Showing papers on "Enzyme assay published in 1979"

Fundamentals of Enzyme Kinetics

Abstract: Basic Principles of Chemical Kinetics Introduction to Enzyme Kinetics "Alternative" Enzymes Practical Aspects of Kinetics Deriving Steady-state Rate Equations Reversible Inhibition and Activation Tight-binding and Irreversible Inhibitors Reactions of More than One Substrate Use of Isotopes for Studying Enzyme Mechanisms Effect of pH on Enzyme Activity Temperature Effects on Enzyme Activity Regulation of Enzyme Activity Multienzyme Systems Fast Reactions Estimation of Kinetic Constants Standards for Reporting Enzymology Data Solutions and Notes to Problems Index

Assay for and enzymatic formation of an ethylene precursor, 1-aminocyclopropane-1-carboxylic acid.

Abstract: A simple and sensitive chemical assay was developed for 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene. The assay is based on the liberation of ethylene from ACC at pH 11.5 in the presence of pyridoxal phosphate, MnCl2 and H2O2. This assay was used to detect ACC in extracts of tomato fruits (Lycopersicon esculentum Mill.) and to measure the activity of a soluble enzyme from tomato fruit that converted S-adenosylmethionine (SAM) to ACC. The enzyme had a Km of 13 μM for SAM, and conversion of SAM to ACC was competitively and reversibly inhibited by aminoethoxyvinylglycine (AVG), an analog of rhizobitoxine. The Ki value for AVG was 0.2 μM. The level of the ACC-forming enzyme activity was positively correlated with the content of ACC and the rate of ethylene formation in wild-type tomatoes of different developmental stages. Mature fruits of the rin (non-ripening) mutant of tomato, which only produce low levels of ethylene, contained much lower levels of ACC and of the ACC-forming enzyme activity than wild-type tomato fruits of comparable age.

A study of the adipose conversion of suspended 3T3 cells by using glycerophosphate dehydrogenase as differentiation marker

Abstract: The adipose conversion of 3T3 cells has been examined in stabilized suspension cultures. In 3T3-F442A cells, glycerophosphate dehydrogenase (sn-glycerol-3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8), a key enzyme in triglyceride synthesis, increases in specific activity by more than 5000-fold and can be used as a sensitive and precise measure of the conversion. The conversion depends on an adipogenic factor present in the serum, and this factor can be assayed by the cellular enzyme response. If the cells are growing at the time they receive the adipogenic factor, the enzyme response does not become detectable until after 3 days, during which the cells first enter a resting state. If the cells are resting at the time they receive the adipogenic factor, the enzyme activity begins to increase in 24 hr or less. Only resting cells seem susceptible to the reprogramming of their differentiated state necessary for the adipose conversion. Once the conversion begins, the increase in enzyme activity is exponential over at least 2 orders of magnitude. When cells in a resting state begin the adipose conversion, their biosynthetic processes are accelerated: the rate of protein synthesis increases, they accumulate cell protein, and they may replicate their DNA and divide. The cell multiplication is not essential for adipose conversion but is a form of clonal selection that increases the proportion of adipose cells relative to nonadipose cells.

Conjugation of Glucose Oxidase from Aspergillus niger and Rabbit Antibodies Using N-Hydroxysuccinimide Ester of N-(4-Carboxycyclohexylmethyl)-Maleimide

Abstract: Glucose oxidase from Aspergillus niger was conjugated with rabbit immunoglobulin G or its monovalent fragments (Fab'). The enzyme was treated with N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide to introduce maleimide groups, which were then allowed to react with thiol groups of reduced IgG or Fab'. More than 40% of immunoglobulin G, Fab' and enzyme used could be conjugated without self-coupling. The enzyme activity decreased about 26 and 15% upon conjugation with immunoglobulin G and Fab', respectively, and the ability of antibody to bind to antigen was well preserved in conjugates. Conjugate preparations purified by gel filtration contained little free form of immunoglobulin G, Fab' or enzyme. Both the cross-link and enzyme activity in Fab' conjugate were stable at pH 6 - 7 at 4°C for at least 6 months.

Purification of rat-liver microsomal UDP-glucuronyltransferase. Separation of two enzyme forms inducible by 3-methylcholanthrene or phenobarbital.

Abstract: Glucuronidation reactions catalysed by rat liver microsomal UDP-glucuronyltransferase are differentially inducible by 3-methylcholanthrene and phenobarbital. To elucidate the molecular basis of this functional heterogeneity the enzyme was purified from livers of rats pretreated with the inducing agents. Using cholate solubilization, chromatography on Bio-Gel A-1.5m and on DEAE-cellulose in the presence of the nonionic detergent Brij 58, two enzyme forms could be separated. Both forms were subsequently purified to apparent homogeneity by affinity chromatography on UDP-hexanolamine Sepharose 4B. 3-Methylcholanthrene-inducible enzyme activity towards 1-naphthol, 4-nitrophenol, 3-hydroxybenzo(a)pyrene and N-hydroxy-2-naphthylamine copurified with one enzyme form (enzyme 1). In contrast phenobarbital-inducible enzyme activity towards morphine, chloramphenicol and 4-hydroxybiphenyl was associated with the other enzyme fraction (enzyme 2). Sodium dodecylsulfate/polyacrylamide gels showed similar molecular weights of 54000 for enzyme 1 and 56000 for enzyme 2. The results suggest the presence of at least two forms of UDP-glucuronyltransferase in rat liver. Factors affecting enzyme activity in purified and membrane-bound states are discussed.

Induction of indoleamine 2,3-dioxygenase in mouse lung during virus infection

Abstract: Indoleamine 2,3-dioxygenase [indoleamine: oxygen 2,3-oxidoreductase (decyclizing)] activity in the supernatant fraction (30,000 X g, 30 min) of mouse lung homogenate increased approximately 120-fold after infection with PR8 influenza virus. Both specific and total enzyme activities started to increase linearly from the 5th day after infection, reached the highest level around the 11th day, and then gradually decreased to normal values in about 3 weeks. Other enzymes in the lung, such as certain lysosomal enzymes and monoamine oxidase, did not change significantly throughout the experiments. The time course of the increase in the enzyme activity was quite different from that of virus replication in the lung (a peak by the 3rd day and persistence until the 9th day) or that of serum antibody content (started to rise on the 9th day). Rather, it appeared to be closely related to the infiltrations of mononuclear and lymphocytic cells. When mice were exposed to a higher dose of virus and did not recuperate, the time course of the increase of the enzyme activity was essentially identical to that seen with a low concentration of virus. A maximum stimulation of the enzyme activity in the lung occurred on the 9th day after infection; the increase was approximately 100-fold. However, serum antibody content was slight and virus titer in the lung remained high.

Purification and properties of a prolyl endopeptidase from rabbit brain.

Abstract: — An enzyme with the specificity of a prolyl endopeptidase was purified about 880-fold from rabbit brain. The enzyme hydrolyzes peptidylprolyl-peptide and peptidylprolyl-amino acid bonds. Several biologically active peptides such as angiotensin, bradykinin, neurotensin. substance P and thyrotropin releasing hormone are degraded by hydrolysis of the bond between the carboxyl group of proline and the adjacent amino acid or ammonia respectively. The enzyme is activated by dithiothreitol and inhibited by heavy metals and thiol blocking agents. The serine protease inhibitor phenylmethanesulfonylfluoride has no effect on activity; however, inhibition was obtained with diisopropylfluorophosphate. Prolyl endopeptidase has a molecular weight of about 66,000 and a pH optimum of about 8.3. A new chromogenic substrate, N-benzyloxycarbonylglycyl-L-prolylsulfamethoxazole, was used for determination of enzyme activity. The substrate is hydrolyzed to N-benzyloxycarbonylglycyl-L-proline and free sulfamethoxazole which can be conveniently determined by a colorimetric procedure.

Catechol-O-methyltransferase: thermolabile enzyme in erythrocytes of subjects homozygous for allele for low activity

Abstract: Low catechol-O-methyltransferase (COMT) activity (less than 8 units per milliliter) in the human erythrocyte is inherited as an autosomal recessive trait (COMTL). The average half-life of COMT in erythrocyte lysates incubated at 48 degrees C was significantly shorter in lysates from three subjects with low enzyme activity than in lysates from three subjects with high enzyme activity (12.5 +/- 0.9 minutes compared with 21.2 +/- 1.4 minutes, P less than .01). When the ratios of COMT activities in lysates heated at 48 degrees C for 15 minutes to enzyme activities in unheated samples were used as a measure of enzyme thermostability in blood samples from 316 randomly selected subjects, the ratios were significantly less for subjects with low enzyme activity than for subjects with higher enzyme activity. The presense of thermolabile COMT in blood of individuals homozygous for COMTL raises the possibility that the locus COMT may represent the structural gene for the human enzyme.

Purification of Streptomyces chromofuscus Phospholipase D by Hydrophobic Affinity Chromatography on Palmitoyl Cellulose

Abstract: Phospholipase D [phosphatidylcholine cholinehydrolase, EC 3.1.4.4] excreted from Streptomyces chromofuscus was purified from the culture supernatant by precipitation with acetone and column chromatographies on palmitoylated gauze (Pal-G), DEAE-cellulose, and Sephadex G-150 with an overall recovery of 46% and 1000-fold increase in specific activity. The purified enzyme preparation showed a single band on sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis. The enzyme had a molecular weight of about 50,000 by gel filtration on Sephadex G-150 or about 57,000 by SDS-polyacrylamide disc gel electrophoresis and an isoelectric point (pI) of pH 5.1 on isoelectric focusing. The enzyme hydrolyses lecithin, lysolecithin, sphingomyelin, and cephalin; the relative reaction velocities and Km's for choline-phospholipids were 87% and 1.43 mM for lecithin, 100% and 1.67 mM for lysolecithin, and 22% and 0.56 mM for sphingomyelin. The enzymatic reaction was optimal at pH 8, and its velocity was appreciably increased by either detergent (Triton X-100, deoxycholate), Ca2+ or both detergent and Ca2+. Diethyl ether stimulated the enzymatic activity by 30%; SDS and EDTA inhibited the activity. Bovine serum albumin, Triton X-100, and lipids (lecithin, lysolecithin, phosphatidic acid, lysophosphatidic acid, palmitic acid, and oleic acid) inhibited adsorption of the purified enzyme onto palmitoyl cellulose (Pal-C) and affected both the enzyme activity and stability: albumin and Triton X-100 increased the activity and enhanced the heat-stability; lysophospholipids decreased the activity but other lipids increased the activity; all the lipids lowered the heat-stability. The enzyme adsorbed on Pal-C was active, although its activity was about one-ninth of that of free enzyme, and was protected from heat-inactivation. Thus this enzyme appears to possess a hydrophobic site distinct from its catalytic site and to be adsorbed onto Pal-C through the hydrophobic site. Albumin, Triton X-100, and lipids seem to bind to the hydrophobic site and to have an appreciable effect on the enzyme activity and stability.

A study of enzyme activities in a dosage series of the long arm of chromosome one in maize

Abstract: The enzyme activity levels of alcohol, malate, isocitrate, glucose-6-phosphate and 6-phosphogluconate dehydrogenases were determined in mature maize scutella in a series of one to four doses of the long arm of chromosome 1, produced by the B-A translocation 1La. Although the Adh structural locus was varied, ADH levels did not exhibit a gene-dosage effect. The levels of G6PDH, 6PGDH and IDH were negatively correlated with the dosage of 1L. MDH was unresponsive. The esterase-8 enzyme, whose structural locus was demonstrated to be elsewhere in the genome, was also negatively correlated with 1L dosage. The portion of the B chromosome involved in the translocation was shown to have no effect on the enzyme levels. Measurements of cell size and hydrolysable DNA per mg dry weight revealed no change in the number of cells through the one, two and three dose series. The topic of enzyme alterations in aneuploids is reviewed.

Proline Oxidase and Water Stress-induced Proline Accumulation in Spinach Leaves.

Abstract: Spinach (Spinacia oleracea L.) leaf discs accumulated free proline when exposed to polyethylene glycol solutions of water potential less than -10 bars. At -20 bars, the accumulation was 11 micromoles per gram original fresh weight in a 24-hour period.When the leaf organelles were separated on a sucrose gradient, a proline oxidase was detected in the mitochondrial fraction. Isolated mitochondria were used for the study of the properties of the enzyme which was assayed by both oxygen uptake measurement and reduction of 2,6-dichlorophenol-indophenol in the presence of phenazine methosulfate. There was a stoichiometry of one-half mole of oxygen uptake per mole of Delta(1)-pyrroline-5-carboxylate production in the enzymic reaction. The enzyme had an optimal activity at pH 8.0 to 8.5 and an apparent K(m) value of 0.028 molar for proline. MgCl(2) and flavin adenine dinucleotide were required for maximal activity. Addition of sucrose, mannitol, or polyethylene glycol to reduce the water potential of the reaction mixture to as low as -20 bars resulted in little inhibition. The enzyme preparation was unable to reduce NAD to NADH, and NAD did not inhibit the enzyme activity. The enzyme preparation reduced cytochrome c in the presence of KCN. Triton X-100 at low concentration strongly inhibited the enzyme activity. The enzyme was apparently linked to the mitochondrial electron transport system. The in vitro activity of the enzyme under optimal assay conditions was high enough to prevent proline accumulation under water stress condition; presumably this activity was restrained in vivo.

Angiotensin-I-converting enzyme and gallium scan in noninvasive evaluation of sarcoidosis.

Abstract: Angiotensin-converting enzyme assays and gallium-scan results were obtained from 27 patients with biopsy-proven, clinically active sarcoidosis. Twenty-three of these patients had elevated converting enzyme levels, and 22 had positive gallium-scan results. Three of four patients with normal or borderline-elevated levels of angiotensin-converting enzyme also had positive gallium-scan results. Of 156 nonsarcoid patients (pulmonary and other diseases), 27 were found to have elevated serum converting enzyme levels, and 25 of these had negative gallium-scan results. These results indicate that the combination of an assay of angiotensin-converting enzyme and gallium scan increases diagnostic specificity from 83% to 99% without sacrificing sensitivity. We conclude that the concurrent use of angiotensin-converting enzyme assay and gallium scan is of value in the diagnosis of sarcoidosis.

Rhamnose-induced propanediol oxidoreductase in Escherichia coli: purification, properties, and comparison with the fucose-induced enzyme.

Abstract: Escherichia coli are capable of growing anaerobically on L-rhamnose as a sole source of carbon and energy and without any exogenous hydrogen acceptor. When grown under such condition, synthesis of a nicotinamide adenine dinucleotide-linked L-lactaldehydepropanediol oxidoreductase is induced. The functioning of this enzyme results in the regeneration of nicotinamide adenine dinucleotide. The enzyme was purified to electrophoretic homogeneity. It has a molecular weight of 76,000, with two subunits that are indistinguishable by electrophoretic mobility. The enzyme reduces L-lactaldehyde to L-1,2-propanediol with reduced nicotinamide adenine dinucleotide as a cofactor. The Km were 0.035 mM L-lactaldehyde and 1.25 mM L-1,2-propanediol, at pH 7.0 and 9.5, respectively. The enzyme acts only on the L-isomers. Strong substrate inhibition was observed with L-1,2-propanediol (above 25 mM) in the dehydrogenase reaction. The enzyme has a pH optimum of 6.5 for the reduction of L-lactaldehyde and of 9.5 for the dehydrogenation of L-1,2-propanediol. The enzyme is, according to the parameters presented in this report, indistinguishable from the propanediol oxidoreductase induced by anaerobic growth on fucose.

Purification and properties of an inducible beta-galactosidase isolated from the yeast Kluyveromyces lactis.

Abstract: beta-Galactosidase (EC 3.2.1.32) was purified 80-fold from the yeast Kluyveromyces lactis induced for this enzyme by growth on lactose. When the purified enzyme was subjected to electrophoresis on an acrylamide gel in the presence of sodium dodecyl sulfate, one protein with an apparent molecular weight of 135,000 was observed. The enzyme has a sedimentation coefficient of 9.6S. This beta-galactosidase and the one from Escherichia coli are not antigenically related. Maximal enzyme activity requires Na+ and Mn2+ and a reducing agent. beta-Galactosidase has Km values of 12 to 17 and 1.6 mM for lactose and o-nitrophenyl-beta-D-galactoside, respectively. The hydrolase and transgalactosylase activities of the enzyme are similar to those of E. coli beta-galactosidase. Images

The Growth of Pseudomonas putida on Chlorinated Aliphatic Acids and its Dehalogenase Activity

Abstract: SUMMARY: Two strains of Pseudomonas putida, S3 and P3, were shown to contain dehalogenase activity against monochloroacetate, dichloroacetate, 2-monochloropropionate and 2,2#-dichloro-propionate but differed markedly in their levels of enzyme activity. Strain S3 had activities of less than 1 μmol substrate converted (mg protein)−1 h−1 and was unable to grow on any of nine chlorinated compounds tested. Strain P3 had enzyme activities 10 to 40 times greater than those of strain S3 but was capable of growth only on 2-monochloropropionate and 2,2#-dichloropropionate. In strain P3, dehalogenase activity was induced by a number of chlorinated compounds other than those that acted as growth substrates. Strain P3 dehalogenase activity dehalogenated C-2 substituted compounds. The evidence of the dehalogenase activity profiles in chemostat cultures and from thermal denaturation experiments suggested that there was more than one dehalogenase enzyme in P. putida strain P3. In crude extract, the enzyme activity was optimal at pH 7.9 to 8.1 and apparent K m values were in the millimolar range for the four major substrates, monochloroacetate, dichloroacetate, 2-monochloropropionate and 2,2#-dichloropropionate.