Showing papers on "Enzyme assay published in 1982"

Enzyme activity in soil: Location and a possible role in microbial ecology

Abstract: The activity of any particular enzyme in soil is a composite of activities associated with various biotic and abiotic components, e.g. proliferating cells, latent cells, cell debris, clay minerals, humic colloids and the soil aqueous phase. The location of the enzyme is at least partially determined by such factors as the size and solubility of its substrate, the species of microorganism, and the physical and chemical nature of the soil colloids. However, enzymes may change location with time, for example, many hydrolases are intracellular sensu stricto but are also found associated with cell debris and clay and organic colloids. There are difficulties in quantifying the various activities, but this may be possible by employing different types of assays, the prudent use of controls and the study of crude enzyme extracts from soil. Enzymes bound to clay and humic colloids (the immobilized or accumulated enzyme fraction) have a residual activity not found in enzymes free in the soil aqueous phase. However, the mere adsorption of enzymes to soil surfaces does not guarantee subsequent activity, and it appears that some mechanism of association with the humic polymer offers the best form of protection, yet permits the retention of enzyme activity. The catalytic activity of extracellular enzymes is discussed and a possible relationship between soil microorganisms, exogenous substrates and immobilized enzymes is suggested.

Delta-aminolevulinic acid dehydratase assay.

Abstract: gamma-Aminolevulinic acid (ALA) dehydratase catalyzes the synthesis of porphobilinogen (PBG) from two molecules of ALA. A semimicro method for the colorimetric determination of ALA dehydratase is presented and applied to various tissues. The enzyme activity in adult male rat liver was 2.22 and 1.94 mumol PBG formed/g liver/h for homogenates assayed with or without dithiothreitol, respectively. The assay was linear for at least 2.5 h and for up to 2.5 mg tissue per assay. The Km for ALA was 4.0 X 10(-4)M and the pH optimum was 6.2-6.4. The effects of activators and inhibitors on enzyme activity are discussed.

Metabolism of Thienamycin and Related Carbapenem Antibiotics by the Renal Dipeptidase, Dehydropeptidase-I

Abstract: Thienamycin (THM), the N-formimidoyl thienamycin derivative MK0787, and related carbapenem antibiotics were metabolized extensively in mice, rats, rabbits, dogs, rhesus monkeys, and chimpanzees. Urinary recovery of THM ranged from a low of 5% in dogs to 58% in rhesus monkeys. Renal clearance rates in dogs and chimpanzees were unusually low, less than glomerular filtration rates. The reduction in clearance of THM and MK0787 from plasma of rats and rabbits after ligation of renal arteries indicate that the kidneys are responsible for 35 and 92%, respectively, of metabolic drug clearance. Degradation was detected only in kidney homogenates. The enzyme activity was membrane bound and sensitive to inhibitors of Zn-metalloenzymes such as EDTA. A renal dipeptidase, dehydropeptidase-I (DHP-I), EC 3.4.13.11, was found to be responsible for the metabolism of the THM-class antibiotics, which exhibit a structural homology to dehydropeptides. A parallel increase in specific activity against THM and the substrate of DHP-I, glycyldehydrophenylalanine, was observed during solubilization and purification of the enzyme from porcine and human renal cortex. DHP-I was found to catalyze the hydrolysis of the beta-lactam ring in THM and MK0787. The products of the enzyme reaction were identical by high-powered liquid chromatography to their respective metabolites found in the urine. Nonbasic N-acylated THM and natural N-acylated carbapenems (epithienamycins and olivanic acids) were degraded 4- to 50-fold faster than THM when exposed to the enzymatic hydrolysis of DHP-I. Good correlations were obtained between the increased susceptibility of the carbapenem antibiotics to DHP-I as measured in the in vitro enzyme assay and the generally lower recoveries of active antibiotic in the urine of test animals. Despite this unusual degree of metabolism localized in the kidney, the plasma half-life of MK0787 and its efficacy against experimental systemic infections in animals remain satisfactory.

Purification and properties of inducible penicillin beta-lactamase isolated from Pseudomonas maltophilia.

Abstract: Two types of beta-lactamase were found in the cell-free extract from Pseudomonas maltophilia GN12873. One was an inducible penicillin beta-lactamase, and the other was an inducible cephalosporin beta-lactamase. The purified penicillin beta-lactamase gave a single protein band on polyacrylamide gel electrophoresis. The isoelectric point was 6.9, and the approximate molecular weight was 118,000 by gel filtration and 26,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that this enzyme consisted of four subunits. For the hydrolysis of penicillin G, the optimal pH was 8.0 and the optimal temperature was 35 degrees C. The enzyme activity was inhibited by cephamycin derivatives, carpetimycins A and B, iodine, and HgCl2, but not by clavulanic acid. Furthermore, beta-lactamase activity was almost completely inhibited by EDTA but was recovered by the addition of zinc ion. The enzyme showed a unique substrate profile, hydrolyzing N-formimidoyl thienamycin at a significant rate.

Regulation of synthesis and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by low density lipoprotein and 25-hydroxycholesterol in UT-1 cells

Abstract: UT-1 cells are a clone of Chinese hamster ovary cells that were selected to grow in the presence of compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]. These cells have 100- to 1,000-fold more immunoprecipitable reductase than normal. The enzyme activity is rapidly decreased when low density lipoprotein (LDL) or 25-hydroxycholesterol is added to the culture medium. In this current study, a quantitative immunoprecipitation assay was used to determine whether LDL and 25-hydroxycholesterol inhibit the synthesis or stimulate the degradation of reductase in UT-1 cells. Each of these agents inhibited the incorporation of [35S]methionine into immunoprecipitable reductase by more than 98%. Pulse-chase experiments showed that reductase was degraded with a half-life of 10-13 hr in UT-1 cells and that the rate of degradation of preformed enzyme was increased 3-fold by the addition of either LDL or 25-hydroxycholesterol. We conclude that the predominant mechanism by which LDL and 25-hydroxycholesterol decrease reductase activity in UT-1 cells is a profound suppression of synthesis of the enzyme.

Monoclonal Antibodies That Inhibit Enzyme Activity of 3-Methylcholanthrene-induced Cytochrome P-450

Abstract: Somatic cell hybrids were made between mouse myeloma cells and spleen cells derived from BALB/c mice immunized with liver microsomal cytochrome P-450 purified from rats treated with 3-methylcholanthrene (MC-P-450). Thirty-seven independent hybrid clones among 66 tested produced monoclonal antibodies to the MC-P-450 as measured by radioimmunoassay. More than 10 of the monoclonal antibodies formed were positive for MC-P-450 with respect to protein binding measured by radioimmunoassay, precipitation of the enzyme caused by antibody binding and enzyme aggregation, and inhibition of enzymatic activity. Analysis by gel electrophoresis indicated that a single microsomal protein band interacted with the antibody and that this band comigrated with MC-P-450. These monoclonal antibodies interacted with the major form of cytochrome P-450 from β-naphthoflavone-induced rats as well as with MC-P-450 but did not bind, precipitate, or inhibit the activity of the major form of cytochrome P-450 from phenobarbital-treated rats. The monoclonal antibodies inhibited 7-ethoxycoumarin deethylase and benzo( a )pyrene hydroxylation activity of the purified MC-P-450 with varying degrees, up to 90%, the latter as measured by the aryl hydrocarbon hydroxylase assay for phenol production. Analysis of benzo( a )pyrene metabolism by high-pressure liquid chromatography indicated that the monoclonal antibodies inhibited the enzyme activity of the purified MC-P-450 at all of the positions at which oxidation occurs. The monoclonal antibodies also inhibited both aryl hydrocarbon hydroxylase and 7-ethoxycoumarin deethylase of liver microsomes from 3-methylcholanthrene-treated rats by 70%, indicating that these activities are functions affected by antibody binding to a common or identical antigenic site on cytochrome(s) P-450 which account for 70% of the total activity in these microsomes. Microsomes from control or phenobarbital-treated rats were unaffected, suggesting that their enzyme activity is a function of a cytochrome P-450 other than that sensitive to the MC-P-450 directed antibody and that the latter P-450 is absent in these microsomes. High-pressure liquid chromatographic analysis of antibody inhibition of benzo-( a )pyrene metabolism by microsomes from 3-methylcholanthrene-treated rats showed an inhibition of phenol and diol formation that ranged from 46 to 72%. 1,6-Quinone production was not affected, which suggests that this metabolite was formed by another cytochrome P-450 isozyme or nonenzymatically. The purity, specificity, and potential immortality of their hybridoma cell source will make the monoclonal antibodies extraordinarily useful for the study of substrate and inducer specificity and in the identification and quantitative assay of multiple forms of the cytochrome P-450 and the determination of their content and function in different tissues, species, and individuals.

Lipoprotein lipase secretion by human monocyte-derived macrophages.

Abstract: Human monocyte-derived macrophages in culture produced lipoprotein lipase. Although freshly isolated blood monocytes did not secrete much lipase activity, 1 d in culture was sufficient to trigger measureable enzyme production. During 3 wk in culture, maximal activity was attained after 7 d. At all times, the culture medium contained more enzyme activity than did a serum-heparin eluate or a detergent extract of the cell layer. The lipase activity was stimulated by serum and was inhibited by preincubation with antiserum to bovine lipoprotein lipase or when assayed at a high salt concentration. Furthermore, the enzyme bound to a heparin-Sepharose affinity column at physiological ionic strength. Cells cultured from a subject with primary lipoprotein lipase deficiency secreted no detectable enzyme. Since macrophages are prominent components of atherosclerotic lesions in man, their ability to synthesize and secrete lipoprotein lipase may be important to atherogenesis.

Pyrophosphorylases in Solanum tuberosum: III. PURIFICATION, PHYSICAL, AND CATALYTIC PROPERTIES OF ADPGLUCOSE PYROPHOSPHORYLASE IN POTATOES.

Abstract: ADPglucose pyrophosphorylase from potato (Solanum tuberosum L.) tubers has been purified by hydrophobic chromatography on 3 aminopropyl-sepharose (Seph-C3-NH2). The purified preparation showed two closely associated protein-staining bands that coincided with enzyme activity stains. Only one major protein staining band was observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The subunit molecular weight was determined to be 50,000. The molecular weight of the native enzyme was determined to be 200,000. The enzyme appeared to be a tetramer consisting of subunits of the same molecular weight. The subunit molecular weight of the enzyme is compared with previously reported subunit molecular weights of ADPglucose pyrophosphorylases from spinach leaf, maize endosperm, and various bacteria. ADPglucose synthesis from ATP and glucose 1-P is almost completely dependent on the presence of 3-P-glycerate and is inhibited by inorganic phosphate. The kinetic constants for the substrates and Mg2+ are reported. The enzyme Vmax is stimulated about 1.5- to 3-fold by 3 millimolar DTT. The significance of the activation by 3-P-glycerate and inhibition by inorganic phosphate ADPglucose synthesis catalyzed by the potato tuber enzyme is discussed.

Effect of pH on enzyme stability in soils

Abstract: The pH stability of urease, acid phosphatase, alkaline phosphatase and phosphodiesterase in soils was investigated by first incubating a soil sample at the indicated pH (1–13) for 24 h and then measuring the activity at its optimal pH under standardized conditions. Generally, the decline in enzyme activity in a pH-profile near the optimum pH range was due to a reversible reaction that involved ionization or deionization of acidic or basic groups in the active centre of the enzyme-protein. Irreversible inactivation of the enzyme was particularly evident at the lower and higher ranges of acidic and alkaline conditions. Results showed that the pH stability of soil enzymes was highly dependent on the soils being assayed. The variation among soils may be attributed to the diversity of vegetation, micro-organisms and soil fauna as sources contributing to the enzyme activity and to the protective sites which allowed entrapment of the enzyme within colloidal humus and organo-mineral complexes. Adherence of the enzyme-protein to the humic-clay fractions would allow some resistance to pH denaturation.

Renal Na-K-ATPase: its role in tubular sodium and potassium transport

Abstract: Na-K-ATPase, the enzymatic equivalent of the sodium:potassium pump, is found in large amounts in the kidney, and this organ has figured prominently both as a source for the purification of the enzyme and as a target for the study of its properties. Located on the basolateral aspect of tubule cells, renal Na-K-ATPase plays a key role in the active translocation of Na and K across this membrane as well as in the "secondary active" transport of a number of other solutes. The activity of renal Na-K-ATPase varies in parallel with sustained changes in Na or K transport, indicating the participation of this enzyme in the chronic adaptation of the kidney to altered Na reabsorption or K secretory load. Because of its slow turnover, however, the role of Na-K-ATPase in the modulation of acute changes in cation transport is unclear. Several hormones and vanadate influence renal Na-K-ATPase activity, and their importance as potential physiologic regulators of this enzyme is examined. Most of the information on the renal enzyme has been obtained from studies using homogenates or subcellular fractions thereof, but more recently the development of tubule microdissection and microanalytic methods has made possible the study of Na-K-ATPase in single nephron segments. This approach has opened new possibilities for evaluating the role of this enzyme in kidney function by facilitating correlation of enzyme activity with transport events in the same structure and by enabling us to focus the study of Na-K-ATPase on discrete anatomic subdivisions of the functionally heterogeneous nephron.

Ethylene Biosynthesis and Cadmium Toxicity in Leaf Tissue of Beans (Phaseolus vulgaris L.).

Abstract: Stress ethylene production in bean ( Phaseolus vulgaris L., cv. Taylor9s Horticultural) leaf tissue was stimulated by Cd 2+ at concentrations above 1 micromolar. Cd 2+ -induced ethylene biosynthesis was dependent upon synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. Activity of ACC synthase and ethylene production rate peaked at 8 h of treatment. The subsequent decline in enzyme activity was most likely due to inactivation of the enzyme by Cd 2+ , which inhibited ACC synthase activity in vitro at concentrations as low as 0.1 micromolar. Decrease in ethylene production rate was accompanied by leakage of solutes and increasing inhibition of ACC-dependent ethylene production. Ca 2+ , present during a 2-hour preincubation, reduced the effect of Cd 2+ on leakage and ACC conversion. This suggests that Cd 2+ exerts its toxicity through membrane damage and inactivation of enzymes. The possibility of an indirect stimulation of ethylene biosynthesis through a wound signal from injured cells is discussed.

Aminoglycoside inhibition of a renal phosphatidylinositol phospholipase C.

Abstract: Aminoglycosides are able to inhibit phosphatidylinositol phospholipase C activity in a soluble fraction of rat renal tissue. The Km of the enzyme with respect to phosphatidylinositol is 0.53 mM and Vmax was 0.671 mumol of phosphatidylinositol cleaved per hr per mg of protein. The enzyme is calcium activated and inhibited by ethylene glycol bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid. Streptomycin, amikacin, kanamycin, tobramycin, gentamicin and neomycin inhibit enzyme activity in the same rank order as their ability to produce nephrotoxicity. The 50% inhibitory concentrations ranged from 0.03 mM for neomycin to 0.38 mM for streptomycin. Lineweaver Burk plots indicate competitive inhibition with a possible relation to the calcium activation. It is possible that aminoglycosides may inhibit phosphatidylinositol phospholipase C in vivo and that this inhibition may be related to the nephrotoxicity of aminoglycosides.

Purification of ornithine decarboxylase from kidneys of androgen-treated mice.

Abstract: Ornithine decarboxylase has been purified to homogeneity from kidneys of androgen-treated mice. Such kidneys have an enzyme content 2 orders of magnitude greater than that of other mammalian tissues such as induced rat liver, and only a 10350-fold purification was needed for purification. The enzyme preparation gave a single band on isoelectric focusing and on polyacrylamide gel electrophoresis under native and denaturing conditions. These bands corresponded to the enzyme activity and to the migration of ornithine decarboxylase labeled by reaction with alpha-(difluoromethyl) [5-14C]ornithine, a specific inhibitor. The enzyme has a Mr of about 100 000 and is a dimer of subunit Mr 53 000. The Km for L-ornithine was 75 micron and for pyridoxal phosphate, 0.3 micron. The preparation had a specific activity of 50 mumol of CO2 produced min-1 mg-1 and bound a stoichiometric amount of the irreversible inhibitor, alpha-(difluoromethyl)ornithine (one molecule per subunit). The purified enzyme was unstable even in the presence of 2.5 mM dithiothreitol and 40 micron pyridoxal phosphate unless 0.02% Brij 35 was added. In the presence of this detergent, the enzyme could be stored with little loss of activity.

Direct evidence that cholesterol ester hydrolase from adrenal cortex is the same enzyme as hormone-sensitive lipase from adipose tissue.

Abstract: Several properties of the cytosolic cholesterol ester hydrolase from bovine adrenal cortex were investigated and those properties were compared directly with those of the well-characterised hormone-sensitive lipase, the rate-limiting enzyme in adipose tissue lipolysis. Properties examined included: (a) activity against different substrates; (b) susceptibility to inhibition by NaF, Hg2+ ions and diisopropyl fluorophosphonate; (c) subunit molecular weight as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate; (d) ability to serve as a substrate for cyclic AMP-dependent protein kinase; (e) effect of phosphorylation on enzyme activity; and (f) degradation pattern of polypeptides following limited proteolysis. In all respects the two enzymes exhibited essentially identical characteristics. It is therefore concluded that the same protein, or two very similar proteins, catalyses the hydrolysis of cholesterol esters in adrenal cortex and lipolysis in adipose tissue. The implication of this finding is discussed in relation to the hormonal control of steroidogenesis in adrenal cortex and of lipolysis in adipose tissue.

Method for quantitatively determining a particular substrate catalyzed by a multisubstrate enzyme

Abstract: A method for control of relative enzyme activity for analytical purposes. The activity of a multisubstrate enzyme may be controlled as a function of the electrical potential applied to the enzyme. The enzyme is preferrably incorporated into a thin layer electrochemical cell laminate having exterior membrane layers and an interior enzyme layer. A control electrode located within the enzyme layer applies an electrical potential to the enzyme. An intermediate electron transfer agent may be used to transfer electrons to and from the enzyme and the control electrode.

Electrochemical measuring apparatus comprising enzyme electrode

Abstract: An immobilized enzyme electrode effective in measurement of the substrate concentration of the enzyme and in conversion from enzyme reaction energies into electric energies. The immobilized enzyme, of an oxidase system, such as glucose oxidase, amino acid oxidase, xanthine oxidase or the like and a metal oxide capable of constituting a redox system which is reduced through coupling with these enzyme reactions and is electrochemically oxidized (anodic oxidation) are combined with each other. The use of the enzyme electrode allows the determination quantity of the enzyme substrate as extremely low as approximately 10 -5 to 10 -6 mole/l in concentration.

Achievement of high rates of in vitro synthesis of 1,4-beta-D-glucan: activation by cooperative interaction of the Acetobacter xylinum enzyme system with GTP, polyethylene glycol, and a protein factor.

Abstract: Regulatory properties of a cellulose synthase (UDP-forming)(UDPglucose:1,4-beta-D-glucan 4-beta-D-glucosyltransferase, EC 2.4.1.12) have been demonstrated by using enzyme preparations derived from cells of Acetobacter xylinum. Preparation of a particulate fraction in the presence of 20% (wt/vol) polyethylene glycol-4000 (PEG-4000) yields enzyme with activity 3- to 10-fold higher than that previously reported. The enzyme prepared in this fashion also shows a further marked, specific activation by GTP. The Ka for GTP is 34 microM. Guanosine 5'-[gamma-thio]triphosphate, an analog of GTP, is even more effective than GTP (Ka for guanosine 5'-[gamma-thio]triphosphate = 17 microM). A large number of other nucleotides and nucleotide derivatives were tested with no effect. Enzyme prepared in the absence of PEG-4000 does not respond to GTP because it lacks a protein factor necessary for GTP activation. PEG-4000 promotes the interaction of the protein factor with the enzyme. The factor itself has no synthase activity nor does it stimulate activity of the enzyme in the absence of GTP. In the presence of GTP, protein factor, and PEG-4000, initial rates of enzyme activity 200 times greater than those previously reported can be achieved. Such rates exceed 40% of the in vivo rate of cellulose synthesis from glucose.

Purification and Properties of β-Glucosidase from Aspergillus terreus

Abstract: A β-glucosidase (EC 3.2.1.21) from the fungus Aspergillus terreus was purified to homogeneity as indicated by disc acrylamide gel electrophoresis. Optimal activity was observed at pH 4.8 and 50°C. The β-glucosidase had Km values of 0.78 and 0.40 mM for p-nitrophenyl-β-d-glucopyranoside and cellobiose, respectively. Glucose was a competitive inhibitor, with a Ki of 3.5 mM when p-nitrophenyl-β-d-glucopyranoside was used as the substrate. The specific activity of the enzyme was found to be 210 IU and 215 U per mg of protein on p-nitrophenyl-β-d-glucopyranoside and cellobiose substrates, respectively. Cations, proteases, and enzyme inhibitors had little or no effect on the enzyme activity. The β-glucosidase was found to be a glycoprotein containing 65% carbohydrate by weight. It had a Stokes radius of 5.9 nm and an approximate molecular weight of 275,000. The affinity and specific activity that the isolated β-glucosidase exhibited for cellobiose compared favorably with the values obtained for β-glucosidases from other organisms being studied for use in industrial cellulose saccharification.

Lysyl oxidase: preparation and role in elastin biosynthesis.

Abstract: Publisher Summary This chapter describes purification and assay methods utilized for the study of aortic lysyl oxidase and its activity toward elastin substrates. Lysyl oxidase is mostly insoluble in neutral salt buffers in a variety of connective tissues, although enzyme activity is present in the growth medium of fibroblast cell cultures as a soluble protein. The enzyme can be solubilized from connective tissue sources by buffers containing 4–6 M urea. Most purification schemes that have evolved begin with the ureasolubilized fraction and generally share other purification steps as well. There are two assay methods for lysyl oxidase: tritium release assay and enzyme-coupled spectrophotometric assays. In tritium release assay chick embryo aortas are incubated in organ culture with [6- 3 H]lysine in the presence of BAPN. The salineinsoluble, elastin-rich fraction is isolated from the pulsed aortas after incubation in organ culture. Oxidative deamination of the E-carbon of radioactive peptidyl lysine in this insoluble substrate releases a tritium ion that forms tritiated water by exchange during the assay. Tritiated water is distilled in vacuo from assay mixtures and quantified by liquid scintillation spectrometry. In enzyme-coupled spectrophotometric assays the production of n-butyraldehyde by lysyl oxidase is coupled to the reduction of NAD + by aldehyde dehydrogenase, measuring the change in absorption at 340 nm.

Purification of pyruvate formate-lyase from Streptococcus mutans and its regulatory properties.

Abstract: Pyruvate formate-lyase (EC 2.3.1.54) from Streptococcus mutans strain JC2 was purified in an anaerobic glove box, giving a single band on disk and sodium dodecyl sulfate electrophoresis. This enzyme was immediately inactivated by exposure to the air. Enzyme activity was unstable even when stored anaerobically, but the activity was restored by preincubating the inactivated crude enzyme with S-adenosyl-L-methionine, oxamate, and reduced for ferredoxin or methylviologen. On the other hand, the purified enzyme was not reactivated. Either D-glyceraldehyde 3-phosphate or dihydroxyacetone phosphate strongly inhibited this enzyme. The inhibitory effects of these compounds were largely influenced by enzyme concentration. The inhibition of these triose phosphates in cooperation with the reactivating effect of ferredoxin and the fluctuations of both the enzyme and the triose phosphate levels may efficiently regulate the pyruvate formate-lyase activity in S. mutans in vivo.