Showing papers on "Enzyme assay published in 1975"

Induction of the Polyamine-biosynthetic Enzymes in Mouse Epidermis by Tumor-promoting Agents

Abstract: A single topical application of 10 mg of crotol oil or 17 nmoles of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) resulted in a rapid, transient stimulation of mouse epidermal ornithine decarboxylase activity The activity reached a peak (230-fold greater than control after TPA) at 4 to 5 hr after croton oil or TPA treatment and returned to control level by 12 hr The stimulation of S-adenosyl-L-methionine decarboxylase activity was less pronounced, reaching a peak of activity (6- to 7-fold greater than control) at 9 to 12 hr after TPA or croton oil and slowly declining to control level The stimulation of both enzyme activities was dependent on the dose of TPA applied and correlated well with the promoting ability of these doses on mouse skin Phorbol, the nonpromoting parent alcohol of TPA, did not affect the enzymes activities Cycloheximide pretreatment abolished the increase in enzyme activities after TPA application By measuring the decline of enzyme activity following cycloheximide treatment, enzyme half-lives of 17 and 41 min were obtained for ornithine and S-adenosyl-L-methionine decarboxylase, respectively 5-Azacytidine pretreatment prevented the stimulation of enzyme activities by TPA, while actinomycin D had no effect Cordycepin (3'-deoxyadenosine) partially blocked the rise in enzyme activities

Chinese hamster ovary cells selected for resistance to the cytotoxicity of phytohemagglutinin are deficient in a UDP-N-acetylglucosamine--glycoprotein N-acetylglucosaminyltransferase activity.

Abstract: Several clones of Chinese hamster ovary cells resistant to the cytotoxicity of the phytohemagglutinin from Phaseolus vulgaris show decreased binding of 125I-labeled phytohemagglutinin and contain decreased levels of a UDP-N-acetylglucosamine--glycoprotein N-acetylglucosaminyltransferase (EC 2.4.1.51; UDP-2-acetamido-2-deoxy-D-glucose:glycoprotein 2-acetamido-2-deoxy-D-glucosyltransferase) activity when compared to wild-type cells. The decrease in transferase activity varies from 45% to 96%, depending on the exogenous acceptor used in the enzyme assay. No differences between lectin-resistant and wild-type cells were noted for several other glycosyltransferases. The absence of a particular N-acetylglucosaminyltransferase in the lectin-resistant cells apparently results in defective glycosylation of lectin-binding glycoproteins on the cell surface. A phytohemagglutinin-resistant clone which shows decreased binding of 125I-labeled phytohemagglutinin but does not exhibit the enzyme deficiency has also been isolated.

Demonstration of Enolase Activity Connected to the Brain‐Specific Protein 14‐3‐2

Abstract: An enzyme activity, enolase (2-phospho-D-glycerate hydro-lyase, E.C. 4.2.1.11), was shown to be connected to the brain-specific protein 14.3.2. Specific staining methods were used on immuno precipitates of the protein. The enzyme activity was strongly inhibited by the addition of fluoride in the presence of phosphate. Metal ion binding capacity of the protein was shown by means of an auto radiographic method.

An affinity-column procedure using poly(L-proline) for the purification of prolyl hydroxylase. Purification of the enzyme from chick embryos.

Abstract: An affinity column procedure is reported for purifying prolyl hydroxylase. The procedure is based on the affinity of the enzyme for its competitive polypeptide inhibitor, and involves affinity chromatography in a column containing poly(L-proline) of molecular weight 30000 linked to agarose, and the elution of the enzyme from the column with poly(L-proline) of molecular weight 5700. The enzyme is finally separated from this polyproline by gel filtration. The procedure was employed for purifying prolyl hydroxylase from an ammonium sulphate fraction of chick embryo extract. The recovery of enzyme activity varied in ten enzyme preparations from 50 to 82%, and the purified preparations synthesized from 59.3 to 91.5 mumol hydroxyproline per mg enzyme per h at 37 degrees C with a saturating concentration of (Pro-Pro-Gly)5 as substrate. The enzyme was pure when examined by polyacrylamide gel electrophoresis as a native protein or in the presence of sodium dodecyl sulphate, and the amino acid composition of the enzyme agreed with that reported previously with only minor exceptions. The molecular weight of the enzyme measured by equilibrium in an analytical ultracentrifuge was 240000, indicating that the enzyme had been isolated in the tetramer form.

Multiple molecular forms of purified human erythrocyte acetylcholinesterase.

Abstract: 1 Human erythrocyte acetylcholinesterase was solubilized by Triton X-100 and purified by affinity chromatography to a specific activity of 3800 IU/mg of protein The yield of the purified enzyme was 25–45% 2 Gel filtration on Sepharose 4-B in the presence of Triton X-100 revealed one peak of enzyme activity with a Stokes' radius of 87 nm Density gradient centrifugation in 01% Triton X-100 showed one peak of enzyme activity with an S4 value of 63S 3 Isoelectric focusing in Triton X-100 resolved the enzyme into five molecular forms with isoelectric points of 455, 468, 481, 498 and 518 Upon incubation with neuraminidase the enzyme activity in the first four forms was decreased with a concommitant increase in activity in the form with the higher isoelectric point 4 After removal of excess Triton X-100 on Bio-Gel HTP, polyacrylamide gel electrophoresis showed seven bands of protein and corresponding bands of enzyme activity Density gradient centrifugation of the detergent-depleted enzyme at high ionic strength revealed five multiple molecular forms with S4 values of 63 S, 102 S, 122 S, 142 S and 163 S At low ionic strength, higher aggregates were observed in addition to the other forms Dodecylsulfate-polyacrylamide gel electrophoresis gave one subunit only with an apparent molecular weight of 80000 5 These results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X-100, exists in various forms differing in net charge but of apparently similar molecular dimensions After removal of the detergent, forms with different molecular sizes are observed

Pitfalls in the use of lead nitrate for the histochemical demonstration of adenylate cyclase activity.

Abstract: The biochemistry of the lead histochemical technique for demonstrating adenylate cyclase was studied. The enzyme activity of fat cell plasma membranes, using 5'-adenylyl-imidodiphosphate (AMP-PNP) as substrate, was completely inhibited at 1 times 10- minus 4 M Pb(NO3)2 and yet at 4 times 10- minus 3 M Pb(NO3)2 precipitate could be demonstrated by electron microscopy on both sides of plasma membrane vesicles. No lead-diphosphoimide or lead-phosphate precipitate could be visualized by electron microscopy when the lead was reduced to a level (2 times 10- minus 5 M) which caused only 50% inhibition of the enzyme. A solubility product coefficient of 1 times 10- minus 10 M was found necessary to allow precipitation of lead-phosphate complex in the adenylate cyclase medium. Varying the ratio of substrate or dextran relative to the lead failed to protect the inhibition of the enzyme. Increasing concentrations of beta-mercaptoethanol restored the basal and stimulated activity of adenylate cyclase but also prevented the precipitation reaction. Lead at 2 times 10- minus 3 M caused the nonenzymatic hydrolysis of AMP-PNP, resulting in the production of small but significant quantities of cyclic AMP and substantial amounts of AMP. This hydrolysis was inhibited by alloxan but unaffected by dextran of NaF. The adenylate cyclase activity of pancreatic islet homogenates and of fat pad capillaries was completely inhibited by lead concentrations equal to or less than those used in histochemical studies (Howell, S. L., and M. Whitfield. 1972. J. Histochem. Cytochem. 20:873-879. and Wagner, R. C., P. Kreiner, R. J. Barrnett, and M. W. Bitensky. 1972. Proc. Natl. Acad. Sci. U.S.A. 69:3175-3179.). The present study shows that the lead histochemical method cannot be used for localization of adenylate cyclase because of the inhibition of the enzyme and artifacts produced by high lead concentrations and the inability to produce a visible precipitate at low lead concentrations which only partially inhibit the enzyme.

Enzyme variation in Eimeria species of the chicken

Abstract: A method for the biochemical identification of protozoa belonging to the genus Eimeria is described for the first time. Starch gel electrophoresis of the enzymes lactate dehydrogenase, glucose phosphate isomerase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase from parasite extracts revealed both intra- and inter-species differences when 11 strains representative of 6 species of Eimeria were examined. Oocysts were the most accessible parasite stage for investigation but sporozoites and merozoites of an embryo-adapted strain of E. tenella were also examined for enzyme activity.

Biochemical and histochemical changes in energy supply enzyme pattern of muscles of the rat during old age.

Abstract: Senile muscles of the rat (28–36 months) show loss of overall activity of glycolytic and aerobic enzymes. However, there is a differential loss and shift of enzyme activity pattern in the three types of muscles. The extensor digitorum longus (EDL) and diaphragm show a decrease of ratios of glycolytic to aerobic-oxidative enzymes. This shift to a more oxidative type of metabolism is not observed in the soleus muscle. Decrease of enzyme activities is least marked in the diaphragm muscle. Biochemical analysis shows a trend to levelling out of metabolic differences between the different muscle types. This trend of ‘dedifferentiation’ is most marked when comparing EDL and soleus, least marked when comparing EDL and diaphragm muscle. The histochemical analysis shows a shift from the original mixed to a more uniform pattern of muscle fibres in the EDL and soleus muscle; this levelling-out of differences between enzymatic activities of different muscle fibres is not observed in the diaphragm muscle. Preferential atrophy and loss of ATPase activity in type II muscle fibres in the soleus muscle and the occurrence of ‘type grouping’ are further characteristic features of senile muscle change. The findings show general (i.e. loss of enzyme activities) and differential trends of biochemical and histochemical enzyme changes in different types of muscles.

Rapid isolation of highly active RNA polymerase from Escherichia coli and its subunits by matrix-bound heparin.

Abstract: 1. RNA polymerase from Escherichia coli is selectively and strongly retained by a heparin-substituted agarose and can be eluted therefrom by a neutral buffer containing 0.6 M salt. The method is applicable to relatively crude preparations of the enzyme on a preparative scale giving highly purified RNA polymerase in excellent yield. The enzyme obtained by this procedure shows the highest specific activity so far reported and is pure and enriched in factor sigma as indicated by dodecylsulfate gel electrophoresis. 2. Based on the differential affinity of the subunits of the enzyme for the heparin-carrying gel matrix, a method for separation of alpha, beta' + beta and sigma subunits by application of urea and salt-containing buffers is described. Upon recombination and dialysis with urea-free buffer 40-50% of the enzyme activity is restored.

Regulation of Ribulose 1,5-Diphosphate Carboxylase by Substrates and Other Metabolites Further Evidence for Several Types of Binding Sites

Abstract: Ribulose 1,5-diphosphate carboxylase (RuDPCase, EC 4.1.1.39) isolated from spinach leaves is metabolically regulated at 10 mm Mg 2+ and low CO 2 concentrations by its substrates (RuDP and CO 2 ) and by effectors which include 6-phosphogluconate (6-PGluA), NADPH, and fructose 1,6-diphosphate (FDP), but not fructose 6-phosphate. Physiological concentrations of RuDP severely inhibit the enzyme activity when the enzyme has not been preincubated with HCO 3 − and Mg 2− , and this inactivity persists for 20 minutes or longer after 1 mm HCO 3 − and 10 mm Mg 2+ are added. Maximum activity requires that the preincubation mixture also include either 0.01 mm 6-PGluA or 0.5 mm NADPH. When the enzyme, following preincubation with HCO 3 − and Mg 2+ , is presented with RuDP plus either 6-PGluA or FDP, competitive inhibition is observed with respect to RuDP. The K i value for 6-PGluA is 0.02 mm and the K i value for FDP is 190 μm. NADPH or 3-phosphoglycerate (PGA) at physiological concentrations does not have any effect when presented simultaneously with RuDP. Other studies on the order of addition of substrates and effectors, concentration effects, and kinetics provide additional information that serves as a basis for a proposed model of allosteric regulation combined with competitive inhibition. In this model, there are catalytic sites at which the substrates and 6-PGluA and FDP can bind, and at least four allosteric regulatory sites, which we designate I, A 1 , A 2 , and A 3 . RuDP binds very tightly to site I (in the absence of Mg 2+ or HCO 3 − ), causing a conformational change in the protein to an inactive form which persists for as long as 20 minutes in the subsequent presence of Mg 2+ and 1 mm HCO 3 − . Mg 2+ and HCO 3 − (or CO 2 ) bind to site A 3 (in the absence of RuDP), holding the enzyme in an active form which has a much lower affinity for RuDP at site I, so that when physiological levels of RuDP are then added, only part of the enzyme activity is lost. This active form of the enzyme can bind 6-PGluA or FDP at site A 1 and NADPH at site A 2 during preincubation with Mg 2+ and HCO 3 − . With optimal levels of bound effectors, 6-PGluA or NADPH, enzyme activity is fully maintained, even when RuDP is subsequently added. Without one of these effectors present, addition of RuDP following preincubation reduces enzyme activity to about 40% at the levels of substrates and effectors studied. FDP is a much poorer effector, and this is ascribed to a possible binding of FDP at site I, as well as at site A 1 . The physiological role of this regulation is discussed, particularly with respect to protection of “C-3” plants against oxidation of RuDP to phosphoglycolate.

Properties of Chitin Synthetase from Coprinus cinereus

Abstract: SUMMARY: Preparations of chitin synthetase (EC. 2.4.1.16) from the stipe of the toadstool Coprinus cinereus were characterized. Microsomal fractions had a very high specific activity. The chitin synthetase had been solubilized by treatment with digitonin, with an increase in specific activity and in stability. Optimum conditions for enzyme activity were determined. These preparations needed only a divalent cation and UDP-N-acetylglucosamine for chitin synthesis and showed no primer requirement. The sole glycosyl product was characterized as macro-molecular chitin.

Reversible inactivation of the isocitrate dehydrogenase of Escherichia coli ML308 during growth on acetate.

Abstract: During aerobic growth of Escherichia coli ML308 on acetate as sole carbon source, the apparent synthesis of isocitrate dehydrogenase was repressed relative to cultures on other carbon sources, such as glucose, which do not employ the glyoxylate bypass as an anaplerotic sequence. When cells were removed from an acetate medium, or when compounds were added which made the operation of the glyoxylate bypass unnecessary, the activity of isocitrate dehydrogenase rapidly increased 3- to 4-fold but fell again on restoration to an acetate medium. Changes in activity were rapid and, furthermore, could be demonstrated in the absence of protein synthesis. It is thus improbable that the mechanism involved degradation or de novo synthesis of the enzyme protein. Oxaloacetate and glyoxylate showed concerted inhibition of isocitrate dehydrogenase which could be relieved by dialysis. Because extracts of low enzyme activity, derived from acetate-metabolizing cells, could not be stimulated by dialysis or by addition of a wide range of metabolites, it is unlikely that low molecular weight, freely dissociable effectors were responsible for stimulation or inhibition of activity. Control of isocitrate dehydrogenase permitted the efficient utilization of acetate as sole source of carbon and energy but perserved the capacity of the cell to respond rapidly to an improvement in nutritional conditions. A limited survey showed that the mechanism is common but not universal among strains of E. coli and occurs in at least one strain each of Klebsiella aerogenes, Salmonella typhimurium and Serratia marcescens.

Regulation of ornithine decarboxylase in 3T3 cells by putrescine and spermidine: indirect evidence for translational control.

Abstract: Addition of putrescine of spermidine prevents the increase in ornithine decarboxylase activity in cultures of 3T3 cells brought about by pituitary growth factors and results in a rapid, specific, and reversible reduction of enzyme activity in cultures previously stimulated by the growth factors. These effects are not due to polyamine toxicity and do not require other organic medium components. The amines apparently share a single carrier-mediated transport system in 3T3 cells. Methylglyoxal bis(guanylhydrazone), an inhibitor of spermidine synthesis from putrescine was found to also inhibit uptake of each amine. Studies with this drug indicate that each amine is effective without further metabolism. Since ornithine decarboxylase activity decays more rapidly in the presence of each polyamine after addition of camptothecin, the major locus of amine action appears to be in the cytoplasm. However, direct inhibition of the enzyme in vivo by assimilated amines appears to account for at most a small part of the reduction in activity, a conclusion supported by the inability to recover activity in vitro. Also, neither amine seems to act by accelerating enzyme inactivation. When amines are removed from the medium, the subsequent recovery of enzyme activity is totally prevented by trichodermin, an inhibitor of protein synthesis, but is only slightly reduced by camptothecin. It is suggested that both putrescine and spermidine reduce ornithine decarboxylase activity by selectively inhibiting translation.

Properties, Inducibility, and an Improved Method of Analysis of Aryl Hydrocarbon Hydroxylase in Cultured Human Lymphocytes

Abstract: The properties and inducibility of aryl hydrocarbon hydroxylase (AHH) in cultured human lymphocytes were studied, and a sensitive method of analysis has been developed. In agreement with other reports, peripheral blood lymphocytes per se had no activity and required pretreatment in culture with a mitogen for conversion to lymphoblasts to possess AHH activity. This activity had an absolute requirement for reduced nicotinamide adenine dinucleotide phosphate. Under our conditions of incubation, reduced nicotinamide adenine dinucleotide (1.3 mM) or ethylenediaminetetraacetic acid disodium salt (1 mM), when added to reduced nicotinamide adenine dinucleotide phosphate-fortified incubation mixture, caused about 30% increase in activity and MnCl2 caused an appreciable inhibition. The reaction was linear with the number of cells, with up to 16.8 X 10-6 cells contained in the incubation mixture, and had a pH optimum of 8.5. The enzyme was fairly stable at -70 degrees and retained about 90% of the original activity for 15 days. The enzyme activity in the mitogen-stimulated lymphocyte cultures appeared slowly and reached a maximum at 48 to 72 hr of incubation at 37 degrees, after which it considerably decayed with time. Viability of the cells in culture also decayed considerably after 72 hr and reached about 50% level at 120 hr. The enzyme was inducible with 3-methylcholanthrene and dibenz(a,h)anthracene. Dibenz(a,h)anthracene was a much more potent inducer than 3-methylcholanthrene and evoked a response in cells that would be considered noninducible with 3-methylcholanthrene. By using purified lymphocytes and Roswell Park Memorial Institute Medium 1640, providing larger surface area during culture, harvesting the cells at the time of minimum cell death and maximum AHH activity, and assaying AHH at its pH optimum of 8.5 instead of 7.6, it was possible to enhance the sensitivity of the assay an average of 17-fold.