Showing papers on "Enzyme assay published in 1985"

Glutathione biosynthesis; gamma-glutamylcysteine synthetase from rat kidney.

Abstract: Publisher Summary Glutathione is synthesized by the consecutive actions of γ-glutamylcysteine synthetase and glutathione synthetase. The most highly purified preparations of the γ-glutamylcysteine synthetase enzyme have been obtained from rat kidney and rat and sheep erythrocytes. This chapter presents the isolation procedure of this enzyme. The assay method (ADP formation) is discussed. The enzyme activity is measured in reaction mixtures containing L-glutamate, L-α-aminobutyrate, and ATP by a coupled enzyme procedure in which the rate of formation of ADP, in the presence of pyruvate kinase, lactate dehydrogenase, phosphoenolpyruvate, and NADH, is obtained from the decrease in the absorbance of NADH at 340 nm. The physical properties of the enzyme are reviewed in the chapter. The molecular weight of the rat kidney enzyme is about 104,000. The enzyme dissociates under denaturing conditions to yield two subunits of molecular weights about 73,000 and 27,700. Treatment of the enzyme with dithiothreitol followed by gel electrophoresis under nondenaturing conditions leads to partial dissociation into subunits.

Human indolylamine 2,3-dioxygenase. Its tissue distribution, and characterization of the placental enzyme

Abstract: The presence of indolylamine 2,3-dioxygenase was examined in human subjects by determining its activity with L-tryptophan as substrate. Enzyme activity was detected in various tissues, and was relatively high in the lung, small intestine and placenta. Human indolylamine 2,3-dioxygenase, partially purified from the placenta, had an Mr of about 40 000 by gel filtration and exhibited a single pI of 6.9. The human enzyme required a reducing system, ascorbic acid and Methylene Blue, for maximal activity and was able to oxidize D-tryptophan, 5-hydroxy-L-tryptophan as well as L-tryptophan, but kinetic studies indicated that the best substrate of the enzyme was L-tryptophan.

Purification and properties of carboxypeptidase G2 from Pseudomonas sp. strain RS-16. Use of a novel triazine dye affinity method.

Abstract: A folate-degrading enzyme, carboxypeptidase G2, has been purified on a large scale from Pseudomonas sp. strain RS-16. Homogeneous enzyme was obtained by a three-step procedure involving ion-exchange chromatography and a novel triazine dye (affinity) chromatography step which utilizes Zn2+ to promote adsorption of the enzyme. Enzyme was selectively eluted by the use of a chelating agent (EDTA) and a step change in pH. The enzyme is a dimeric protein (Mr 83000) with two identical subunits of 41800 and contains four atoms of zinc per enzyme molecule, which are required for full activity. The enzyme follows Michaelis-Menten kinetics with Km values of 4.0 μM for folate, 8.0 uM for methotrexate and 34.0 μM for 5-methyltetrahydrofolate, the predominant form of reduced folate found in plasma.

Glucocorticoid Regulation of Alkaline Phosphatase in the Osteoblastic Osteosarcoma Cell Line ROS 17/2.8*

Abstract: Dexamethasone increased alkaline phosphatase levels up to 7-fold in the osteoblast-like rat osteosarcoma cell line ROS 17/2.8. This effect was associated with reduced cell growth and took place over several days in culture. The increase in enzyme activity was dose dependent, (half-maximum near 1 nm, with a hormone specificity suggesting glucocorticoid receptor mediation). Dexamethasone also increased enzyme activity in ROS 2/3 cells, but not in two nonosteoblastic osteosarcoma cell lines, indicating that among these cell lines, the effect is specific for osteoblast-like cells. Moreover, enzyme activity in both control and dexamethasone-treated cells correlated directly with levels of radioimmunoassayable bone-type isoenzyme. Increases in alkaline phosphatase activity in response to dexamethasone were detectable after about 5 h and were inhibited by both actinomycin D and cycloheximide. Thus glucocorticoids appear to increase de novo enzyme synthesis in ROS 17/2.8 cells. Finally, the cAMP-elevating agents ...

Soluble metalloendopeptidase from rat brain: action on enkephalin-containing peptides and other bioactive peptides.

Abstract: A soluble metalloendopeptidase identified in rat brain, preferentially cleaves bonds in peptides having hydrophobic amino acid residues in the P1, P2, and P3' positions. (The nomenclature of T. Schechter and A. Berger is used to describe the interaction between enzyme and substrate. The amino acid residues in the substrate are designated as P1, P2, P3 etc. in the N-terminal direction and P1', P2', P3' etc. in the C-terminal direction from the bond undergoing cleavage. The corresponding subsites in the enzyme are identified by the letter S.) The degradation of a series of biologically active peptides and their affinity toward the enzyme was studied. Dynorphin-(1-8), alpha-neo-endorphin, and beta-neo-endorphin are rapidly hydrolyzed to form leu-enkephalin, whereas bovine adrenal medulla dodecapeptide is hydrolyzed to form met-enkephalin. The enzyme, however, does not cleave a larger precursor molecule of metenkephalin, such as bovine adrenal medulla docosapeptide. Several other bioactive peptides are also cleaved at sites consistent with our previously reported specificity studies. Met- and leu-enkephalin are resistant to hydrolysis. The binding affinity [as expressed by inhibitory constant (Ki) or Michaelis-Menten constant (Km) values] of several bioactive peptides such as dynorphin-(1-8), beta-neo-endorphin, neurotensin, angiotensin I, and bradykinin was found to be in the micromolar range. These peptides were also rapidly hydrolyzed by the enzyme, showing, as a result, high specificity constants (kcat/Km values). The highest enzyme activity was found in brain, testis, and in the anterior and posterior lobes of the pituitary, while the activity in such tissues as spleen, liver, kidney, lung, adrenals, and thyroid amounted to only 10-20% of that found in brain. This distribution of enzyme activity, together with its preference for oligopeptides as substrates, its ability to generate leu- and met-enkephalin from several larger peptide precursors, and its affinity toward several other bioactive peptides, suggests that the enzyme functions in the metabolism of neuropeptides.

N-Acetylation of L-aspartate in the nervous system: differential distribution of a specific enzyme

Abstract: L-Aspartate N-acetyltransferase, a nervous system enzyme that mediates the synthesis of N-acetyl-L-aspartic acid, has been characterized. In the presence of acetyl-CoA, L-aspartate was acetylated 10-fold more efficiently than L-glutamate, and the acetylation of aspartylglutamate was not detectable. Within the nervous system, a 10-fold variation in the enzyme activity was observed, with the brainstem and spinal cord exhibiting the highest activity (10-15 pmol/min/mg tissue) and retina the lowest detectable activity (1-1.5 pmol/min/mg). No enzyme activity was detected in pituitary, heart, liver, or kidney. The enzyme activity was found to be membrane-associated and was solubilized by treatment with Triton X-100.

Human adipose tissue lipoprotein lipase: changes with feeding and relation to postheparin plasma enzyme.

Abstract: An assay procedure using three different methods to recover lipoprotein lipase (LPL) activity from biopsy specimens of human adipose tissue has been developed. Elution of enzyme from small pieces of tissue was performed at 4 and 37 degrees C using a physiological buffer containing heparin and serum. Extraction of enzyme from a tissue homogenate was carried out in the presence of detergent (sodium deoxycholate and Nonidet P-40), which markedly improved the recovery of enzyme activity. It is suggested that elution at 4 degrees C represents extracellular enzyme activity only and therefore theoretically is the closest measure of physiologically active LPL on vascular endothelium, whereas elution at 37 degrees C, in addition, reflects some intracellular enzyme secreted during the incubation period. In female subjects of various relative body weights activity eluted at 37 degrees C as well as detergent-extracted activity were highly correlated with the extracellular activity eluted at 4 degrees C (r = 0.9). Furthermore, all three parameters correlated strongly with LPL activity in post-heparin plasma, suggesting that they are valid indices of physiologically active LPL. The regression of LPL activity in plasma after a 60-min heparin infusion on adipose tissue LPL yielded higher correlation coefficients for activities recorded after elution at 4 and 37 degrees C (r = 0.725 and 0.754, respectively) than for detergent extraction (r = 0.607). Moreover, the increment of adipose tissue LPL after feeding was approximately twice as high for the activity eluted at 4 and 37 degrees C (34%) as for detergent-extracted activity (19%).(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of glutamine synthetase and glutamate dehydrogenase in trifolium subterraneum l. and allium cepa l: effects of mycorrhizal infection and phosphate nutrition

Abstract: Summary Activities of glutamate dehydrogenase and glutamine synthetase were determined using crude extracts of roots and shoots of mycorrhizal and non-mycorrhizal plants of Trifolium subterraneum L. and Allium cepa L., grown at different levels of fertilizer phosphate. Glutamate dehydrogenase activity was low in all tissues [0.1 to 1.6 μmol NAD(P)H oxidized min−1 gFW−1 tissue] and there was no consistent effect of mycorrhizal infection or phosphate nutrition on this activity. Glutamine synthetase (GS) activity (assayed by the transferase method) was in the range 1 to 40/iimol γ-glutamyl hydroxamate produced min−1 gFW−1. In general, activity of this enzyme was low in phosphate-deficient plants and was increased both by mycorrhizal infection and by improved phosphate supply. In T. subterraneum routine assays of GS were done on roots only. The effects of mycorrhizal infection in increasing enzyme activity in roots were similar whether natural soil inoculum (containing a mixture of several mycorrhizal fungi) or inoculum of Glomus mosseae Nichol. & Gerd. was used. Both increased phosphate supply and mycorrhizal infection increased nodulation of clover plants as well as GS activity, so that it was difficult to relate changes in GS activity to the interacting effects of mycorrhizal infection and phosphate nutrition. Onions had low GS activity in uninfected roots, compared with shoots. Again improved phosphate supply resulted in increased enzyme activity in both roots and shoots. However, the patterns of interaction between phosphate supply, P concentration in tissues, mycorrhizal infection and enzyme activity were different in the two tissues. In shoots, as expected, the effects were consistent with an indirect effect of mycorrhizal infection on enzyme activity, via improved P nutrition. In roots there appeared to be a ‘fungal effect’ superimposed on the phosphate effect. This was investigated by manipulating the amount of fungal tissue in mycorrhizal roots via differences in propagule density of G. mosseae in soil. Results were again consistent with the hypothesis that the mycorrhizal fungi contributed GS activity to the symbiotic root system. Fungal structures were separated from roots following digestion in cellulase and pectinase. GS activity was high in fungal tissue from young roots (29 to 31 d), but low in older infections (55 d). The high activity could not have been caused by contamination of fungal tissue by root cells. The digestion technique reduced GS activity in uninfected and infected root segments, so that results obtained with separated fungi are not quantitatively comparable with those obtained from extracts of fresh tissues. We conclude that vesicular-arbuscular mycorrhizal fungi are able to assimilate ammonium via GS. This ability would be important in increased uptake of nitrogen which is an inevitable prerequisite for increased growth following relief of phosphate stress. It is also consistent with the recent findings by others that hyphae of G. mosseae can absorb and translocate 15NH+4

Polyamine catabolism in higher plants: characterization of pyrroline dehydrogenase

Abstract: Both mono- and dicotyledonous species catabolize putrescine to γ-aminobutyric acid (GABA), but by two different pathways. GABA is the major labeled product in pea shoots and oil leaves fed with a 2–4 h pulse of [l,4-14C]-putrescine (Put) or [1,4- tetramethylene-14C]-spermidine (Spd), respectively.In the presence of 1-10μM gabaculine, a specific inhibitor of GABA:pyruvate-transaminase, the label appearing in GABA increase 2 to 7-fold, which indicates that the transmination reaction is a major fate of GABA formed from Put or Spd In vivo. The conversions to GABA were demonstrated in vitro in coupled assays involving diamine oxidase from pea or polyamine oxidase from oat, and pyrroline dehydrogenase (PYRR-DH). The latter enzyme from either pea or oat is strictly NAD-dependent and is specific for pyrroline. The optimal temperature (40–45°C) and pH (7.5–8.0) are similar to those of bacterial PYRR-DH. In all cases the enzyme was inhibited by the NAD analogs thionicotinamide and aminopyridine dinucleotide (0.1—1.0 mM). In addition to pea and oat, PYRR-DH was also detected in corn, barley, soybean and broadbean Di-and polyamine oxidase are released by enzymes which degrade the cell wall, while PYRR-DH remains associated with the protoplast.

Purification of the calmodulin-sensitive adenylate cyclase from bovine cerebral cortex.

Abstract: A calmodulin-sensitive adenylate cyclase was purified 3000-fold from bovine cerebral cortex using DEAE-Sephacel, calmodulin-Sepharose, and two heptanediamine-Sepharose column steps. The purified enzyme activity was stimulated by calmodulin, forskolin, 5'-guanylyl imidodiphosphate, and NaF. The molecular weight of the protein component was estimated as 328 000 with a smaller form of Mr 153 000 obtained in the presence of Mn2+. The most highly purified preparations contained major polypeptides of 150 000, 47 000, and 35 000 daltons on sodium dodecyl sulfate (SDS) gels. Photoaffinity labeling of the preparation with azido[125I]iodocalmodulin gave one product of 170 000 daltons on SDS gels. It is proposed that the catalytic subunit of the calmodulin-sensitive enzyme is 150 000 +/- 10 000 daltons and that the enzyme exists as a complex of one catalytic subunit and the stimulatory guanyl nucleotide regulatory complex. These data are consistent with the previous report that the catalytic subunit of this enzyme has a molecular weight of 150 000 +/- 10 000 [Andreasen, T.J., Heideman, W., Rosenberg, G.B., & Storm, D.R. (1983) Biochemistry 22,2757].

Coenzyme A metabolism

Abstract: The metabolism of coenzyme A and control of its synthesis are reviewed. Pantothenate kinase is an important rate-controlling enzyme in the synthetic pathway of all tissues studied and appears to catalyze the flux-generating reaction of the pathway in cardiac muscle. This enzyme is strongly inhibited by coenzyme A and all of its acyl esters. The cytosolic concentrations of coenzyme A and acetyl coenzyme A in both liver and heart are high enough to totally inhibit pantothenate kinase under all conditions. Free carnitine, but not acetyl carnitine, deinhibits the coenzyme A-inhibited enzyme. Carnitine alone does not increase enzyme activity. Thus changes in the acetyl carnitine-to-carnitine ratio that occur with nutritional states provides a mechanism for regulation of coenzyme A synthetic rates. Changes in the rate of coenzyme A synthesis in liver and heart occurs with fasting, refeeding, and diabetes and in heart muscle with hypertrophy. The pathway and regulation of coenzyme A degradation are not understood.

Hormone processing and membrane-bound proteinases in yeast

Abstract: A search for maturating peptidases of the precursor protein of the mating hormone (pheromone) alpha-factor of Saccharomyces cerevisiae was performed using short model peptides representing those sequences of the precursor protein, where cleavage is thought to occur in vivo. This search was done in a mutant lacking several of the unspecific vacuolar peptidases. The chromogenic peptide Cbz-Tyr-Lys-Arg-4-nitroanilide led to the detection of a membrane-bound enzyme called proteinase yscF. Cleavage of the synthetic peptide derivative occurs after the basic amino acid pair, a proposed signal for hormone processing. Optimum pH for the reaction is 7.2. The enzyme does not cleave after single basic amino acid residues indicating that it is distinct from trypsin-like proteinases. Proteolytic activity is enhanced by Triton X-100. The enzyme is strongly inhibited by EGTA, EDTA and mercurials but insensitive to phenylmethylsulfonyl fluoride. The enzyme activity is strongly dependent on Ca2+ ions. In a mutant (kex2), which accumulates an over-glycosylated alpha-factor precursor, no proteinase yscF activity can be found. Membrane-bound peptidase activity possibly involved in removal of the arginyl and lysyl residues remaining at the carboxy terminus of the alpha-factor pheromone peptide after the initial cut of the precursor molecule could be identified by using the model peptides Cbz-Tyr-Lys-Arg and Cbz-Tyr-Lys.

Immobilized enzyme probes for determining inhibitors

Abstract: The rapid growth in the number of enzymes and the reactions associated with them, ensures that a wide variety of enzyme catalyzed analysis procedures are now available. The development of immobilized enzymes probes (I.E.P.) is related to advances in both immobilization technology and in the improvement of numerous sensing devices. Potentiometric, amperometric, enthalpimetric, and chemiluminescent sensors have been employed as transducers for enzyme electrodes. Most of them have been designed for organic and biological substrates for which simple analyses were not available. Nevertheless, few electrodes have been constructed for inhibitor determination. Other immobilized enzyme systems developed for analysis of enzyme inhibitors will be treated under reactor systems since they are generally used in a flow-through reactor configuration. On the other hand, if some type of electrode is used to measure the rate of an enzyme catalyzed reaction which takes place in solution, this is not a true enzyme electrode. The concept of an enzyme electrode was introduced by Hicks and Updike. The basic features of an I.E.P. are a thin enzyme layer held in close proximity to the active surface...

Persistence of denitrifying enzyme activity in dried soils.

Abstract: The effects of air drying soil on denitrifying enzyme activity, denitrifier numbers, and rates of N gas loss from soil cores were measured. Only 29 and 16% of the initial denitrifying enzyme activity in fresh, near field capacity samples of Maury and Donerail soils, respectively, were lost after 7 days of air drying. The denitrifying activity of bacteria added to soil and activity recently formed in situ were not stable during drying. When dried and moist soil cores were irrigated, evolution of N gas began, and it maximized sooner in the dried cores. This suggests that the persistence of denitrifying enzymes permits accelerated denitrification when dried soils are remoistened. Enzyme activity increased significantly in these waterlogged cores, but fluctuations in enzyme activity were small compared with fluctuations in actual denitrification rate, and enzyme activities were always greater than denitrification rates. Apparent numbers of isolatable denitrifiers (most-probable-number counts) decreased more than enzyme activity as the soils were dried, but after the soils were rewetted, the extent of apparent growth was not consistently related to the magnitude of N loss. We hypothesize that activation-inactivation of existing enzymes by soil O2 is of greater significance in transient denitrification events than is growth of denitrifiers or synthesis of new enzymes.

Peptidyl-Gly cine α-Amidation Activity in Tissues and Serum of the Adult Rat*

Abstract: Bioactive peptides frequently terminate in a carboxyl- terminal a-amide. The tissue distribution of enzymatic activity capable of converting [125I]D-Tyr-Val-Gly into [125I]DTyr- Val-NH2 has been determined. Assay conditions have been established so that enzyme activity can be measured in crude homogenates. In adult male rats, the highest concentrations of activity are found in the anterior and neurointermediate lobes of the pituitary. Lower concentrations of activity are found in the hypothalamus, submandibular glands, and the rest of the brain. Enzyme activity is also easily assayed in serum. Taking into account the mass of each tissue, the submandibular glands and the brain are the major tissue sources of enzymatic activity; serum contains more enzymatic activity than is found in the pituitary gland. In all tissues and in serum, enzyme activity is stimulated by the addition of copper sulfate and ascorbate and is dependent on molecular oxygen. This activity is, therefore, referred to as peptidyl glycine ...

Antigen-induced increase in protein kinase C activity in plasma membrane of mast cells

Abstract: Bridging of cell-bound IgE antibody molecules on colony-stimulating factor-dependent mouse mast cell line (PT-18) cells by multivalent antigen induces phospholipid methylation, a transient rise in intracellular cAMP, intracellular mobilization and uptake of Ca2+, and formation of diacylglycerol followed by histamine release. Exposure of the sensitized cells to antigen also induces a substantial increase in protein kinase C activity in the plasma membrane, which is accompanied by a slight decrease in the enzyme in cytosol. Protein kinase C activity in the membrane fraction reached maximum within 30 sec after antigen challenge and then gradually declined. The increase of the enzyme activity in the membrane could not be explained by a shift of the enzyme from cytosol, and it suggested that bridging of IgE-receptor may induce a modulation of existing enzyme to a state of higher catalytic activity. Phorbol 12-myristate 13-acetate also induced a rapid but persistent increase in protein kinase C activity in the membrane fraction of mast cells. However, the increase in the enzyme activity in the membrane was accompanied by a marked decrease in the enzyme in cytosol.

Control by ethylene of arginine decarboxylase activity in pea seedlings and its implication for hormonal regulation of plant growth.

Abstract: Activity of arginine decarboxylase in etiolated pea seedlings appears 24 hours after seed imbibition, reaches its highest level on the 4th day, and levels off until the 7th day This activity was found in the apical and subapical tissue of the roots and shoots where intensive DNA synthesis occurs Exposure of the seedlings to ethylene greatly reduced the specific activity of this enzyme The inhibition was observed within 30 min of the hormone application, and maximal effect-90% inhibition-after 18 hours Ethylene at physiological concentrations affected the enzyme activity; 50% inhibitory rate was recorded at 012 microliters per liter ethylene and maximal response at 12 microliters per liter Ethylene provoked a 5-fold increase in the K(m) (app) of arginine decarboxylase for its substrate and reduced the V(max) (app) by 10-fold However, the enzyme recovered from the inhibition and regained control activity 7 hours after transferral of the seedlings to ethylene-free atmosphere Reducing the endogenous level of ethylene in the tissue by hypobaric pressure, or by exposure to light, as well as interfering with ethylene action by treatment with silver thiosulfate or 2,5-norbornadiene, caused a gradual increase in the specific activity of arginine decarboxylase in the apical tissue of the etiolated seedlings On the basis of these findings, the possible control of arginine decarboxylase activity by endogenous ethylene, and its implication for the hormone effect on plant growth, are discussed

Characterization of pituitary calcium-activated, phospholipid-dependent protein kinase: redistribution by gonadotropin-releasing hormone

Abstract: We report the presence in the rat pituitary of a calcium-activated, phospholipid-dependent protein kinase (C kinase), originally described by Takai et al. [Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. & Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695]. Enzyme activity is absolutely dependent on the simultaneous presence of Ca2+ and phospholipid--in particular, phosphatidylserine. The presence of small amounts of unsaturated diacylglycerol greatly increases the apparent affinity of the enzyme for Ca2+ and phosphatidylserine. Pituitary C kinase is mostly soluble (70%) and partly particulate (30%). Although the soluble form of the enzyme can be detected in a crude cytosol preparation, the particulate form is detectable only after solubilization and anion-exchange chromatography. Administration of a gonadotropin-releasing hormone (Gn-RH) agonist analog, [D-Ser(But)6]des-Gly10-Gn-RH-N-ethylamide, to ovariectomized rats resulted in elevated serum luteinizing hormone levels (245%) accompanied by a decrease in the cytosolic form of the enzyme (60%) and an increase in the particulate form (300%) after 5 min. This apparent activation of the particulate form seems to result from translocation of a soluble C kinase to the membrane. Several endogenous substrate proteins for C kinase ranging from 16 to 100 kDa were identified in pituitary cytosol. Pituitary C kinase might be involved in signal-transduction mechanisms in Gn-RH action, in particular, and in other hypophysiotropic hormones, in general, which operate by means of stimulation of phosphoinositide turnover during which diacylglycerol is liberated.