Showing papers on "Enzyme assay published in 1973"

Familial Hypercholesterolemia: Identification of a Defect in the Regulation of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity Associated with Overproduction of Cholesterol

Abstract: The homozygous form of the autosomal dominant disorder, familial hypercholesterolemia, is characterized by the presence in children of profound hypercholesterolemia, cutaneous planar xanthomas, and rapidly progressive coronary vascular disease that usually results in death before age 30 years. Cultured skin fibroblasts from three unrelated subjects with this disorder showed 40- to 60-fold higher activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), the rate-controlling enzyme in cholesterol biosynthesis, when compared with fibroblasts of seven control subjects. Enhanced enzyme activity resulted from a complete absence of normal feedback suppression by low-density lipoproteins, which led to a marked overproduction of cholesterol by the mutant cells. The demonstration of apparently identical kinetic properties of the reductase activity of control and mutant cells, coupled with the evidence that this enzyme is normally regulated not by allosteric effectors but by alterations in enzyme synthesis and degradation, suggests that the primary genetic abnormality does not involve the structural gene for the enzyme itself, but a hitherto unidentified gene whose product is necessary for mediation of feedback control by lipoproteins. The fibroblasts of two obligate heterozygotes, the parents of one of the homozygotes, showed a pattern of enzyme regulation intermediate between that of controls and homozygotes.

Regulation of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Human Fibroblasts by Lipoproteins

Abstract: The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), the rate-limiting enzyme of hepatic cholesterol biosynthesis, is suppressed in human fibroblasts cultured in the presence of serum. This enzyme activity increases by more than 10-fold after the removal of serum from the medium. The rise in enzyme activity requires de novo protein synthesis and is not accompanied by changes in the activities of several other cellular enzymes. The factor responsible for the suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured fibroblasts is present in the sera of at least four mammalian species, and in human serum it is found in the low-density lipoproteins. Human high-density lipoproteins, very low-density lipoproteins from chicken egg yolk, and the fraction of human serum containing no lipoproteins do not suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Immunochemical demonstration of increased accumulation of tyrosine hydroxylase protein in sympathetic ganglia and adrenal medulla elicited by reserpine.

Abstract: Chronic administration of reserpine to rats increases, in sympathetic ganglia and adrenal medulla, the activity of tyrosine hydroxylase (EC 1.14.3.x), the enzyme catalyzing the rate-limiting step in the biosynthesis of catecholamines. Immunochemical titration of the enzyme in both adrenal gland and innervated superior cervical ganglia demonstrates that enhanced enzyme activity is entirely attributable to accumulation of more specific enzyme protein and not activation of preexistent enzyme molecules.

The unlabeled antibody enzyme method of immunohistochemistry molecular immunocytochemistry of antibodies on the erythrocyte surface

Abstract: The number of discrete unit stains on the surface of sheep erythrocytes enumerated by electron microscopy after preembedding staining using 125I antierythrocyte antibody (anti-E) and the unlabeled antibody enzyme method was correlated with enzyme activity and radioactivity of lysed cell suspensions. Purified anti-E was applied in a concentration range resulting in the binding of 10-2,600,000 molecules/cell. Sheep serum antirabbit immunoglobulin and peroxidase-antiperoxidase complex were used in excess. With a new light-scattering assay for peroxidase the sensitivity of measurement of enzyme activity on suspensions of cell stromata exceeded that of radioactivity assay allowing measurement of as little as 5 x 10–13 mmole antibody/ml. Discrete unit stains on cell sections were visualized to 1.5 x 104 anti-E/cell. Above this number, staining occurred in patches of increasing size but was still discontinuous to ∼3 x 104 anti-E bound/cell. Above 2-3 x 104 anti-E bound/cell continuous staining of the membrane wa...

Phenol hydroxylase from yeast

Abstract: An enzyme, able to carry out an NADPH-dependent hydroxylation of monocyclic phenols, was purified 20–30-fold from Trichosporon cutaneum grown on phenol or resorcinol as a major carbon source The purified enzyme was homogeneous upon analytical disc electrophoresis The enzyme is a bright-yellow protein with an absorption spectrum typical of flavoproteins Its molecular weight is 148000, and its estimated FAD content is approximately 1 mole per mole enzyme The purified enzyme has essentially the same broad substrate specificity as crude preparations In addition to phenol it also hydroxylates the three isomeric diphenols and a number of phenol derivatives to their corresponding o-diols Km values for the three substrates of the holoenzyme are all of the order 10 μM : Km (phenol) = 18 μM, Km(NADPH) = 71 μM The absorption spectrum of the holoenzyme is modified in the presence of phenol FAD could be resolved from the purified enzyme by preparative disc electrophoresis Reassembly of the holoenzyme required SH-groups The enzyme activity is unaffected by chelators of iron and copper but it is inhibited by heavy metals The inhibition by p-chloromercuribenzoate is readily reversed by dithiothreitol Among oxidizing agents, hydrogen peroxide and peroxidase depressed the enzyme activity, whereas catalase was without effect Among reducing agents ascorbate depressed enzyme activity Sodium dithionite and sodium borohydride bleached the enzyme with concomitant loss of activity After reduction with dithionite, the enzyme was rapidly re-oxidized, re-gaining its original activity After reduction with borohydride re-oxidation was very slow However, the enzyme could be re-activated by incubation with FAD The enzyme is very sensitive to inorganic salts, nitrogen bases and detergents Chloride is quite deleterious whereas phosphate seems to stabilize the enzyme

The nature of the electrophoretically separable multiple forms of rat liver monoamine oxidase.

Abstract: 1. Treatment of a partly purified preparation of rat liver monoamine oxidase with the chaotropic agent sodium perchlorate caused the enzyme to migrate as a single band of activity of polyacrylamide-gel electrophoresis, whereas the untreated enzyme separated into a number of bands. 2. Treatment with the chaotropic agent caused no loss of enzyme activity towards benzylamine, dopamine or tyramine. 3. The activities of the untreated preparation towards different substrates were inhibited to different extents by heat treatment and by some inhibitors. No such differences could be detected after the enzyme preparation had been treated with sodium perchlorate. 4. Lipid material, which could be separated by gel filtration, was liberated from the enzyme preparation by sodium perchlorate treatment. 5. The molecular weight of the treated enzyme was found to be 380000±38000. 6. Perchlorate treatment altered the solubility of the enzyme. 7. A continuous assay method for monoamine oxidase is described.

Serum Dopamine-β-Hydroxylase Activity: Sibling-Sibling Correlation

Abstract: Dopamine-β-hydroxylase activity is released into the blood with catecholamines from the adrenal medulla and sympathetic nerves. This enzyme activity has been measured in the blood of 317 normal children and 227 normal adults. A significant sibling-sibling correlation of serum dopamine-β-hydroxylase values was found in the 94 sibling pairs tested. Frequency distributions of serum enzyme values in both children and adults suggest the existence of two populations with regard to serum activity of this enzyme.

Insulin and the regulation of adipose tissue acetyl-coenzyme A carboxylase.

Abstract: Rat epididymal fat-pads were incubated for 30min with glucose (2mg/ml) in the presence or absence of insulin. A twofold or greater increase in acetyl-CoA carboxylase activity was observed in extracts from insulin-treated tissue provided that assays were performed rapidly after extraction. This effect of insulin was evident whether or not extracts were prepared with albumin, and was not noticeably diminished by the presence of citrate or albumin or both in the assay. Incubation of extracts before assay led to activation of acetyl-CoA carboxylase and a marked diminution in the insulin effect. The enzyme in extracts was very sensitive to reversible inhibition by palmitoyl-CoA even in the presence of albumin (10mg/ml); inhibition persisted on dilution of enzyme and inhibitor. It is suggested that the observed activation of acetyl-CoA carboxylase by insulin may reflect changes in enzyme activity in the fat-cell resulting from the reduction of long-chain fatty-acyl-CoA that occurs in the presence of insulin. Activation of the enzyme with loss of the insulin effect on incubation of the extracts may be due to the slow dissociation of long-chain fatty acyl-CoA from the enzyme.

Ionic properties of an essential histidine residue in pig heart lactate dehydrogenase

Abstract: 1. Pig heart lactate dehydrogenase is inhibited by addition of one equivalent of diethyl pyrocarbonate. The inhibition is due to the acylation of a unique histidine residue which is 10-fold more reactive than free histidine. No other amino acid side chains are modified. 2. The carbethoxyhistidine residue slowly decomposes and the enzyme activity reappears. 3. The essential histidine residue is only slightly protected by the presence of NADH but is completely protected when substrate and substrate analogues bind to the enzyme-NADH complex. The protection is interpreted in terms of a model in which substrates can only bind to the enzyme in which the histidine residue is protonated and is thus not available for reaction with the acylating agent. 4. The apparent pK(a) of the histidine residue in the apoenzyme is 6.8+/-0.2. In the enzyme-NADH complex it is 6.7+/-0.2. 5. Acylated enzyme binds NADH with unchanged affinity. The enzyme is inhibited because substrates and substrate analogues cannot bind at the acylated histidine residue in the enzyme-NADH complex.

Adrenergic-adenosine 3',5'-monophosphate regulation of serotonin N-acetyltransferase activity and the temporal relationship of serotonin N-acetyltransferase activity synthesis of 3H-N-acetylserotonin and 3H-melatonin in the cultured rat pineal gland.

Abstract: The regulation of serotonin N-acetyltransferase actiyity has been studied in cultured pineal glands. Addition of l-norepinephrine (NE) to cultures produces a 10- to 30-fold increase in enzyme activity after six hours of treatment. There is a plateau in enzyme activity for another six hours followed by a spontaneous decrease in enzyme activity to base-line values. NE can stimulate the enzyme over a range of 10-4 to 10-9 M concentratioin. Exposure to NE does not have to be constant for a long-term response. Exposure for as little as 15 minutes to NE produces about a 50% maximal response at six hours. The initial increase in N-acetyltransferase activity is blocked by cycloheximide. Cycloheximide treatment after a gland has been stimulated with NE does not cause a precipitous fall in enzyme activity but does prevent a further increase. Aliphatic amines. indoleamines. an imidazolamine and tyramine are ineffective in stimulating the enzyme. The relative potency of the compounds that stimulate enzyme activity is: l-NE DL-isoproterenol > l-epinephrine > DL-octopamine > d-norepinephrine > 3,4-dihydroxyphenylamine > l-3,4-dihydroxyphenylalanine. The stimulation of enzyme activity is blocked by propranolol and is enhanced by phentolamine. This indicates that the receptor involved is a beta adrenergic receptor and that the response to beta adrenergic stimulation can be influenced by an alpha adrenergic mechanism. The effects of NE on N-acetyltransferase activity are mimicked by dibutyryl cyclic adenosine monophosphate (AMP), which is more effective than theophylline. Cyclic AMP is only slightly effective. The effects of dibutyryl cyclic AMP are blocked by cycloheximide but not by cyclic AMP. The stimulation of N-acetvltransferase by either NE or dibutyryl cyclic AMP is coincident in time and magnitude with the stimulation of the total production of 3H-N-acetylserotonin and 3H-melatonin by glands incubated with 3H-tryptophan. This study describes a striking number of similarities between the factors regulating pineal adenyl cyclase, cyclic AMP, radiolabeled melatonin production from radiolabeled tryptophan, and serotonin N-acetyl-transferase activity in cultured pineal glands. The findings add support to the hypothesis that melatonin production is regulated by the amount of N-acetylserotonin made available for O-methylation and that N-acetylserotonin production by serotonin N-acetyl-transferase is regulated by an adrenergic-cyclic AMP mechanism.

Properties of a 5′-nucleotidase purified from mouse liver plasma membranes

Abstract: 1. Extraction of a mouse liver plasma-membrane fraction with a detergent buffer, N-dodecylsarcosinate-Tris buffer (sarcosyl-Tris buffer), solubilized 90% of the protein and 70% of the 5'-nucleotidase activity. 2. The proteins of the sarcosyl-Tris buffer extract were fractionated by a rate-zonal centrifugation in a sucrose-detergent gradient. The major protein peak sedimented ahead of phospholipids, which mainly remained in the overlay. Glycoproteins were separated ahead of the protein peak. 3. The 5'-nucleotidase activity peak was associated with 5% of the protein applied to the gradient, and contained relatively few protein bands. 4. The 5'-nucleotidase was purified further by gel filtration on Sepharose and Sephadex columns equilibrated with sarcosyl-Tris buffer, to give a single glycoprotein band on sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. The purified enzyme was lipid-free. 5. Electrophoresis in polyacrylamide gels in sarcosyl-Tris buffers showed that the enzymic activity was coincident with the protein band. 6. The molecular weight suggested for the enzyme activity by gel filtration or centrifugation in sucrose gradients was 140000-150000. Sometimes, a minor enzyme peak of lower molecular weight was obtained. 7. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate indicated that as the polyacrylamide concentration was increased from 5 to 15%, the apparent molecular weight of the enzyme decreased from 130000 to 90000. 8. The evidence that 5'-nucleotidase is composed of two active and similar, if not identical, glycoprotein subunits and the role of detergent in effecting the separation of membrane proteins and glycoproteins are discussed. 9. Substrate requirements, pH optima and the nature of inhibition by an analogue of adenosine diphosphate are reported.

Microbial Assimilation of Methanol

Abstract: 1 An NAD-dependent alcohol dehydrogenase has been isolated from the yeast Candida boidinii grown on methanol. A study of the properties of the enzyme has shown that it is very similar to the alcohol: NAD oxidoreductase from baker's yeast. The alcohol dehydrogenase from Candida boidinii does not catalyze the oxidation of methanol and is constitutively formed. 2 A mutant, 48, has been isolated which is unable to grow on methanol as sole carbon and energy source. This strain lacks the methanol-oxidizing enzyme but not NAD-dependent alcohol dehydrogenase. 3 Cells grown on glucose or ethanol do not contain the methanol-oxidizing enzyme. During adaptation from glucose to methanol, enzyme activity appears before growth on methanol begins. 4 The methanol-oxidizing enzyme has been partially purified. It catalyzes the oxidation of methanol to formaldehyde and hydrogen peroxide, and is independent of the addition of a hydrogen acceptor. The prosthetic group of this enzyme is FAD. The molecular weight was calculated to be 600000, one subunit has a molecular weight of 74000. The optima of pH and temperature for enzyme activity are 7.5–9.5 and 30°C, respectively. It is unstable in acidic pH. The enzyme is not specific for methanol. It also oxidizes lower primary alcohols. The Km value for methanol is 2.0 mM and that for ethanol 4.5 mM.

Multiple N-acetyltransferases and drug metabolism. Tissue distribution, characterization and significance of mammalian N-acetyltransferase.

Abstract: Investigations in the rabbit have indicated the existence of more than one N-acetyltransferase (EC 2315) At least two enzymes, possibly isoenzymes, were partially characterized The enzymes differed in their tissue distribution, substrate specificity, stability and pH characteristics One of the enzymes was primarily associated with liver and gut and catalysed the acetylation of a wide range of drugs and foreign compounds, eg isoniazid, p-aminobenzoic acid, sulphamethazine and sulphadiazine The activity of this enzyme corresponded to the well-characterized polymorphic trait of isoniazid acetylation, and determined whether individuals were classified as either ;rapid' or ;slow' acetylators Another enzyme activity found in extrahepatic tissues readily catalysed the acetylation of p-aminobenzoic acid but was much less active towards isoniazid and sulphamethazine The activity of this enzyme remained relatively constant from individual to individual Studies in vitro and in vivo with both ;rapid' and ;slow' acetylator rabbits revealed that, for certain substrates, extrahepatic N-acetyltransferase contributes significantly to the total acetylating capacity of the individual The possible significance and applicability of these findings to drug metabolism and acetylation polymorphism in man is discussed

New methods for binding enzyme molecules into a water‐insoluble matrix: Properties after insolubilization

Abstract: New methods of crosslinking enzyme molecules inside a matrix with or without an inactive protein are described. Enzyme activity yields range between 30 and 80% of the activity of the untreated preparations. Even fragile enzyme systems, for instance those using mobile cofactors, can be efficiently immobilized. Increased resistance towards heat denaturation and proteolysis results.

Nonreversible d-Glyceraldehyde 3-Phosphate Dehydrogenase of Plant Tissues.

Abstract: Preparations of TPN-linked nonreversible d-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.9), free of TPN-linked reversible d-glyceraldehyde 3-phosphate dehydrogenase, have been obtained from green shoots, etiolated shoots, and cotyledons of pea (Pisum sativum), cotyledons of peanut (Arachis hypogea), and leaves of maize (Zea mays). The properties of the enzyme were similar from each of these sources: the Km values for d-glyceraldehyde 3-phosphate and TPN were about 20 μm and 3 μm, respectively. The enzyme activity was inhibited by l-glyceraldehyde 3-phosphate, d-erythrose 4-phosphate, and phosphohydroxypyruvate. Activity was found predominantly in photosynthetic and gluconeogenic tissues of higher plants. A light-induced, phytochrome-mediated increase of enzyme activity in a photosynthetic tissue (pea shoots) was demonstrated. Appearance of enzyme activity in a gluconeogenic tissue (endosperm of castor bean, Ricinus communis) coincided with the conversion of fat to carbohydrate during germination. In photosynthetic tissue, the enzyme is located outside the chloroplast, and at in vivo levels of triose-phosphates and pyridine nucleotides, the activity is probably greater than that of DPN-linked reversible d-glyceraldehyde 3-phosphate dehydrogenase. Several possible roles for the enzyme in plant carbohydrate metabolism are considered.

Regulation of phosphoenolpyruvate carboxylase of Zea mays by metabolites.

Abstract: Crude preparations of phosphoenolpyruvate carboxylase obtained from aetiolated seedlings of Zea mays are unstable but can be stabilized with glycerol. At the pH optimum of 8.3, the K(m) value for phosphoenolpyruvate is 80mum. When assayed at 30 degrees C, the enzyme shows normal Michaelis-Menten kinetics, but when assayed at 45 degrees C sigmoid kinetics are exhibited. At pH7.0 the enzyme is inhibited by a number of dicarboxylic acids and by glutamate and aspartate. d and l forms of the hydroxy acids and amino acids are inhibitory and the kinetics approximate to simple non-competitive inhibition. The same compounds produce less inhibition at pH7.6 than at pH7.0 and the kinetics of inhibition are more complex. The enzyme is activated by P(i), by SO(4) (2-) and by a number of sugar phosphates. Maximum activation occurs at acid pH values, where enzyme activity is lowest. The enzyme is activated by AMP and inhibited by ADP and ATP so that the response to energy charge is of the R type and is thus at variance with Atkinson's (1968) concept of energy charge. The physiological significance of the response to metabolites is discussed.

Purification and properties of a mouse liver plasma-membrane glycoprotein hydrolysing nucleotide pyrophosphate and phosphodiester bonds.

Abstract: 1. A mouse liver plasma-membrane preparation was solubilized in an N-dodecylsarcosinate-Tris buffer, pH7.8, and the proteins and glycoproteins were separated by a rate-zonal centrifugation in sucrose-detergent gradients. 2. A peak of alkaline phosphodiesterase activity which sedimented ahead of the 5'-nucleotidase peak was associated with a major glycoprotein component of the plasma membrane. 3. The phosphodiesterase activity was then purified further by gel filtration and gave a single glycoprotein band after electrophoresis on polyacrylamide gels. The apparent molecular weight of the polypeptide at pH7.4 and 8.9 was 128000-130000 and was independent of the polyacrylamide concentration. Electrophoresis in gels containing deoxycholate showed that the protein band was coincident with phosphodiesterase activity. 4. After two-dimensional immunoelectrophoresis, with agarose containing rabbit anti-(mouse plasma-membrane) antiserum as second dimension, the enzyme showed one component which was also coincident with the phosphodiesterase activity. 5. An amino acid composition of the glycoprotein is presented. Carbohydrate analysis indicated the presence of glucosamine, neutral sugars and sialic acid. 6. The enzyme was also a nucleotide pyrophosphatase, as shown by a similar enrichment during purification of activity towards ATP, NAD(+), UDP-galactose and UDP-N-acetylglucosamine. The phosphodiesterase activity, measured by using dTMP p-nitrophenyl ester as substrate, was competitively inhibited by nucleotide pyrophosphate substrates. The enzyme showed little or no activity towards RNA, cyclic AMP, AMP, ADP and glycerylphosphorylcholine. 7. The significance of this enzyme activity in the plasma membrane is discussed.

Homogeneous Enzyme Immunoassay for Opiates in Urine

Abstract: An assay technique, homogeneous enzyme immunoassay, is described for the quantitative determination of morphine derivatives in biological fluids. An enzyme is labeled with morphine. When the enzyme-labeled morphine is bound by antimorphine antibodies, the enzyme is rendered inactive. Free morphine competes with enzyme—morphine for antibody binding sites preventing inhibition of enzyme activity. Enzyme activity is thus directly related to the concentration of free morphine. See PDF for Equation Where the enzyme used is lysozyme, the assay can detect 0.5 µg of morphine per milliliter of urine with >95% confidence (CV, 5.0% at 0.5 µg/ml morphine). The technique gave nearly twice as many confirmed positives as did thin-layer chromatography in a comparative study of urines from a methadone maintenance program. The immediacy of the results (assay time: <1 min) permits use of the assay in hospital emergency rooms, law-enforcement facilities, and methadone treatment programs.

Nitrate Reductase Activity and Polyribosomal Content of Corn (Zea mays L.) Having Low Leaf Water Potentials

Abstract: Desiccation of 8- to 13-day-old seedlings, achieved by withholding nutrient solution from the vermiculite root medium, caused a reduction in nitrate reductase activity of the leaf tissue. Activity declined when leaf water potentials decreased below −2 bars and was 25% of the control at a leaf water potential of −13 bars. Experiments were conducted to determine whether the decrease in nitrate reductase activity was due to reduced levels of nitrate in the tissue, direct inactivation of the enzyme by low leaf water potentials, or to changes in rates of synthesis or decay of the enzyme. Although tissue nitrate content decreased with the onset of desiccation, it did not continue to decline with tissue desiccation and loss of enzyme activity. Nitrate reductase activity recovered when the plants were rewatered with nitrate-free medium, suggesting that the nitrate in the plant was adequate for high nitrate reductase activity. The rate of decay of nitrate reductase activity from desiccated tissue was essentially identical to that of the control, in vivo or in vitro , regardless of the rapidity of desiccation of the tissue. Direct inactivation of the enzyme by the low water potentials was not detected. Polyribosomal content of the tissue declined with the decrease in water potential, prior to the decline in nitrate reductase activity. Changes in ribosomal profiles occurred during desiccation, regardless of whether the tissue had been excised or not and whether desiccation was rapid or slow. Reduction in polyribosomal content did not appear to be associated with changes in ribonuclease activity. Nitrate reductase activity and the polyribosomal content of the tissue recovered upon rewatering, following the recovery in water potential. The increase in polyribosomal content preceded the increase in nitrate reductase activity. Recovery of enzyme activity was prevented by cycloheximide. Based on these results, it appears that nitrate reductase activity was affected primarily by a decrease in the rate of enzyme synthesis at low leaf water potentials.