarose hydrolysis using the purified enzyme was agarotetraose. Using SDS/PAGE, the purified α-agarase was detected as a single band with a molecular mass of 180 kDa. After the affinity-chromatography step, however, the native molecular mass was approximately 360 kDa, suggesting that the native enzyme is a dimer which is dissociated to active subunits by anion-exchange chromatography. The isolectric point was estimated to be 5.3. Enzyme activity was observed using agar as the substrate over the pH range 6.0–9.0 with a maximum value at pH 7.2 in Mops or Tris buffer. The enzyme was inactivated by prolonged treatment at a pH below 6.5, or by temperatures over 45°C or by removing calcium. In addition, a β-galactosidase specific for the end products of the α-agarase was present in the α-agarase affinity-chromatography fraction, probably as part of a complex with this enzyme. The degradation of agarose by this agarase complex yielded a mixture of oligosaccharides in the agarotetraose series and the agarotriose series, the latter consisting of oligosaccharides with an odd number of galactose residues.

169 citations


Journal ArticleDOI
TL;DR: Endothelin-converting enzyme, a key enzyme in the production of a potent vasoconstricting peptide, endothelin, was purified to homogeneity from rat lung microsomes using a sensitive and convenient assay method using the 125I-endothelins-1 receptor binding assay for purification studies.

157 citations


Journal ArticleDOI
TL;DR: Results suggest that bacterial expression of potato tuber cDNAs could be used to study the role and interaction of the subunits of the native ADP-glucose pyrophosphorylase.

155 citations


Journal ArticleDOI
TL;DR: It is reported that cPLA2 displays a relatively low, CoA-independent transacylase activity which produces phosphatidylcholine from lysophospholipase substrate, consistent with the formation of an acyl-enzyme intermediate.

151 citations


Journal ArticleDOI
TL;DR: The examination of the glucanase structure showed that the lower apparent molecular mass of the protein is due to anomalous migration in sodium dodecyl sulfate gels and not to posttranslational processing of the polypeptide chain.
Abstract: One of the major proteins of the Saccharomyces cerevisiae cell wall, a beta-glucanase (BGL2 gene product), has been isolated and purified to homogeneity under conditions for preserving enzyme activity. The study of enzyme properties of the protein revealed that it is an endo-beta-1,3-glucanase and not an exoglucanase as reported previously (F. Klebl and W. Tanner, J. Bacteriol. 171:6259-6264, 1989). The examination of the glucanase structure showed that the lower apparent molecular mass of the protein (29 kDa) compared with what was calculated from the amino acid sequence of the enzyme (33.5 kDa) is due to anomalous migration in sodium dodecyl sulfate gels and not to posttranslational processing of the polypeptide chain. Of two potential N glycosylation sites at Asn-202 and Asn-284, only the latter site is glycosylated. The overproduction of the beta-glucanase from the high-copy-number plasmid brought about a significant decrease in the growth rate of transformed yeast cells.

Journal ArticleDOI
Seoung Yong Lee1, Joon Shick Rhee1
TL;DR: Extracellular lipase was produced by the solvent-resistant strain Pseudomonas putida 3SK during the late logarithmic growth phase and was purified 21-fold with 5.3% recovery by ion exchange chromatography and gel filtration chromatography.

Journal ArticleDOI
TL;DR: The amino acid sequence deduced from the cDNA shows significant homology with a 4-alpha-glucanotransferase from Streptococcus and is discussed in terms of the function of D-enzyme in potato starch metabolism.

Journal ArticleDOI
TL;DR: Activity of the serum enzyme was further characterized with a new HA-substrate gel procedure and it was found that this circulating enzyme activity is different from the liver-derived activity.

Journal ArticleDOI
TL;DR: Results show that sequon occupancy as well as steric effects at residue 19 are important for the development of an active conformer of this enzyme, the first example of a lysosomal hydrolase that requiresSequon occupancy for the synthesis of a catalytically active enzyme.

Journal ArticleDOI
TL;DR: The purification of an endonuclease from human spleen cell nuclei is reported that is likely to be responsible for DNA digestion in apoptosis and it seems likely that this enzyme is involved in the apoptotic degradation of DNA in human lymphocytes.
Abstract: A major event in apoptosis is the digestion of chromatin into oligonucleosomal fragments. However, the enzymes responsible for the DNA degradation have not been well characterized. Here we report the purification of an endonuclease from human spleen cell nuclei that is likely to be responsible for DNA digestion in apoptosis. Enzyme activity was measured by a sensitive fluorometric assay, which assesses the conversion of plasmid DNA from a supercoiled to an open form. The endonuclease was extracted from isolated nuclei with NaCl between 100 and 350 mM and was further purified by chromatography on columns of phosphocellulose, Superdex 75, and chelating Sepharose (Zn2+ form). By gel filtration, the apparent molecular mass was 22-26 kDa; on SDS-polyacrylamide gel electrophoresis, the purified enzyme showed a single 27-kDa band. The enzyme required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity. It was inhibited by Zn2+ (100% inhibition at 50 microM) and by high (> 10 mM) concentrations of Ca2+. Aurintricarboxylic acid, spermine, p-(hydroxymercuri)benzoate, and N-ethylmaleimide were also endonuclease inhibitors. No inhibition was observed with iodoacetamide, G-actin, or nucleoside 3',5'-bisphosphates. An optimum pH of 8.0 was found. When added to human CCRF-CEM lymphoblast nuclei, that do not contain the endonuclease, the purified splenic enzyme digested the chromatin into the mono- and oligonucleosomal fragments that are characteristic of apoptosis. On the basis of this result, and the observation that the activators and inhibitors of the purified endonuclease closely parallel those that affect apoptosis, it seems likely that this enzyme is involved in the apoptotic degradation of DNA in human lymphocytes.

Journal ArticleDOI
TL;DR: Human 15-lipoxygenase was expressed to high levels (approx. 20% of cellular protein) in a baculovirus/insect cell expression system and the N-terminal amino acid sequence was found to be identical to that predicted from the cDNA.

Journal ArticleDOI
01 Jul 1993-Placenta
TL;DR: The NO synthase enzyme of the villous vasculature appears to correspond to the type III calcium-calmodulin dependent endothelial isoform as part of the ongoing studies of the regulation of NO synthesis in this circulation.

Journal ArticleDOI
TL;DR: A spectrophotometric assay method is described in which iodonitrotetrazolium chloride is used as a final electron acceptor and may be used in the determination of enzyme activity either in tissue homogenates or as a marker of the mitochondrial fraction in cell fractionation procedures.

Journal ArticleDOI
TL;DR: Two distinct NADH oxidases, corresponding to H 2O2-forming and H2O-forming enzymes were purified to homogeneity from Streptococcus mutans and their basic properties determined.
Abstract: SUMMARY: Two distinct NADH oxidases, corresponding to H2O2-forming and H2O-forming enzymes were purified to homogeneity from Streptococcus mutans and their basic properties determined. The H2O2-forming enzyme was a tetramer with a subunit molecular mass of about 56 kDa and required flavin adenine dinucleotide (FAD) for full activity. The enzyme had an isoelectric point of 6.6 and exhibited optimal activity at pH 6.0 The H2O-forming enzyme was a monomer with a molecular mass of 50 kDa and activity independent of exogenously added flavin. The enzyme had an isoelectric point of 4.8 and exhibited optimal activity between pH 7.0 and 7.5 Both enzymes oxidized NADH (Km 0.05 and 0.025 mM for the H2O2- and H2O-forming enzyme, respectively) but not NADPH and contained 1 mol of FAD per monomer. Spectra of the oxidized enzymes exhibited maxima at 271, 383 and 449 nm for the H2O2-forming enzyme and 271, 375 and 447 nm for the H2O-forming enzyme. Antibodies raised against the H2O2-forming enzyme or the H2O-forming enzyme reacted with their corresponding antigen, but did not cross-react. The amino-terminal regions of the two enzymes had completely different amino acid sequences.

Journal ArticleDOI
TL;DR: An aerobic Gram-positive sporeforming bacterium was isolated from an alkaline hot spring at Wondo Genet, Ethiopia and in an optimized culture medium it produced maximum activity of protease at 55°C and pH 9.5.
Abstract: An aerobic Gram-positive sporeforming bacterium was isolated from an alkaline hot spring at Wondo Genet, Ethiopia. In an optimized culture medium it produced maximum activity of protease at 55°C and pH 9.5. The protease activity against casein was 65 units/ml. Enzyme activity was detected between 30–70°C and pH of 4.5–11.5. The enzyme had a half-life of 55 and 30 min at 60° and 70°C, respectively. The isolate hydrolysed 90, 60 and 50% of the skin, feather and horn used in the optimized medium within 120 h.

Journal ArticleDOI
TL;DR: Observations provide the first clear evidence that the ND4 gene product is essential for Complex I activity and give some insights into the function and the structural relationship of this polypeptide to the rest of the enzyme.
Abstract: In most eukaryotic cells, the respiratory chain NADH dehydrogenase (Complex I) is a multimeric enzyme under dual (nuclear and mitochondrial) genetic control. Several genes encoding subunits of this enzyme have been identified in the mitochondrial genome from various organisms, but the functions of these subunits are in most part unknown. We describe here a human cell line in which the enzyme lacks the mtDNA-encoded subunit ND4 due to a frameshift mutation in the gene. In this cell line, the other mtDNA-encoded subunits fail to assemble, while at least some of the nuclear-encoded subunits involved in the redox reactions appear to be assembled normally. In fact, while there is a complete loss of NADH:Q1 oxidoreductase activity, the NADH:Fe(CN)6 oxidoreductase activity is normal. These observations provide the first clear evidence that the ND4 gene product is essential for Complex I activity and give some insights into the function and the structural relationship of this polypeptide to the rest of the enzyme. They are also significant for understanding the pathogenetic mechanism of the ND4 gene mutation associated with Leber's hereditary optic neuropathy.

Journal ArticleDOI
TL;DR: Wild-type enzyme produced in the presence of tunicamycin was also inactive, indicating that glycosylation is required for realization of enzyme activity, however, active enzyme could be deglycosylated with only minimal loss of activity.

Journal ArticleDOI
TL;DR: The data suggest that the continuous influx of calcium with A23187 is responsible for the extensive inactivation of the 5-lipoxygenase.

Journal ArticleDOI
TL;DR: The results indicate that the in vivo induction of hepatic 2E1 protein by ethanol involves increased enzyme synthesis rather than decreased enzyme degradation.

Journal ArticleDOI
TL;DR: The data suggest that protein synthesis is needed for selenium repletion to exert control on glutathione peroxidase activity, and eliminate the possibility of differential transcription rates as an explanation for the observed reduction in mRNA.

Journal ArticleDOI
TL;DR: The enzyme system concerning volatile ester formation in strawberry Fragaria ananassa×Duchessne var. Chandler was studied in this paper, where protein with alcohol acyltransferase activity was purified about 29-fold from Chandler strawberry fruits by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography.
Abstract: The enzyme system concerning volatile ester formation in strawberry Fragaria ananassa×Duchessne var. Chandler was studied. Protein with alcohol acyltransferase activity was purified about 29-fold from Chandler strawberry fruits by ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The enzyme activity had a pH optimum of 8.0 and an optimum temperature of 35 o C. The apparent M r estimated by gel filtration was 70 000. The enzyme was tested for its preference in using different acyl-CoAs and alcohols. Maximum activity was obtained using acetyl-CoA and hexyl alcohol as substrates. A clear correlation was observed between substrate preference and volatile esters present in strawberry var. Chandler

Journal ArticleDOI
TL;DR: It is demonstrated that most anticarcinogens, in particular flavone, coumarin and alpha-angelicalactone, enhance GST activity in liver and intestine, mainly by induction of class alpha and mu isozymes.
Abstract: Effects of dietary naturally occurring anticarcinogens; quercetin, flavone, ellagic acid, ferulic acid, tannic acid, curcumin, coumarin, alpha-angelicalactone, fumaric acid and Brussels sprouts on male Wistar rat hepatic and intestinal (i) glutathione S-transferases (GST) enzyme activity, (ii) GST isozyme levels and (iii) glutathione (GSH) content were investigated. GST enzyme activity was significantly increased by all anticarcinogens tested, except fumaric acid, at least at one of the five sites investigated: proximal, middle, distal small intestine, large intestine and liver. Only alpha-angelicalactone gave an enhanced GST enzyme activity at all five sites. Large intestinal GST enzyme activity was increased only by quercetin (175%) and alpha-angelicalactone (138%). Concomitant changes in GST isozyme levels occurred. Class alpha GSTs were induced in 50% of the cases, especially in liver and upper parts of the intestine by quercetin, flavone, coumarin and alpha-angelicalactone. GST class pi levels were enhanced only at one site by quercetin, coumarin and alpha-angelicalactone. GST class mu changed in 14% of the cases, most profoundly in proximal and middle small intestine by flavone, coumarin and alpha-angelicalactone. Tannic acid and fumaric acid gave a significant raise in class alpha GSTs at almost all sites, whereas overall GST enzyme activity hardly changed. GSH was increased at various sites in 14% of the cases by Brussels sprouts, quercetin, flavone and alpha-angelicalactone. These data demonstrate that most anticarcinogens, in particular flavone, coumarin and alpha-angelicalactone, enhance GST activity in liver and intestine, mainly by induction of class alpha and mu isozymes.

Journal ArticleDOI
TL;DR: It is shown that an enzyme activity displaying identical properties is present in the cell medium of uninfected corticotroph AtT-20 cells and that its level is increased following stimulation of secretion by the secretagogue 8-bromo cyclic AMP.
Abstract: Prohormone convertase-1 (PC1), an endopeptidase that is structurally related to the yeast subtilisin-like Kex2 gene product, has been proposed to be involved in mammalian tissue-specific prohormone processing at pairs of basic residues. To better study this enzyme, a rat somatomammotroph cell line, GH4C1, was infected with vaccinia virus recombinants of murine PC1 (mPC1) and human PC1 (hPC1). An enzymically active form of each protein was secreted into the cell medium and partially purified by anion-exchange chromatography. The 80-85 kDa enzyme was shown to be Ca(2+)-dependent and exhibited a pH optimum of 6.0 when assayed against a synthetic fluorogenic substrate, acetyl-Arg-Ser-Lys-Arg-4-methylcoumaryl-1-amide. mPC1 and hPC1 displayed identical cleavage selectivity towards a number of fluorogenic substrates, and those incorporating an Arg at the P4 site were most favoured. Synthetic peptides, encompassing the junction between the putative pro-region and the active enzyme, and between the pro-region and the biologically active parathyroid hormone, were shown to be recognized and cleaved specifically at the pair of basic residues by both enzymes. Group-specific proteinase inhibitors such as metal ion chelators and p-hydroxymercuribenzoate, but not phenylmethanesulphonyl fluoride and pepstatin, strongly inhibit the PC1-associated activity. In addition, it is shown that an enzyme activity displaying identical properties is present in the cell medium of uninfected corticotroph AtT-20 cells and that its level is increased following stimulation of secretion by the secretagogue 8-bromo cyclic AMP.

Journal ArticleDOI
TL;DR: Analysis of HuAChE mutants, defective in a single or multiple N-glycosylation sites, by expression in transiently or stably transfected human embryonal 293 kidney cells suggests the following.
Abstract: The role of N-glycosylation in the function of human acetylcholinesterase (HuAChE) was examined by site-directed mutagenesis (Asn to Gln substitution) of the three potential N-glycosylation sites Asn-265, Asn-350 and Asn-464. Analysis of HuAChE mutants, defective in a single or multiple N-glycosylation sites, by expression in transiently or stably transfected human embryonal 293 kidney cells suggests the following. (a) All three AChE glycosylation signals are utilized, but not all the secreted molecules are fully glycosylated. (b) Glycosylation at all sites is important for effective biosynthesis and secretion; extracellular AChE levels in mutants defective in one, two or all three sites amounted to 20-30%, 2-4% and about 0.5% of wild-type level respectively. (c) Some glycosylation mutants display impaired stability, as reflected by increased susceptibility to heat inactivation; substitution of Asn-464 has the most pronounced effect on thermostability. (d) Abrogation of N-glycosylation has no detectable effect on the enzyme activity of HuAChE; all glycosylation mutants, including the triple mutant, hydrolyse acetylthiocholine efficiently, displaying Km, kcat. and kcat./Km values similar to those of the wild-type enzyme. (e) In most mutants, inhibition profiles with edrophonium and bisquaternary ammonium ligands are identical with those of wild-type enzyme; the Asn-350 mutants, however, exhibit a slight decrease in their affinity towards these ligands. (f) Elimination of oligosaccharide side chains has no detectable effect on the surface-related ‘peripheral-site’ functions; like the wild-type enzyme, all mutants were inhibited by propidium and by increased concentrations of acetylthiocholine.