Showing papers on "Enzyme assay published in 1974"

Regulation of the pentose phosphate cycle

Abstract: 1. A search was made for mechanisms which may exert a ;fine' control of the glucose 6-phosphate dehydrogenase reaction in rat liver, the rate-limiting step of the oxidative pentose phosphate cycle. 2. The glucose 6-phosphate dehydrogenase reaction is expected to go virtually to completion because the primary product (6-phosphogluconate lactone) is rapidly hydrolysed and the equilibrium of the joint dehydrogenase and lactonase reactions is in favour of virtually complete formation of phosphogluconate. However, the reaction does not go to completion, because glucose 6-phosphate dehydrogenase is inhibited by NADPH (Neglein & Haas, 1935). 3. Measurements of the inhibition (which is competitive with NADP(+)) show that at physiological concentrations of free NADP(+) and free NADPH the enzyme is almost completely inhibited. This indicates that the regulation of the enzyme activity is a matter of de-inhibition. 4. Among over 100 cell constituents tested only GSSG and AMP counteracted the inhibition by NADPH; only GSSG was highly effective at concentrations that may be taken to occur physiologically. 5. The effect of GSSG was not due to the GSSG reductase activity of liver extracts, because under the test conditions the activity of this enzyme was very weak, and complete inhibition of the reductase by Zn(2+) did not abolish the GSSG effect. 6. Preincubation of the enzyme preparation with GSSG in the presence of Mg(2+) and NADP(+) before the addition of glucose 6-phosphate and NADPH much increased the GSSG effect. 7. Dialysis of liver extracts and purification of glucose 6-phosphate dehydrogenase abolished the GSSG effect, indicating the participation of a cofactor in the action of GSSG. 8. The cofactor removed by dialysis or purification is very unstable. The cofactor could be separated from glucose 6-phosphate dehydrogenase by ultrafiltration of liver homogenates. Some properties of the cofactor are described. 9. The hypothesis that GSSG exerts a fine control of the pentose phosphate cycle by counteracting the NADPH inhibition of glucose 6-phosphate dehydrogenase is discussed.

A cytokinin oxidase in Zea mays.

Abstract: An enzyme activity that catalyzes conversion of N6-(Δ2-isopentenyl)adenosine (i6Ado) to adenosine was detected in cultured tobacco tissue by Paces et al. (1971) (Plant Physiol. 48, 775–778). Purification and characteristics of this enzyme in corn kernels have been studied. Only the naturally occurring cytokinins i6Ado and ribosylzeatin serve as substrates; the nucleoside or free base works equally as well. The reaction requires oxygen. An unstable intermediate appears to be the primary reaction product. This product decomposes to adenosine.The enzyme activity is greatest at pH 5–7. It is independent of added magnesium. The molecular weight of the enzyme is about 88 000. The activity of the enzyme in corn kernels increases from the time of pollination to about 21 days.A nucleoside hydrolase is isolated with i6Ado oxidase. The activities can be partially separated by G-150 gel filtration. The hydrolase is less stable than the i6Ado oxidase and frozen preparations lose their activity after 2 months, whereas ...

Purification and properties of cytidine deaminase from normal and leukemic granulocytes.

Abstract: Cytidine deaminase, an enzyme that catalyses the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara-C) and 5-azacytidine (5-azaC), has been partially purified from normal and leukemic human granulocytes. The purification procedure included heat precipitation at 70°C, ammonium sulfate precipitation, calcium phosphate gel ion exchange, and Sephadex G-150 gel filtration. The enzyme has mol wt 51,000, isoelectric pH of 4.8, and maximum activity over a broad pH range of 5-9.5. The enzyme is stabilized by the presence of the sulfhydryl reagent, dithiothreitol. Cytidine deaminase from normal human granulocytes has a greater affinity for its physiologic substrate cytidine (Km = 1.1 × 10−5 M) than for ara-C (8.8 × 10−5 M) or 5-azaC (4.3 × 10−4 M). Halogenated analogues such as 5-fluorocytidine and 5-bromo-2′-deoxycytidine also exhibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine and deoxycytidine. No activity was observed with nucleotides or deoxynucleotides. The relative maximum velocity of the enzyme for cytidine and its nucleoside analogues remained constant during purification, indicating that a single enzyme was responsible for deamination of these substrates. Tetrahydrouridine (THU) was found to be a strong competitive inhibitor of partially purified deaminase with a Ki of 5.4 × 10−8 M. The biochemical properties of partially purified preparations of cytidine deaminase from normal and leukemic cells were compared with respect to isoelectric pH, molecular weight, and substrate and inhibitor kinetic parameters, and no differences were observed. However, normal circulating granulocytes contained a significantly greater concentration of cytidine deaminase (3.52±1.86 × 103/mg protein) than chronic myelocytic leukemia (CML) cells (1.40±0.70 × 103 U/mg protein) or acute myelocytic leukemia (AML) cells (0.19±0.17 × 103 U/mg protein). To explain these differences in enzyme levels in leukemic versus normal cells, the changes in cytidine deaminase levels associated with maturation of normal granulocytes were studied in normal human bone marrow. Myeloid precursors obtained from bone marrow aspirates were separated into mature and immature fractions by Ficoll density centrifugation. Deaminase activity in lysates of mature granulocytes was 3.55-14.2 times greater than the activity found in the lysates of immature cells. Decreased enzyme activity was also found in immature myeloid cells from a patient with CML as compared to mature granulocytes from the same patient. These observations support the conclusion that the greater specific activity of cytidine deaminase in normal mature granulocytes as compared to leukemic cells is related to the process of granulocyte maturation rather than a specific enzymatic defect in leukemic cells.

The kinetics of coupled enzyme reactions. Applications to the assay of glucokinase, with glucose 6-phosphate dehydrogenase as coupling enzyme

Abstract: 1. A theory is developed to account for the kinetics of coupled-enzyme reactions without assuming that the second reaction follows first-order kinetics. 2. A simple procedure is described for applying the theory to the practical design of enzyme assays. 3. The validity of the theory is confirmed for the assay of glucokinase with glucose 6-phosphate dehydrogenase as coupling enzyme. 4. The possibility of extending the theory to three or more coupled reactions is discussed.

Dependence of specific metabolism of benzo(a)pyrene on the inducer of hydroxylase activity.

Abstract: Summary Adult male Sprague-Dawley rats were treated with polycyclic hydrocarbons, phenobarbital (PB), or barbital and the induced hydroxylase enzymes in liver and lung were studied in terms of the specific metabolites of benzo ( a ) pyrene (BP) that were produced in an in vitro incubation system. The metabolite profile produced by phenobarbital- or barbital-induced enzymes differed from that found with the hydrocarbon-induced liver enzymes in that only the production of 4,5-dihydro-4,5-dihydroxy-BP and 3-hydroxy-BP was increased over the control level, while the production of all identified metabolites was increased when the hydrocarbons were used as inducers. An exception was benzo ( e ) pyrene, which was essentially inactive as an inducer for either liver or lung enzymes. PB and barbital did not increase enzyme activity in the lung, but BP, 3-methylcholanthrene (3-MC), and 1,2-benzanthracene did. When the induced liver enzymes were tested for their ability to activate BP to a bacterial mutagen, it was found that the PB-induced enzymes were much more active than the 3-MC-induced enzymes when low levels (less than 1 mg/ml) of enzyme protein were present in the incubation mixtures. At higher levels of enzyme protein, the 3-MC-induced enzymes were more active. Addition of an epoxide hydrase inhibitor, 1,2-epoxy-3,3,3-trichloropropane, to the test mixtures with PB-induced enzymes greatly increased the mutagenic activity but only slightly increased the activity obtained with the 3-MC-induced enzymes. The conclusions were: ( a ) PB and polycyclic hydrocarbons induce enzymes in rat liver with different specificities toward BP; ( b ) the induced enzymes activated BP to a form that was mutagenic for bacteria; ( c ) inhibition of the epoxide hydrase induced by PB allowed the accumulation of a form of BP that was mutagenic for bacteria, probably the 4,5-epoxide.

Kinetics of Allosteric Enzymes

Abstract: Within the cell, hundreds of different chemical reactions go on simultaneously, each requiring different substrates and producing different products. This array of rea,ctions must be carefully regulated in order to maintain the orderly function of a cell. Since all these chemical reactions are catalyzed by enzymes, the regulation of biological processes ultimately requires the regulation of enzymatic activity. Such enzyme regulation can be achieved at two levels : the control of enzyme quantity and the control of enzyme activity (of a given quantity of enzyme). Both induction and repression of enzyme synthesis serve to control the enzyme quantity at the genetic level. In addition, the amount of enzyme can be regulated by the degradation of enzyme molecules. The biochemical and genetic hypotheses involved in these regulatory processes have been extensively reviewed (1-6) and are not considered further. Direct control of enzyme activity can be exerted by the binding of small molecules such as substrates or effectors (activators and inhibitors) to catalytic or regulatory sites on the enzyme. It can also be achieved by modification of the enzyme molecules, through either covalent changes (hydrolysis, phosphorylation, etc) or noncovalent changes (polymerization-depolymerization, etc). This article will concentrate on general theories and experimental manifestations of regulatory enzymes, with special emphasis on equilibrium and kinetic analysis of the molecular interactions involved in the regulatory process. A few i11ustrative examples arc discussed. Regulation through covalent modifications of enzymes and by the interaction of enzymes with membranes is not discussed. A number of recent reviews are available that supplement the material presented here (7-12).

A bleomycin-inactivating enzyme in mouse liver.

Abstract: Affinity chromatography of a bleomycin-inactivating enzyme was studied on Sepharose 4 B-bleomycin A5 and Sepharose 4 B-lysinamide. Mouse liver was homogenized, and the enzyme extracted by ammonium sulfate precipitation and Sephadex G-200 chromatography. Affinity chromatography of the extract on Sepharose 4 B-lysinamide gave 25-fold purified enzyme, Further purification vas not successful because of instability of the enzyme. It hydrolyzed L-lysinamide, L-lysyl-β-naphthyl-amide, L-arginy1-β-naphthylamide, L-lysyl-L-lysine and Iysyl-bradykinin, but not leucinamide or L-leucyl-β-naphthylamide. Hydrolysis of L-lysyl-β-naphthylamide and L-arginyl-β-naphthylamide by the enzyme was competitively inhibited by bleomycin B2. The bleomycin-inactivating enzyme of rat liver was separated from a known aminopeptidase B.

A prolidase deficiency in man with iminopeptiduria.

Abstract: A deficiency in the enzyme, prolidase (EC 3.4.3.7), is described in a patient with iminopeptiduria and clinical findings that suggest a defect in collagen metabolism. The massive iminopeptiduria is unique to this disorder, with daily proline excretion rates of 1.76 g. Fifteen peptides were characterized and quantitated from a deproteinized, amino-acid-free, 24-hr urine collection using column chromatographic procedures. Amino acid analysis revealed the peptides to be di- and tri-peptides containing proline plus another amino acid, “X.” Dansylation revealed “X” to be amino-terminal in each case. Prolidase, an enzyme known to cleave X-proline bonds, was measured by two separate methods. Enzyme activity in the patient's red and white cells was absent to markedly decreased as compared to age-matched controls. Furthermore, the activity of the enzyme in the mother's and maternal grandfather's white cells was less than that found in adult controls, suggesting a heterozygous condition. The maternal grandmother's enzyme activity was normal. The enzyme data is suggestive of an autosomal recessive disorder. This patient represents the first direct demonstration of a prolidase deficiency in man.

Mechanism of the Adherence of Streptococcus mutans to Smooth Surfaces III. Purification and Properties of the Enzyme Complex Responsible for Adherence.

Abstract: Enzymes which possess the ability to cause the adherence of Streptococcus mutans cells to a smooth glass surface were purified 1,100 times by chromatography on agarose gel followed by hydroxylapatite gel. During the purification procedures, the enzymes from strain HS6 (group a) were examined for the synthesis of water-soluble and water-insoluble polysaccharide and the ability to produce adherence. The enzyme preparations producing adherence of the S. mutans cells in the presence of sucrose possessed a molecular size of about 400,000 to 2,000,000 and were composed of approximately equivalent amounts of dextran and levan sucrases and 5 to 30% polysaccharide. The most highly purified preparation contained a negligible amount of contaminating protein as judged by polyacrylamide gel electrophoresis, immunoelectrophoresis, and gel diffusion. In these three tests, the location of the enzyme responsible for the synthesis of insoluble polymer was detected by embedding or covering the enzyme-containing gel with a layer of sucrose-containing agarose gel and observing the formation of insoluble polymer. During purification the ability of all fractions to produce adherence was parallel with the enzyme activity responsible for the synthesis of insoluble polysaccharide from sucrose. About two-thirds of the sucrase enzyme complex in the S. mutans culture fluid synthesized water-soluble polymer. This complex, obtained by filtration through agarose gel, was smaller in molecular size, lower in sugar content, and did not produce adherence, in contrast to the enzyme complex which possessed adherence activity. The inhibition of the enzyme complex synthesizing soluble polymer required more anti-synthetase serum than that required to inhibit the synthesis of water-insoluble polymer. It is not known whether the lack of adherence activity in this enzyme was due to its smaller size and lower sugar content or the absence of unknown factors which are essential for its activity. The carbohydrate in these enzyme preparations, composed of glucose, may represent a primer molecule and/or a remnant of the polymer synthesized by the enzyme. The enzyme activity was not inhibited by anti-dextran globulin.

Guanosine 3′:5′-Monophosphate-Dependent Protein Kinases in Mammalian Tissues

Abstract: The mammalian protein kinase activity stimulated preferentially by low concentrations of guanosine 3':5'-monophosphate (cyclic GMP), but not by adenosine 3':5'-monophosphate (cyclic AMP), was readily assayed in a modified incubation system that contained a neutral phosphate buffer, protein kinase modulator, and arginine-rich histone. Cyclic GMP-dependent protein kinase activity assayed under these conditions was about two to three orders of magnitude higher than that previously detected. The enzyme activity occurred in all guinea pig and rat tissues (lung, heart, aorta, brain, liver, ileum, adipose, and pancreatic islets) examined. The activity can be separated from the cyclic AMP-dependent protein kinase activity, also present in the same tissues, by means of either Sephadex G-200 gel filtration or ammonium sulfate fractionation. The cyclic GMP-dependent enzyme preparations had K(a) values for cyclic GMP ranging from 0.03 to 0.12 muM, compared to the K(a) values for cyclic AMP ranging from 0.6 to 3.8 muM. The presence of phosphate and protein kinase modulator was essential for maximal cyclic GMP-dependent enzyme activity. The occurrence of high levels of cyclic GMP-dependent protein kinase activity in mammalian tissues clearly suggests that it may serve as a "target" enzyme for cyclic GMP, mediating many biological effects of this cyclic nucleotide.

A microassay for uroporphyrinogen I synthase, one of three abnormal enzyme activities in acute intermittent porphyria, and its application to the study of the genetics of this disease.

Abstract: A new spectrofluorometric assay is described for quantitating uroporphyrinogen I synthase (EC 4.3.1.8) activity in volumes of human blood as small as 2 μl. By this sensitive assay the inheritance of the enzyme's activity has been studied and the genetic defect for acute intermittent porphyria has been confirmed to be autosomal dominant in nature. There is a 3-fold range of uroporphyrinogen I synthase activity in erythrocytes in the normal population, with a mean Vmax ± SD of 35.7 ± 8.4 nmol of uroporphyrinogen I formed per ml of erythrocytes per hr, at 37°. One-half this level of enzyme activity (18.0 ± 5.0) is found in erythrocytes from patients with clinically manifest acute intermittent porphyria; and in erythrocytes from those of their relatives, including prepubertal children, who have the latent gene defect for the disease. The Km of erythrocyte enzyme of normal people is 12.3 ± 3.9 μM, whereas the Km of the erythrocyte enzyme of patients with acute intermittent porphyria is 6.2 ± 3.9 μM, as determined on whole blood lysates. Three enzymic changes have now been identified in patients with acute intermittent porphyria; a high level of δ-aminolevulinate synthase activity; a low level of uroporphyrinogen I synthase activity; and a deficiency of steroid Δ4-5α reductase activity.

Hormonal regulation of adipose-tissue acetyl-coenzyme A carboxylase by changes in the polymeric state of the enzyme. The role of long-chain fatty acyl-coenzyme A thioesters and citrate

Abstract: 1. Acetyl-CoA carboxylase activity was measured in extracts of rat epididymal fat-pads either on preparation of the extracts (initial activity) or after incubation of the extracts with citrate (total activity). In the presence of glucose or fructose, brief exposure of pads to insulin increased the initial activity of acetyl-CoA carboxylase; no increase occurred in the absence of substrate. Adrenaline in the presence of glucose and insulin decreased the initial activity. None of these treatments led to a substantial change in the total activity of acetyl-CoA carboxylase. A large decrease in the initial activity of acetyl-CoA carboxylase also occurred with fat-pads obtained from rats that had been starved for 36h although the total activity was little changed by this treatment. 2. Conditions of high-speed centrifugation were found which appear to permit the separation of the polymeric and protomeric forms of the enzyme in fat-pad extracts. After the exposure of the fat-pads to insulin (in the presence of glucose), the proportion of the enzyme in the polymeric form was increased, whereas exposure to adrenaline (in the presence of glucose and insulin) led to a decrease in enzyme activity. 3. These changes are consistent with a role of citrate (as activator) or fatty acyl-CoA thioesters (as inhibitors) in the regulation of the enzyme by insulin and adrenaline; no evidence that the effects of these hormones involve phosphorylation or dephosphorylation of the enzyme could be found. 4. Changes in the whole tissue concentration of citrate and fatty acyl-CoA thioesters were compared with changes in the initial activity of acetyl-CoA carboxylase under a variety of conditions of incubation. No correlation between the citrate concentration and the initial enzyme activity was evident under any condition studied. Except in fat-pads which were exposed to insulin there was little inverse correlation between the concentration in the tissue of fatty acyl-CoA thioesters and the initial activity of acetyl-CoA carboxylase. 5. It is suggested that changes in the concentration of free fatty acyl-CoA thioesters (which may not be reflected in whole tissue concentrations of these metabolites) may be important in the regulation of the activity of acetyl-CoA carboxylase. The possibility is discussed that the concentration of free fatty acyl-CoA thioesters may be controlled by binding to a specific protein with properties similar to albumin.

Tyrosine hydroxylase: activation by nerve stimulation.

Abstract: The synthesis of the sympathetic neurotransmitter, norepinephrine, is accelerated by electrical stimulation of the guinea pig vas deferens. The molecular mechanism responsible for this enhanced formation of transmitter is unknown but has been attributed to an increase in the activity of tyrosine hydroxylase (EC 1.14.16.2; tyrosine 3-monooxygenase) during nerve stimulation. In the present experiments, we found that crude preparations of tyrosine hydroxylase isolated from guinea pig vasa deferentia that were electrically stimulated or depolarized by potassium show an increase in activity compared with enzyme obtained from untreated paired control tissues. This increase in activity is partially antagonized by addition of the Ca++ chelator, ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA), to the assay medium, and can be completely blocked if Ca++ is removed from the potassium-rich medium used to depolarize the intact tissue, before preparation of the enzyme. A similar increase in enzyme activity occurs when Ca++ ions are added directly to enzyme prepared from untreated vasa deferentia. In this instance, the activation is completely reversed by EGTA. The increase in activity produced by addition of Ca++ to the isolated enzyme or by electrical stimulation or potassium depolarization of the tissue before isolation of the enzyme appears to be mediated by changes in the kinetic properties of tyrosine hydroxylase. All treatments appear to activate tyrosine hydroxylase by causing an increase in its affinity for substrate and pteridine cofactor and by decreasing its affinity for the end-product inhibitor, norepinephrine. These results provide direct evidence that the enhanced formation of norepinephrine seen during stimulation of sympathetically innervated tissues arises from an activation of tyrosine hydroxylase. The fact that the activation produced by nerve stimulation is mimicked by Ca++ ions raises the intriguing possibility that the influx or mobilization of Ca++ that accompanies nerve stimulation and that is intimately involved in release of transmitter may also participate in the activation of tyrosine hydroxylase.

Cytochemical localization of phosphatase in barley aleurone cells: The pathway of gibberellic-acid-induced enzyme release

Abstract: Acid phosphatase has been localized by cytochemical techniques in aleurone layers of dry barley (Hordeum vulgare L.) grains, in imbibed half-grains and in isolated layers treated with and without gibberellic acid (GA3). A major fraction of the enzyme activity is located in the cell walls. During imbibition and incubation of layers without GA3 a steady increase of enzyme activity in the inner wall region indicates a continued release of enzyme into the walls, but there is no essential change in the distribution of wall-enzyme sites. On the other hand, when GA3 is present enzyme activity is found for the first time in regions of the wall that become digested during GA3 treatment. These results indicate that the digested wall channels act as preferential routes through which acid phosphatase is released from the aleurone layer. No digested wall channels are formed in the absence of GA3 and, there being no route for release of the enzyme, it accumulates in the inner regions of the wall around aleurone cells. Assays of enzyme activity in vitro support the conclusions based on the histochemical data. They indicate that release of acid phosphatase from aleurone layers is under strict GA3 control, but that some of the increase in acid phosphatase activity in the isolated during incubation is not GA3 dependent.Acid phosphatase is present in the protein matrix of aleurone grains in all stages except the dry grain. Enzyme activity persists in aleurone grains throughout GA3 treatment when enlargement of the grains and mobilization of reserves takes place. It is suggested that this phosphatase hydrolyses phosphate reserves within the aleurone grains.