Showing papers on "Enzyme assay published in 1987"
TL;DR: The results suggest that loss of functional enzyme activity and increased heat lability of enzymes during aging may be due in part to oxidative modification by mixed function oxidation systems.
1,165 citations
TL;DR: The release of nitric oxide from organic nitrates is examined by means of a difference-spectrophotometric method and it is found that the results correlated well with the extent of enzyme activation, suggesting both an enzymatic and a non-enzymatic mode of inhibition of enzyme activity does exist.
891 citations
TL;DR: These animals provide a unique system for studying the consequences of increased dosage of the Cu/Zn-superoxide dismutase gene in Down syndrome and the role of this enzyme in a variety of other pathological processes.
Abstract: Down syndrome, the phenotypic expression of human trisomy 21, is presumed to result from a 1.5-fold increase in the expression of the genes on human chromosome 21. As an approach to the development of an animal model for Down syndrome, several strains of transgenic mice that carry the human Cu/Zn-superoxide dismutase gene have been prepared. These animals express the transgene in a manner similar to that of humans, with 0.9- and 0.7-kilobase transcripts in a 1:4 ratio, and synthesize the human enzyme in an active form capable of forming human-mouse enzyme heterodimers. Cu/Zn-superoxide superoxide dismutase activity is increased from 1.6- to 6.0-fold in the brains of four transgenic strains and to an equal or lesser extent in several other tissues. These animals provide a unique system for studying the consequences of increased dosage of the Cu/Zn-superoxide dismutase gene in Down syndrome and the role of this enzyme in a variety of other pathological processes.
476 citations
TL;DR: Plasma GSHPx, like the erythrocyte enzyme, was found to contain approximately four atoms of selenium per mole of protein and is a glycoprotein, and radioimmunoassay showed that less than 0.13% of the antigenically detectable protein is found in the purified GSHpx from plasma.
399 citations
TL;DR: The enzyme seems to be important in assimilation of ammonia under stress conditions such as dark starvation, high temperature, salinity, water stress, environmental pollution, senescence and other abnormalities.
256 citations
01 Jan 1987
TL;DR: Enzyme linked immunosorbent assays (ELISA) provide sensitive methods for detecting either antigens or antibodies in biological fluids, and as such have found numerous applications, particularly in the field of clinical analysis but also in applications to research in molecular biology.
Abstract: Enzyme linked immunosorbent assays (ELISA) provide sensitive methods for detecting either antigens or antibodies in biological fluids, and as such have found numerous applications, particularly in the field of clinical analysis but also in applications to research in molecular biology (see below, p. 94). The first ELISA techniques were described by Engvall and Perlmann1 and Van Weeman and Schuurs2 working independently in 1971. The principles of ELISA are similar to those of radioimmunoassay techniques3 except that instead of using a radiolabel-linked antigen or antibody to quantitate antigen-antibody reactions, an enzyme label is used instead. Depending on the type of assay to be used (see below), either the antigen or antibody is initially linked to an enzyme to give a conjugate which has both enzymatic and immunological reactivity. The assay is then carried out with either antigen or antibody immobilised on a solid surface. This immobilisation allows for simple washing steps that are necessary to separate reacted labelled reagent from unreacted reagent. Having incubated antigen with antibody, and having carried out appropriate washing steps to remove excess reagents, the enzyme activity remaining in the bound fraction is then quantitated by the addition of a non-chromatic substrate which is converted by the linked enzyme to a highly chromatic product.
242 citations
TL;DR: Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing and recognized not only native but also partially deglycosylated protein.
Abstract: Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing. Results of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated the approximate molecular weight of the native protein to be 85-100-KDa. On the basis of a molecular weight of 85-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 1.2 X 10(4) M-1 cm-1. The isoelectric point of the enzyme, as deduced by chromatofocusing, was about 4.5. The purified enzyme is remarkably stable at 0 degree C. Thermal inactivation studies have shown that the enzyme retained 40% of its activity after being subjected to 68 degrees C for 10 minutes, and the enzyme exhibited a broad temperature optimum with maximum catalytic activity at 58 degrees C. The Km of the enzyme for phytate and p-nitrophenylphosphate is about 40 uM and 265 uM, respectively, with an estimated turnover number of the enzyme for phytate of 220 per sec. Enzymatic deglycosylation of phytase by Endoglycosidase H lowered the molecular weight of native enzyme from 85-100-KDa to about 76-KDa; the digested phytase still retained some carbohydrate as judged by positive periodic acid-Schiff reagent staining of the electrophoresed protein. Immunoblotting of the phytase with monoclonal antibody 7H10 raised against purified native enzyme recognized not only native but also partially deglycosylated protein.
241 citations
TL;DR: The availability of purified beta-ARK should greatly facilitate studies of its role in receptor desensitization, as it is effectively inhibited by Zn2+, digitonin and a variety of salts.
240 citations
TL;DR: This is the first demonstration that superoxide dismutase is glucosylated in erythrocytes and that the glucOSylation leads to the inactivation of the enzyme.
207 citations
TL;DR: Regardless of assay method, post-mortem concentrations of glycogen in bovine sternomandibularis muscle were more variable than in longissimus dorsi samples, and marked decreases in muscle glycogen during the first 48 h post slaughter were revealed.
184 citations
TL;DR: Ketanserine and methiothepin, 5-HT receptor antagonists, did partially block the methylenedioxymethamphetamine-induced loss of tryptophan hydroxylase activity, suggesting a possible role for neurotransmitter release in the acute effects of the drug on enzyme activity.
TL;DR: These studies show that certain synthetic heme analogues possess the ability to simultaneously inhibit as well as induce the enzyme heme oxygenase in liver.
Abstract: Tin(IV)-protoporphyrin (Sn-protoporphyrin) potently inhibits heme degradation to bile pigments in vitro and in vivo, a property that confers upon this synthetic compound the ability to suppress a variety of experimentally induced and naturally occurring forms of jaundice in animals and humans. Utilizing rat liver heme oxygenase purified to homogeneity together with appropriate immunoquantitation techniques, we have demonstrated that Sn-protoporphyrin possesses the additional property of potently inducing the synthesis of heme oxygenase protein in liver cells while, concurrently, completely inhibiting the activity of the newly formed enzyme. Substitution of tin for the central iron atom of heme thus leads to the formation of a synthetic heme analogue that regulates heme oxygenase by a dual mechanism, which involves competitive inhibition of the enzyme for the natural substrate heme and simultaneous enhancement of new enzyme synthesis. Cobaltic(III)-protoporphyrin (Co-protoporphyrin) also inhibits heme oxygenase activity in vitro, but unlike Sn-protoporphyrin it greatly enhances the activity of the enzyme in the whole animal. Co-protoporphyrin also acts as an in vivo inhibitor of heme oxygenase; however, its inducing effect on heme oxygenase synthesis is so pronounced as to prevail in vivo over its inhibitory effect on the enzyme. These studies show that certain synthetic heme analogues possess the ability to simultaneously inhibit as well as induce the enzyme heme oxygenase in liver. The net balance between these two actions, as reflected in the rate of heme oxidation activity in the whole animal, appears to be influenced by the nature of the central metal atom of the synthetic metalloporphyrin.
TL;DR: Nitrile hydratase of Pseudomonas chlororaphis B23 was completely stabilized by the addition of 22 mM n-butyric acid and appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis, analytical ultracentrifuge, and double diffusion in agarose.
Abstract: Nitrile hydratase of Pseudomonas chlororaphis B23 was completely stabilized by the addition of 22 mM n-butyric acid. The enzyme was purified from extracts of methacrylamide-induced cells of P. chlororaphis B23 in eight steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis, analytical ultracentrifuge, and double diffusion in agarose. The enzyme has a molecular mass of about 100 kDa and consists of four subunits identical in molecular mass (approximately 25 kDa). The enzyme contained approximately 4 mol iron/mol enzyme. The concentrated solution of highly purified nitrile hydratase had a pronounced greyish green color and exhibited a broad absorption in visible range with a absorption maxima at 720 nm. A loss of enzyme activity occurred in parallel with the disappearance of the absorption in the visible range under a variety of conditions. The enzyme catalyzed stoichiometrically the hydration of nitrile to amide, and no formation of acid and ammonia were detected. The enzyme was active toward various aliphatic nitriles, particularly, nitriles with 3-6 carbon atoms, e.g. propionitrile, n-butyronitrile, acrylonitrile and cyclopropyl cyanide, served as the most suitable substrates.
TL;DR: Specific acetyl xylan esterase activities were identified in enzyme preparations from both organisms and produced alpha-l-arabinofuranosidase activity that was not associated with endoxylanase activity.
Abstract: Extracellular enzyme preparations from Streptomyces flavogriseus and Streptomyces olivochromogenes cultures grown on cellulose contained primarily cellulase activities, but similar preparations from cultures grown on xylan-containing materials possessed high levels of both cellulase and xylanase activities. Growth conditions that gave high endoxylanase levels also resulted in the production of enzymes involved in the hydrolysis of the nonxylose components of xylan. Specific acetyl xylan esterase activities were identified in enzyme preparations from both organisms. Both organisms also produced alpha-l-arabinofuranosidase activity that was not associated with endoxylanase activity. Other activities produced were alpha-l-O-methylglucuronidase and ferulic acid esterase. The latter enzyme was produced only by S. olivochromogenes and is an activity which has not previously been identified as a component of hemicellulase preparations.
TL;DR: The major effect of proteolysis was to enhance enzyme activity in the absence of added activator while greatly decreasing its sensitivity to the allosteric effectors 3-P-glycerate and inorganic phosphate.
Abstract: ADPglucose pyrophosphorylase from developing endosperm tissue of starchy maize (Zea mays) was purified 88-fold to a specific activity of 34 micromoles α-glucose-1-P produced per minute per milligram protein. Rabbit antiserum to purified spinach leaf ADPglucose pyrophosphorylase was able to inhibit pyrophosphorolysis activity of the purified enzyme by up to 90%. The final preparation yielded four major protein staining bands following sodium dodecyl sulfate polyacrylamide gel electrophoresis. When analyzed by Western blot hybridization only the fastest migrating, 54 kilodaltons, protein staining band cross-reacted with affinity purified rabbit antispinach leaf ADPglucose pyrophosphorylase immunoglobulin. The molecular mass of the native enzyme was estimated to be 230 kilodaltons. Thus, maize endosperm ADPglucose pyrophosphorylase appears to be comprised of four subunits. This is in contrast to the respective subunit and native molecular masses of 96 and 400 kilodaltons reported for a preparation of maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). Proteolytic degradation of maize endosperm ADPglucose pyrophosphorylase appears to occur during incubation of crude extracts at 30°C or during the partial purification of the enzyme according to a previously reported procedure (DB Dickinson, J Preiss 1969 Arch Biochem Biophys 130: 119-128). The progressive appearance of a 53 kilodalton antigenic peptide suggested the loss of a 1 kilodalton proteolytic fragment from the 54 kilodalton subunit. The complete conservation of the 54 kilodalton subunit structure following extraction of the enzyme in the presence of phenylmethylsulfonyl fluoride and/or chymostain was observed. The allosteric and catalytic properties of the partially purified proteolytic degraded versus nondegraded enzyme were compared. The major effect of proteolysis was to enhance enzyme activity in the absence of added activator while greatly decreasing its sensitivity to the allosteric effectors 3-P-glycerate and inorganic phosphate.
TL;DR: The test proved to be a simple and reliable method for early diagnosis and follow-up of tuberculous meningitis and showed substantial increases that coincided with the development of complications.
Abstract: We studied the activity of adenosine deaminase in the cerebrospinal fluid of 40 normal controls and 205 patients who were grouped according to disease (tuberculous, viral, and purulent meningitis; neoplasms; stroke; and miscellaneous). The mean enzyme value was clearly higher for the patients with tuberculous meningitis (15.7 ? 4.3 U/liter) than for the other patients (1.4 ? 1.5 U/liter). The sensitivity of the test for diagnosing tuberculous meningitis was 1 and specificity, 0.99. The enzyme activity, as well as progression of the disease, was studied in 32 patients with tuberculous meningitis. A significant rise in levels of enzyme was observed during the first 10 days of therapy, was followed by a gradual decline, and reached normal values after three to four months of treatment. Two patients showed substantial increases that coincided with the development of complications. The test proved to be a simple and reliable method for early diagnosis and follow-up of tuberculous meningitis.
TL;DR: The results suggest that there is no relationship between the inhibition for angiotensin converting enzyme activity and the tannin activity for the four flavonoids.
Abstract: The leaves of the persimmon Diospyros kaki, have been traditionally used for treatment of hypertensive diseases in Japan. We have studied the inhibitory effects of four flavonoids isolated from the leaves of the persimmon on angiotensin-converting enzyme activity. The four flavonoids astragalin [1], kaempferol-3-O-(2"-O-galloyl)-glucoside [2], isoquercitrin [3], and quercetin-3-O-(2"-O-galloyl)-glucoside [4] inhibited the angiotensin-converting enzyme activity in a dose-dependent fashion. Compounds 1-4 produced 67%, 53%, 33%, and 48% inhibition at a concentration of 300 micrograms/ml, respectively. The 50% inhibitory concentrations (IC50) of 1 and 2 for the angiotensin-converting enzyme were 180 micrograms/ml and 280 micrograms/ml, respectively. On the other hand, 2 and 4 were shown to have tannin activities, but 1 and 3 had no tannin activities. These results suggest that there is no relationship between the inhibition for angiotensin converting enzyme activity and the tannin activity for the four flavonoids.
TL;DR: Two possible pathways for scopolamine biosynthesis are discussed in the light of the hydroxyl enzyme and epoxidase activities found in the partially purified preparation of hyoscyamine 6 beta-hydroxylase.
Abstract: Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of l-hyoscyamine to 6 beta-hydroxyhyoscyamine in the biosynthetic pathway leading to scopolamine [Hashimoto, T. & Yamada, Y. (1986) Plant Physiol. 81, 619-625] was purified 310-fold from root cultures of Hyoscyamus niger L. The enzyme has an average Mr of 41,000 as determined by gel filtration on Superose 12 and exhibited maximum activity at pH 7.8 l-Hyoscyamine and 2-oxoglutarate are required for the enzyme activity, with respective Km values of 35 microM and 43 microM. Fe2+, catalase and a reductant such as ascorbate significantly activated the enzyme. 2-Oxoglutarate was not replaced by any of ten other oxo acids tested, nor was Fe2+ by nine other divalent cations tested. The enzyme was inhibited moderately by EDTA, Tiron and various oxo acids and aliphatic dicarboxylic acids, and strongly by nitroblue tetrazolium and divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Several pyridine dicarboxylates and o-dihydroxyphenyl derivatives inhibited the hydroxylase. Pyridine 2,4-dicarboxylate and 3,4-dihydroxybenzoate are competitive inhibitors with respect to 2-oxoglutarate with the respective Ki values of 9 microM and 90 microM. Several alkaloids with structures similar to l-hyoscyamine were hydroxylated by the enzyme at the C-6 position of the tropane moiety. The enzyme preparation also epoxidized 6,7-dehydrohyoscyamine, a hypothetical precursor of scopolamine, to scopolamine (Km 10 microM). This epoxidation reaction required the same co-factors as the hydroxylation reaction and the epoxidase activities were found in the same fractions with the hydroxylase activities during purification. Two possible pathways for scopolamine biosynthesis are discussed in the light of the hydroxylase and epoxidase activities found in the partially purified preparation of hyoscyamine 6 beta-hydroxylase.
TL;DR: It is reported here that the 37-kDa protein is homologous to the enzyme glyceraldehyde-3-phosphate dehydrogenase and the results suggest that the interaction of the enzyme with the red cell membrane is more complex than previously envisioned.
TL;DR: In this article, the effects of herbicide antidotes CGA-92194 (α-[(1,3-dioxolan-2,yl-methoxy)-imino]benzeneacetonitrile), flurazole [phenylmethyl 2-chloro-4-(trifluoromethyl)-5-thiazolecarboxylate], dichlormid (2,2-dichloro- N,N -di-2-propenylacetamide), and naphthalic anhydride (
TL;DR: This is the first report of such an enzyme in Streptomyces spp.
Abstract: The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Isoenzymes were analyzed by polyacrylamide gel electrophoresis and activity staining on the gels. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward alpha-naphthyl acetate and alpha-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed.
TL;DR: The variation in enzyme activity ratios with extraction methods suggests that hydroperoxide lyase is activated by plant injury and thus may function in a wound response.
Abstract: The metabolism of 13-hydroperoxylinolenic acid was examined in protoplasts and homogenates prepared from mature leaves of spinach (Spinacia oleracea L.). Chloroplast membranes were the principal site for metabolism of the compound by at least two highly hydrophobic enzyme systems, hydroperoxide lyase and hydroperoxide dehydrase, the new name for an enzyme system formerly known as hydroperoxide isomerase and hydroperoxide cyclase. Hydroperoxide lyase was most active above pH 7 and could be separated from hydroperoxide dehydrase by anion exchange chromatography. Hydroperoxide dehydrase, measured by the formation of both α-ketol product and 12-oxo-phytodienoic acid, had its optimum activity in the range of pH 5 to 7. Lyase was more active than dehydrase activity when the enzymes were extracted by homogenization. The reverse was true when the enzyme activities were measured in protoplasts, which are isolated by gentle extraction methods. The variation in enzyme activity ratios with extraction methods suggests that hydroperoxide lyase is activated by plant injury and thus may function in a wound response. In the absence of injury, the normal pathway of fatty acid hydroperoxide metabolism is probably by hydroperoxide dehydrase activity. The molecular weights of both the lyase and dehydrase were approximately 220,000, as estimated by gel filtration.
TL;DR: The presence of ascorbate peroxidase in root nodules from nine species of legumes and from Alnus rubra is confirmed and indicates that this enzyme, rather than catalase, is responsible for most H( 2)O(2) removal outside of peroxisomes inRoot nodules.
Abstract: All aerobic biological systems, including N2-fixing root nodules, are subject to O2 toxicity that results from the formation of reactive intermediates such as H2O2 and free radicals of O2. H2O2 may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN, NaN3, CO, and C2H2 were potent inhibitors of activity. Nonphysiological reductants such as guaiacol, o-dianisidine, and pyrogallol functioned as substrates for the enzyme. No activity was detected with NAD(P)H, reduced glutathione, or urate. Ascorbate peroxidation did not follow Michaelis-Menten kinetics. The substrate concentration which resulted in a reaction rate of ½ Vmax was 70 micromolar for ascorbate and 3 micromolar for H2O2. The high affinity of ascorbate peroxidase for H2O2 indicates that this enzyme, rather than catalase, is responsible for most H2O2 removal outside of peroxisomes in root nodules.
TL;DR: Results suggest that in addition to stimulating tyrosinase synthesis, MSH may also promote an increase in the catalytic efficiency of the enzyme.
TL;DR: A genetic library of Streptococcus mutans GS-5, constructed in an Escherichia coli plasmid vector, was screened for cells which could utilize sucrose as the sole carbon and energy source, and pFRU1, containing a 4.2-kilobase pair insert of S. mutans DNA, was shown to confer this phenotype.
Abstract: A genetic library of Streptococcus mutans GS-5, constructed in an Escherichia coli plasmid vector, was screened for cells which could utilize sucrose as the sole carbon and energy source. The recombinant plasmid pFRU1, containing a 4.2-kilobase pair insert of S. mutans DNA, was shown to confer this phenotype. Further characterization of the gene product encoded by pFRU1 revealed that the enzyme was a beta-D-fructosidase with the highest specificity for the beta (2----6)-linked fructan polymer levan. The enzyme could also hydrolyze inulin [beta (2----1)-linked fructan], sucrose, and raffinose with 34, 21, and 12%, respectively, of the activity observed for levan. The gene (designated fruA) appeared to be expressed under its own control in E. coli, as judged by the lack of influence on gene product activity of induction or repression of the beta-galactosidase promoter adjacent to the insertion site on the cloning vector. The protein was purified to homogeneity, as judged by silver staining of purified protein in denaturing and reducing conditions in polyacrylamide gels, from sonic lysate of E. coli, as well as from culture supernatants of S. mutans GS-5 grown in a chemostat at low dilution rate with fructose as the sole carbohydrate source. Both purified proteins had an apparent molecular mass of 140,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were immunologically related and comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as determined by Western blotting with antisera raised against the cloned gene product, and were identical in all physical and biochemical properties tested. The pH optimum of the enzyme acting on fructan polymers was 5.5, with a significant amount of activity remaining at pH 4.0. The optimum pH for sucrose degradation was broader and lower, with a peak at approximately 4.5. Enzyme activity was inhibited almost completely by Hg2+ and Ag2+, inhibited partially by Cu2+, not inhibited by fluoride ion or Tris, and slightly stimulated by Mn2+ and Co2+. Fructan polymers were attacked exohydrolytically by the enzyme, fructose being the only product released. With sufficient time, both levan and inulin were degraded to completion, with no evidence of product inhibition.
TL;DR: The activation by insulin and other findings indicate that the purified enzyme, which seems to belong to a subtype of low Km cAMP phosphodiesterases that is specifically and potently inhibited by cGMP, cilostamide, other OPC derivatives, and certain cardiotonic drugs, is likely to account for the hormone-sensitive particulate cAMPosphodiesterase activity of rat adipocytes.
TL;DR: Inositol polyphosphate 1-phosphatase shows a sigmoidal dependence upon magnesium ion, with 0.3 mM Mg2+ causing half-maximal stimulation and a Hill plot of the data is linear with a value of n = 1.9, suggesting that the enzyme binds magnesium cooperatively.
TL;DR: Peritoneal exudate produced in rat injected with caseinate contained extracellular phospholipase A2, which hydrolyzed phosphatidylethanolamine (PE) and phosph atidylserine (PS) more effectively thanosphatidylcholine (PC) and was purified about 14,000-fold to near homogeneity.
Abstract: Peritoneal exudate produced in rat injected with caseinate contained extracellular phospholipase A2. The activity required Ca2+ ion and had a pH optimum of 9 (Chang, H.W., Kudo, I., Hara, S., Karasawa, K., & Inoue, K. (1986) J. Biochem. 100, 1099-1101). This phospholipase A2 was purified about 14,000-fold to near homogeneity by the sequential use of column chromatography on Sephadex G-75, Toyopearl HW-65, and TSK ODS-120T reverse-phase HPLC. The final preparation showed a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 13,500. The enzyme hydrolyzed phosphatidylethanolamine (PE) and phosphatidylserine (PS) more effectively than phosphatidylcholine (PC). When 1-acyl-2-[1-14C]inoleoyl-sn-glycero-3-phospholipids were used as a substrate, the apparent Km values were 0.027 mM with PE, 0.032 mM with PS, and 0.1 mM with PC, and the Vmax values were 105 mumol/min/mg with PE, 71 mumol/min/mg with PC. The enzyme activity was inhibited by p-bromophenacyl bromide, dithiothreitol, and mepacrine. The amino acid sequence of the NH2-terminal portion and the amino acid composition of the purified enzyme were determined. They were different from those of rat pancreatic phospholipase A2, but very similar to those of phospholipase A2 secreted from rat platelets.
TL;DR: A 20-residue peptide containing the octadecapropeptide of human clotting factor X has now been shown to strongly stimulate the activity of the enzyme toward a noncovalently linked substrate, raising the possibility that the catalytic activity of other enzymes involved in protein processing may be regulated by a portion of their normal substrates.
TL;DR: A polyphosphoinositide phospholipase C has been identified in highly purified plasma membranes from shoots and roots of wheat seedlings and may participate in signal transduction over the plant plasma membrane.