Showing papers on "Enzyme assay published in 1971"

A sensitive enzymatic assay for dopamine- -hydroxylase.

Abstract: A sensitive and convenient procedure for the determination of dopamine-β-hydroxylase (DBH) activity in homogenates of sympathetically innervated organs is described. The assay uses either phenylethylamine or tyramine as substrate and is based on the sequential conversion of the product of the DBH reaction to a radioactively labeled N-methyl derivative by reaction with partially purified bovine adrenal phenylethanolamine-N-methyltransferase in the presence of S-adenosyl-l-methionine methyl- 14 C. The N-methyl derivatives are separated by solvent extraction and their radioactivity is determined. The identity of the reaction products has been determined chromatographically, and the characteristics of the two enzymatic steps in the assay procedure are defined. Endogenous tissue inhibitors of DBH must be inactivated by cupric ions prior to determination of the enzyme activity. DBE activity in sympathetically innervated tissues of the rat has been measured.

Generation of reduced nicotinamide adenine dinucleotide for nitrate reduction in green leaves.

Abstract: An in vivo assay of nitrate reductase activity was developed by vacuum infiltration of leaf discs or sections with a solution of 02 m KNO 3 (with or without phosphate buffer, pH 75) and incubation of the infiltrated tissue and medium under essentially anaerobic conditions in the dark Nitrite production, for computing enzyme activity, was determined on aliquots of the incubation media, removed at intervals By adding, separately, various metabolites of the glycolytic, pentose phosphate, and citric acid pathways to the infiltrating media, it was possible to use the in vivo assay to determine the prime source of reduced nicotinamide adenine dinucleotide (NADH) required by the cytoplasmically located NADH-specific nitrate reductase It was concluded that sugars that migrate from the chloroplast to the cytoplasm were the prime source of energy and that the oxidation of glyceraldehyde 3-phosphate was ultimately the in vivo source of NADH for nitrate reduction This conclusion was supported by experiments that included: inhibition studies with iodoacetate; in vitro studies that established the presence and functionality of the requisite enzymes; and studies showing the effect of light (photosynthate) and exogenous carbohydrate on loss of endogenous nitrate from plant tissue The level of nitrate reductase activity obtained with the in vitro assay is higher (25- to 20-fold) than with the in vivo assay for most plant species The work done to date would indicate that the in vivo assays are proportional to the in vitro assays with respect to ranking genotypes for nitrate-reducing potential of a given species The in vivo assay is especially useful in studying nitrate assimilation in species like giant ragweed from which only traces of active nitrate reductase can be extracted

Angiotensin-Forming Enzyme in Brain Tissue

Abstract: A renin-like enzyme is present in brain tissue and is independent of kidney and plasma renin. In the presence of homologous substrate it forms angiotensin. Administration of aldosterone significantly decreases this angiotensinforming enzyme activity, while administration of progesterone markedly enhances it.

Enzyme alterations and lipid storage in three variants of tay-sachs disease

Abstract: — In autopsy tissues of 12 cases of Tay-Sachs disease the N-acetyl-β-hexosamini-dase A and B activities were investigated using chromogenic and physiological substrates. In three cases of Tay-Sachs disease, classified as the variant O, the enzyme activities A and B were missing; in eight cases, classified as the variant B, the enzyme activity A was missing. In another case, both enzyme activities wcre shown to be enhanced in brain tissue (‘variant AB’), using a chromogenic substrate. The three enzymic variants showed different glycolipid storage patterns of Tay-Sachs-ganglioside (TSG) and its asialo residue, the trihexosylceramide (THC) in the nervous tissues. Additional storage of kidney globosidc was found in the visceral tissues of the O variant. A decrease of the non-accumulated lipids, especially of those characteristic for myelin, was observed. The quantitative lipid determinations were performed by means of a thin-layer densitometric micromethod (standard deviation 2–5 per cent). Evidence is presented that the different storage patterns result from the corresponding enzyme alterations in the three variants. An essential condition for this statement was the isolation of the storage compounds from Tay-Sachs tissues and their radioactive labelling by the addition of tritium to the double bond in their sphingosine moiety. In a previous investigation it was shown that enzyme A degrades the storage compounds TSG, THC and kidney globoside while enzyme B acts on THC and kidney globoside only. In agreement with this finding, a highly concentrated mixture of both enzymes from normal tissues hydrolyses the main storage compound, the Tay-Sachs-ganglioside. This hydrolysis was reduced when corresponding enzyme preparations from tissues of variants of Tay-Sachs disease (including variant AB) acted on TaySachs ganglioside. Some properties of the N-acetyl-β-D-hexosaminidases from normal and from pathological tissues were determined with chromogenic and physiological substrates. The relationship between the enzymes A and B is discussed.

Gaucher's disease: deficiency of 'acid' -glucosidase and reconstitution of enzyme activity in vitro.

Abstract: The spleen from a patient with adult Gaucher's disease was shown to be deficient in a β-glucosidase (EC 3.2.1.21) isoenzyme that has optimal activity at pH 4.0-4.3, and is stimulated by 0.02% Triton X-100. A mixture of spleen homogenates from a control and from the patient contained β-glucosidase activity equivalent to 2-3 times the theoretical expected activity. The increase in enzyme activity occurred at pH 4.0-4.3; the magnitude of the increase was proportional to the amount of each homogenate added. Two factors, one called factor P from the patient's spleen, the other called factor C from the control spleen, were responsible for a reconstitution of β-glucosidase activity in vitro. Factor P is tentatively identified as an acid glycoprotein.

Procollagen Peptidase: An Enzyme Excising the Coordination Peptides of Procollagen

Abstract: A heritable connective tissue disorder of cattle, dermatosparaxis, is characterized by an extreme fragility of the skin and the presence of additional peptides at the N-terminal extremities of the collagen α chains, p-α1 and p-α2. The existence of an enzyme activity is demonstrated in normal connective tissues that is capable of cleaving these additional N-terminal peptides from dermatosparaxic collagen. The activity is demonstratable with dermatosparaxic collagen in solution, as well as with reconstituted dermatosparaxic collagen fibrils polymerized in vitro. It has a pH optimum of about 7.0 and is inhibited by EDTA and mercaptoethanol. Differences in Km and Vmax values exist depending on the substrate utilized, i.e., p-α1 or p-α2; and the presence of additional amounts of one substrate, p-α1, alters the concentration requirement for the second substrate, p-α2. The product of the excision reaction with p-α1 as substrate is an equimolar amount of normal α1 monomer; the product when p-α2 is substrate is an equimolar amount of normal α2 monomer. The enzyme is present in normal calf skin, tendon, aorta, cartilage, and lung; it can be demonstrated in the skin of rats and humans. The enzyme activity is absent in dermatosparaxic connective tissues, thus suggesting that dermatosparaxis is caused by the absence of a normal enzyme function rather than by the production of an abnormal collagen.

Inactivation of fructose-1,6-diphosphatase by glucose in yeast.

Abstract: Fructose-1,6-diphosphatase was derepressed in Saccharomyces cerevisiae by incubation in media containing non-sugar carbon sources. Addition of glucose to a derepressed culture led to a rapid loss of the measurable activity of the enzyme. Fructose and mannose also produced inactivation, but 2-deoxyglucose was ineffective. Experiments with cycloheximide indicated that the inactivation does not require protein synthesis. It was also shown that the process is not energy-dependent. The reappearance of the enzyme was dependent on an energy source and was prevented by cycloheximide. These results suggest that fructose diphosphatase inactivation is irreversible and that reappearance of enzyme activity implies de novo synthesis. Screening of different genera of yeasts has shown that the inactivation of fructose diphosphatase is a relatively widespread phenomenon.

Effects of colchicine on collagenase in cultures of rheumatoid synovium

Abstract: Rheumatoid synovium, cultured in vitro in media not enriched with serum, synthesized and released a specific collagenase. This enzyme appeared in measurable quantities only after three or more days of culture. Enzyme activity correlated well with hydroxyproline content in the culture media, indicating that extracellular matrix collagen in synovial explants was being degraded. Collagenase activity was found to be 2 to 10 times greater when colchicine (0.1 μg/ml) was added to the culture media. This stimulation of enzyme was dependent upon cells which could metabolize glucose and synthesize RNA; it was not dependent upon continued addition of colchicine to fresh culture media. Colchicine was found to depress 14CO2 production from glucose-6-14C, whereas lactate formation and 14CO2 evolution from glucose-1-14C were less markedly depressed. Mitotic figures were not seen in cells of tissue cultures in the absence of serum, nor was mitotic arrest observed in colchicine-treated tissue. The effect of colchicine was not mediated by decreased degradation of the enzyme in culture nor by stimulation of release of stored enzyme, but was associated with an increase in radioactive leucine incorporation into several proteins synthesized by the cell, including collagenase.

The purification and properties of microsomal palmitoyl-coenzyme A synthetase

Abstract: The isolation and purification of palmitoyl-CoA synthetase from rat liver microsomes is described. Several methods suitable for enzyme assay are described. The general properties and kinetic parameters of the purified enzyme were determined and are discussed in relationship to microsomal fatty acid activation.

Inducible heme oxygenase in the kidney: a model for the homeostatic control of hemoglobin catabolism

Abstract: We have recently identified and characterized NADPH-dependent microsomal heme oxygenase as the major enzymatic mechanism for the conversion of hemoglobin-heme to bilirubin-IXalpha in vivo. Enzyme activity is highest in tissues normally involved in red cell breakdown, that is, spleen, liver, and bone marrow, but it usually is negligible in the kidney. However, renal heme oxygenase activity may be transiently increased 30- to 100-fold following hemoglobinemia that exceeded the plasma haptoglobin-binding capacity and consequently resulted in hemoglobinuria. Maximal stimulation of enzyme activity in rats is reached 6-16 hr following a single intravenous injection of 30 mg of hemoglobin per 100 g body weight; activity returns to basal levels after about 48 hr. At peak level, total enzyme activity in the kidneys exceeds that of the spleen or liver. Cyclohexamide, puromycin, or actinomycin D, given just before, or within a few hours after, a single intravenous injection of hemoglobin minimizes or prevents the rise in renal enzyme activity; this suggests that the increase in enzyme activity is dependent on continued synthesis of ribonucleic acid and protein. The apparent biological half-life of renal heme oxygenase is about 6 hr. These observations indicate that functional adaptation of renal heme oxygenase activity reflects enzyme induction either directly or indirectly by the substrate, hemoglobin. Filtered rather than plasma hemoglobin appears to regulate renal heme oxygenase activity. Thus, stabilization of plasma hemoglobin in its tetrameric form with bis (N-maleimidomethyl) ether, which diminishes its glomerular filtration and retards it plasma clearance, results in reduced enzyme stimulation in the kidney, but enhances its activity in the liver. These findings suggest that the enzyme is localized in the tubular epithelial cells rather than in the glomeruli and is activated by luminal hemoglobin. Direct support for this concept was obtained by the demonstration of heme oxygenase activity in renal tubules isolated from rabbits that had been injected with hemoglobin.

The enzymatic degradation of hemoglobin to bile pigments by macrophages.

Abstract: Recent studies have identified and characterized the enzymatic mechanism by which hemoglobin-heme is converted to bilirubin. Under physiologic conditions the enzyme system, microsomal heme-oxygenase, is most active in the spleen followed by the liver and bone marrow, all of which are tissues that normally are involved in the sequestration and metabolism of red cells. Indirect evidence suggested that the reticuloendothelial system is important in this process. To test this hypothesis, conversion of heme to bilirubin was studied in macrophages obtained by chemical or immunological means from the peritoneal cavity or from the lungs of rodents. Homogenates of pure populations of these cells were devoid of heme-oxygenase activity, unless before harvesting the macrophages had been exposed to methemalbumin, microcrystalline hemin, or hemoglobin in vivo. In macrophages exposed to heme pigments, the specific activity of heme-oxygenase was far in excess of that in the spleen or liver. Enzyme activity was also present in the granulomatous tissue surrounding subcutaneous hematomas. The heme-oxygenase system in macrophages resembles that in the spleen and liver in that it is localized in the microsomal fraction, has an absolute requirement for molecular oxygen and NADPH, is inhibited by carbon monoxide, and has a similar K(m). These findings indicate that cells of the reticuloendothelial system, presumably including the Kupffer cells of the liver and the macrophages of the spleen, possess the enzymatic machinery for converting hemoglobin-heme to bilirubin. The reaction is a mixed function oxidation, probably involving cytochrome P450 as the terminal oxidase. Enzyme activity in macrophages is capable of regulatory adaptation in response to substrate loads. In the standard assay system for the enzyme, disappearance of heme always was in excess of the amount of bilirubin formed, suggesting the simultaneous presence of alternate routes of heme degradation not involving bilirubin as an end product or intermediate.

Host-Pathogen Interactions: II. Parameters Affecting Polysaccharide-degrading Enzyme Secretion by Colletotrichum lindemuthianum Grown in Culture.

Abstract: The effect of a number of physiological variables on the secretion of polysaccharide-degrading enzymes by culture-grown Colletotrichum lindemuthianum (Saccardo and Magnus) Scribner was determined. The number of spores used to inoculate cultures grown on isolated bean hypocotyl cell walls affects the time after inoculation at which enzyme secretion occurs, but has no significant effect on the maximal amount of enzyme ultimately secreted. Cell walls isolated from bean leaves, first internodes, or hypocotyls (susceptible to C. lindemuthianum infection), when used as carbon source for C. lindemuthianum growth, stimulate the fungus to secrete more alpha-galactosidase than do cell walls isolated from roots (resistant to infection). The concentration of carbon source used for fungal growth determines the final level of enzyme activity in the culture fluid. The level of enzyme secretion is not proportional to fungal growth; rather, enzyme secretion is induced. Maximal alpha-galactosidase activity in the culture medium is found when the concentration of cell walls used as carbon source is 1% or greater. A higher concentration of cell walls is necessary for maximal alpha-arabinosidase activity. Galactose, when used as the carbon source, stimulates alpha-galactosidase secretion but, at comparable concentrations, is less effective in doing so than are cell walls. Polysaccharide-degrading enzymes are secreted by C. lindemuthianum at different times during growth of the pathogen on isolated cell walls. Pectinase and alpha-arabinosidase are secreted first, followed by beta-xylosidase and cellulase, then beta-glucosidase, and, finally, alpha-galactosidase.

Conversion of N6-(Δ2-Isopentenyl)adenosine to Adenosine by Enzyme Activity in Tobacco Tissue

Abstract: By using [8-(14)C]-N(6)-(Delta(2)-isopentenyl)adenosine as a substrate, we detected the presence of enzyme activity in a crude extract of tobacco tissue that converts this cytokinin into adenosine. The extract also contains strong hydrolase activity that converts adenosine to adenine.

Genetic Aspects of Increase in Rat Liver Aldehyde Dehydrogenase Induced by Phenobarbital

Abstract: In the supernatant fraction of homogenized rat liver, the activity of aldehyde dehydrogenase that is dependent on nicotinamide adenine dinucleotide (E.C. 1.2.1.3) is increased up to tenfold after administration of phenobarbital for 3 days. The effect is genetically controlled and is inherited as an autosomal dominant characteristic. The mechanism is apparently unrelated to other druginduced increases in enzyme activity such as that which occurs in the hepatic microsomal systems for drug metabolism.

pH dependence of the activity of beta-galactosidase from Escherichia coli.

Abstract: The “specific reactivity” of β-galactosidase from Escherichia coli for different substrates has been studied at the optimal pH value The pH dependence of the enzyme activity has been reinvestigated in highly controlled conditions with respect to Mg2+ and Na+ concentrations and ionic strength The different kinetic parameters kcat, km and the kcat/km ratio have been determined with o-nitrophenyl-β-d-galactoside and o-nitrophenyl-β-d-fucoside, at different pH values between 516 and 10, for both Mg2+-enzyme and Mg2+-free enzyme, since a residual activity has been found in the absence of Mg2+ The activity of both types of enzyme is controlled by a protonated group which ionizes in the alkaline range and by at least one unprotonated group which ionizes in the acidic range This latter group has a pK smaller than 6 in both types of enzyme; it is plausible to assume that this group is a carboxylate In the alkaline range, the pK of the involved group shifts from about 65 in Mg2+-free enzyme to 84 in the Mg2+ enzyme Either the substrate or the Mg binding induces this shift in the ionization of the group The β-galactosidase catalyzed reactions proceed via two intermediary complexes For o-nitrophenyl-β-d-galactoside substrate the limiting process is not the same for the Mg2+ enzyme and for the Mg2+-free enzyme

Thyroid Hormone Induced Changes in the Enzyme Activity Pattern of Energy-Supplying Metabolism of Fast (Whit) Slow (Red), and Heart Muscle of the Rat

Abstract: Influeces of thyroid hormones onthe activity levels of glyucogen phosphorylase, hexokinase, trisoephosphate dehydrogenease, critrate synthase, 3-hydroxyacyl-CoA dehydrogenease, mitochondrial glycerolphosphate dehydrogenase and the contnt of cytochrome a3 were examined in a “white” (m. rectus femoris), “red” (m. soleus) and heart muscle of male rats. Of the enzymes examined, glycerolphosphate dehydrogenase and hexokinase, revaled most significant increases, with comparable extent and simultaneous time of onset. The pattern o reaction was, however, different in the three types of muscle. In white muscle, an increase of hexokinase, but not of glycerolphosphate dehydrogenase was found. In red muscle, the two enzymes increased in parallel and in heart muscle the increase in glycerolphosphate dehydrogenase was the largest and exceeded by far that of hexokinse. In skeletal muscles it could be shown that the increase of hexokinase activity was limited to hexokinase-isoenzyme II, hexokinase I being unaffected. Histochemical staining for glycerolphosphate dehydrogenase revealed that the activity of this enzyme is predominalty demonstrable in the subsarcolemmal mitochondria. The increased activities of hexokinase in the skeletal muscles and of glycerophosphate dehydrogenase in red and heart muscle are interpreted as a coordinate process of a “long-term metabolic regulation” which is related to an elvelatd aerobic carbohydrate catabolism of muscle tissue and to an enhanced rate of basal metabolism.