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1.
Affinity chromatography of nicotinamide–adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide
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1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD(+) through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD(+) (probably through the 8 position of the adenine residue) to a number of different spacer-arm-agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD(+) derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD(+). Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD(+)-binding site of this enzyme. Problems associated with the use of immobilized derivatives for enzyme binding and mechanistic studies are briefly discussed. 相似文献
2.
Nicotinate, quinolinate and nicotinamide as precursors in the biosynthesis of nicotinamide–adenine dinucleotide in barley 总被引:1,自引:0,他引:1
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1. The relative efficiencies of nicotinate, quinolinate and nicotinamide as precursors of NAD(+) were measured in the first leaf of barley seedlings. 2. In small amounts, both [(14)C]nicotinate and [(14)C]quinolinate were quickly and efficiently incorporated into NAD(+) and some evidence is presented suggesting that NAD(+) is formed from each via nicotinic acid mononucleotide and deamido-NAD. 3. [(14)C]Nicotinamide served equally well as a precursor of NAD(+) and although significant amounts of [(14)C]NMN were detected, most of the [(14)C]NAD(+) was derived from nicotinate intermediates formed by deamination of [(14)C]nicotinamide. 4. Radioactive NMN was also a product of the metabolism of [(14)C]nicotinate and [(14)C]quinolinate but most probably it arose from the breakdown of [(14)C]NAD(+). 5. In barley leaves where the concentration of NAD(+) is markedly increased by infection with Erysiphe graminis, the pathways of NAD(+) biosynthesis did not appear to be altered after infection. A comparison of the rates of [(14)C]NAD(+) formation in infected and non-infected leaves indicated that the increase in NAD(+) content was not due to an increased rate of synthesis. 相似文献
3.
Biospecific affinity chromatography of an adenosine 3′:5′-cyclic monophosphate-stimulated protein kinase (protamine kinase from trout testis) by using immobilized adenine nucleotides
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1. Two adenine nucleotides, 8-(6-aminohexyl)aminoadenosine 3':5'-cyclic monophosphate and 8-(6-aminohexyl)amino-AMP, were synthesized. Their structures were established in particular by using mass spectroscopy. 2. Free cyclic AMP and 8-(6-aminohexyl)amino cyclic AMP both stimulate protamine kinase activity at low concentrations, but are inhibitory at concentrations above 0.1mm. AMP is an inhibitor of enzymic activity, whereas neither 8-(6-aminohexyl)amino-AMP nor the earlier synthesized N(6)-(6-aminohexyl)-AMP is inhibitory. 3. The nucleotides were coupled to Sepharose 4B and used for biospecific chromatography of partially purified protamine kinase. Enzyme applied at high buffer concentrations to the cyclic AMP-Sepharose material was retarded and thereby purified tenfold. At low buffer concentrations the enzyme was adsorbed to the affinity material, and was subsequently released by a pulse of the inhibitor AMP, yielding a 50-100-fold purification. Enzyme applied to immobilized 8-(6-aminohexyl)amino-AMP or N(6)-(6-aminohexyl)-AMP was eluted together with the main protein peak in the void volume. 4. Protamine kinase eluted from 8-(6-aminohexyl)amino cyclic AMP-Sepharose was no longer activated by cyclic AMP. Results from sucrose gradient centrifugation suggest that a dissociation of the enzyme took place on the immobilized nucleotide. 5. Further information on the mass spectroscopy has been deposited as Supplementary Publication SUP 50026 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5. 相似文献
4.
The role of nicotinamide–adenine dinucleotide phosphate-dependent malate dehydrogenase and isocitrate dehydrogenase in the supply of reduced nicotinamide–adenine dinucleotide phosphate for steroidogenesis in the superovulated rat ovary
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1. Superovulated rat ovary was found to contain high activities of NADP-malate dehydrogenase and NADP-isocitrate dehydrogenase. The activity of each enzyme was approximately four times that of glucose 6-phosphate dehydrogenase and equalled or exceeded the activities reported to be present in other mammalian tissues. Fractionation of a whole tissue homogenate of superovulated rat ovary indicated that both enzymes were exclusively cytoplasmic. The tissue was also found to contain pyruvate carboxylase (exclusively mitochondrial), NAD-malate dehydrogenase and aspartate aminotransferase (both mitochondrial and cytoplasmic) and ATP-citrate lyase (exclusively cytoplasmic). 2. The kinetic properties of glucose 6-phosphate dehydrogenase, NADP-malate dehydrogenase and NADP-isocitrate dehydrogenase were determined and compared with the whole-tissue concentrations of their substrates and NADPH; NADPH is a competitive inhibitor of all three enzymes. The concentrations of glucose 6-phosphate, malate and isocitrate in incubated tissue slices were raised at least tenfold by the addition of glucose to the incubation medium, from the values below to values above the respective K(m) values of the dehydrogenases. Glucose doubled the tissue concentration of NADPH. 3. Steroidogenesis from acetate is stimulated by glucose in slices of superovulated rat ovary incubated in vitro. It was found that this stimulatory effect of glucose can be mimicked by malate, isocitrate, lactate and pyruvate. 4. It is concluded that NADP-malate dehydrogenase or NADP-isocitrate dehydrogenase or both may play an important role in the formation of NADPH in the superovulated rat ovary. It is suggested that the stimulatory effect of glucose on steroidogenesis from acetate results from an increased rate of NADPH formation through one or both dehydrogenases, brought about by the increases in the concentrations of malate, isocitrate or both. Possible pathways involving the two enzymes are discussed. 相似文献
5.
The activities of nicotinamide mononucleotied adenylyltransferase and of nicotinamide–adenine dinucleotide kinase in the livers of rats subjected to different hormonal treatments
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1. The activities of NMN adenylyltransferase and of NAD(+) kinase have been measured in the livers of adrenalectomized or alloxan-diabetic rats and in the livers of rats treated with glucagon, pituitary growth hormone or thyroxine. 2. The activities of these enzymes have been compared with the effects of the same treatments on the nicotinamide nucleotide concentrations in the liver. 3. Alloxandiabetes (+37%) and thyroxine (+27%) both increased the activity of NMN adenylyltransferase. The other treatments were without effect on this enzyme. 4. Only thyroxine increased the activity of NAD(+) kinase significantly (+26%) although both adrenalectomy and glucagon tended to increase its activity. 5. The activity of NAD(+) glycohydrolase was measured in the livers of diabetic rats, and in the livers of rats treated with either growth hormone or thyroxine. Of these treatments, only growth hormone altered the enzyme activity (+26%, calculated on a total hepatic activity basis). 6. Female rats had a greater hepatic NAD(+)-kinase activity than males but there was no sex difference with respect to NMN adenylyltransferase. 7. The lack of correlation between the maximum potential activity of these three enzymes and the known changes of the nicotinamide nucleotides in each of the hormone conditions is discussed. 相似文献
6.
Relation between the oxidation state of nicotinamide–adenine dinucleotide and the metabolism of spermatozoa
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1. The existing procedures for extraction of oxidized and reduced nicotinamide coenzymes were adapted to spermatozoa to overcome the coenzyme-degrading activity of seminal plasma. 2. The content of total NAD(+) and NADH was determined in the spermatozoa of ram, bull, boar, stallion and cock. NADP(+) and NADPH were not detected in ram spermatozoa. 3. The oxidation state of sperm NAD depended on the seminal plasma, the removal of which produced a change in the percentage oxidation state of the coenzyme, 100x[NAD(+)/(NAD(+)+NADH)], without altering the total content of NAD(+)+NADH. 4. In suspensions of washed ram spermatozoa, incubated anaerobically at 25 degrees C, the percentage oxidation state of NAD declined with increasing spermatozoa concentration. 5. When ram or boar spermatozoa that had been previously washed and resuspended in Ringer phosphate medium, were incubated anaerobically at 25 degrees C with various substances, pronounced effects on the percentage oxidation state of NAD could be observed with l-lactate, pyruvate, oxaloacetate, dihydroxyacetone, formaldehyde and glyceraldehyde; sorbitol and acetoacetate acted only on ram spermatozoa; fructose, glucose, mannose and acetaldehyde acted predominantly on boar spermatozoa. Formaldehyde lowered the (NAD(+)+NADH) content of ram spermatozoa, but none of the other substances had a comparable effect. 6. The percentage oxidation state of sperm NAD was not influenced by exogenous cysteine, cystine, ergothioneine or ascorbate. 7. A highly active sorbitol dehydrogenase could be prepared from ram, but not from boar, spermatozoa. 8. Sorbitol, acetoacetate and 3-hydroxybutyrate effectively supported the respiration of ram, but not boar, spermatozoa. 9. ;Cold shock', resulting from sudden cooling of spermatozoa, abolished motility completely and irreversibly but produced only a slow and partial decrease in the total NAD content. Slight over-heating, sufficient to produce loss of motility, had no adverse effect on the total NAD content. 10. Storage of ram sperm at 14 degrees C produced only a small decrease of NAD after 2 days, but subsequently the loss became greater. 相似文献
7.
Control of the redox state of the nicotinamide–adenine dinucleotide couple in rat liver cytoplasm 总被引:1,自引:0,他引:1
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1. A study has been made of the ability of rat liver in vivo to maintain equilibrium in the combined glyceraldehyde 3-phosphate dehydrogenase, 3-phosphoglycerate kinase and lactate dehydrogenase reactions, i.e. in the system: [Formula: see text] Attempts were made to upset equilibrium. The [lactate]/[pyruvate] ratio was rapidly changed by injection of ethanol or crotyl alcohol, and the value of [ATP]/[ADP][HPO(4) (2-)] was rapidly changed by injection of ethionine or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone. 2. The concentrations of the metabolites occurring in the above equation were measured in freeze-clamped liver. 3. Although the injected agents caused large changes in the concentrations of the individual components, near-equilibrium in the system was maintained, as indicated by the fact that the value of [ATP]/[ADP][HPO(4) (2-)], referred to as the phosphorylation state of the adenine nucleotides, measured directly agreed with the value calculated for equilibrium conditions from the above equation. 4. The results are discussed and taken to confirm that the order of magnitude of the value of the redox state of the cytoplasmic NAD couple in rat liver is controlled by the phosphorylation state of the adenine nucleotide system. 相似文献
8.
Oxidized and reduced nicotinamide–adenine dinucleotide phosphate in tissue suspensions of rat liver
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T. F. Slater 《The Biochemical journal》1967,104(3):833-842
1. The concentrations of NADP and NADPH(2) in homogenates of rat liver (expressed as mug./g. wet wt. of tissue homogenized) were compared with values obtained from intact samples of liver taken from the same female rat. With 0.25m-sucrose alone as the suspending medium, or in combination with tris buffer or 0.01-0.1m-nicotinamide, considerable decreases in the sum of the NADP+NADPH(2) concentrations were occasionally observed during 30min. storage of homogenates at 0 degrees . However, addition of 0.5m-nicotinamide+5mm-tris buffer to 0.25m-sucrose for use as a suspending medium maintained the sum of the NADP+NADPH(2) concentrations in homogenates at the level found in intact tissue for at least 30min. at 0 degrees . 2. The effects of freezing intact tissue and homogenates in liquid nitrogen before the extraction of NADP and NADPH(2) were studied. Freezing alone appears to convert a significant amount (approx. 30%) of liver NADPH(2) into an equivalent amount of NADP in intact tissue. This is discussed in terms of the ;bound NADP' reported by Burch, Lowry & Von Dippe (1963). 3. The intracellular distributions of NADP and NADPH(2) in intracellular fractions of rat liver were studied by using a modified centrifuging scheme that allows extraction of the isolated fractions to be performed within 45min. of killing the animal. Approx. 50% of the total NADP+NADPH(2) was found in the large-particle fractions and the remaining 50% was mostly in the soluble fraction of the cell. 4. Further investigations are reported on the nature of ;bound NADP' in rat liver. Most of this material appears associated with the ;nuclear' (containing nuclei, debris, erythrocytes etc.) or large-mitochondrial fractions, or both, obtained by low-speed centrifuging of rat-liver homogenates. 5. Although in some experiments the variations produced in the concentration of NADPH(2) present in large-particle fractions were followed by similar changes in that of ;bound NADP', in other cases no such direct relationship was obtained. Addition of phenazine methosulphate, for example, consistently lowered the concentration of NADPH(2) yet raised the concentration of ;bound NADP' in rat-liver mitochondrial fractions. 相似文献
9.
Nicotinamide–adenine dinucleotide phosphate enzymes in the mosquito during growth and aging
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1. Optimum conditions were established for determining the activities of the NADP(+)-linked enzymes, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase, in mosquito tissues. 2. The activity of each dehydrogenase was determined in samples of mosquitoes of different ages throughout the life-span. The specific-activity curves attained maximal values in the pupal or early adult period. From these maxima an 81% decrease in glucose 6-phosphate-dehydrogenase and 67% decrease in 6-phosphogluconate-dehydrogenase activities occurred after the tenth day of adult life; a 77% decrease in isocitrate-dehydrogenase activity occurred before the fifth day. 3. The activity differences were found in different body regions as well as in whole organisms. 4. Starvation of the larva or adult did not result in decreases in enzyme activity. 5. These findings support the hypothesis that the activities of enzymes that form NADPH are related to the biosynthetic activity, for the enzyme activities increased during the period of cellular growth and decreased during the aging period. 相似文献
10.
The redox state of free nicotinamide–adenine dinucleotide phosphate in the cytoplasm of rat liver 总被引:9,自引:0,他引:9
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1. The concentrations of the oxidized and reduced substrates of the ;malic' enzyme (EC 1.1.1.40) and isocitrate dehydrogenase (EC 1.1.1.42) were measured in freeze-clamped rat livers. By assuming that the reactants of these dehydrogenase systems are at equilibrium in the cytoplasm the [free NADP(+)]/[free NADPH] ratio was calculated. The justification of the assumption is discussed. 2. The values of this ratio obtained under different nutritional conditions (well-fed, 48hr.-starved, fed with a low-carbohydrate diet, fed with a high-sucrose diet) were all of the same order of magnitude although characteristic changes occurred on varying the diet. The value of the ratio fell on starvation and on feeding with the low-carbohydrate diet and rose slightly on feeding with the high-sucrose diet. 3. The mean values of the ratio were calculated to be between 0.001 and 0.015, which is about 100000 times lower than the values of the cytoplasmic [free NAD(+)]/[free NADH] ratio. 4. The differences in the redox state of the two nicotinamide-adenine dinucleotide couples can be explained on a simple physicochemical basis. The differences are the result of equilibria that are determined by the equilibrium constants of a number of highly active readily reversible dehydrogenases and transaminases and the concentrations of the substrates and products of these enzymes. 5. The decisive feature is the fact that the NAD and NADP couples share substrates. This sharing provides a link between the redox states of the two couples. 6. The application of the method of calculation to data published by Kraupp, Adler-Kastner, Niessner & Plank (1967), Goldberg, Passonneau & Lowry (1966) and Kauffman, Brown, Passonneau & Lowry (1968) shows that the redox states of the NAD and NADP couples in cardiac-muscle cytoplasm and in mouse-brain cytoplasm are of the same order as those in rat liver. 7. The determination of the equilibrium constant at 38 degrees , pH7.0 and I 0.25 (required for the calculation of the [free NADP(+)]/[free NADPH] ratio), gave a value of 3.44x10(-2)m for the ;malic' enzyme (with CO(2) rather than HCO(3) (-) as the reactant) and a value of 1.98x10(-2)m(-1) for glutathione reductase. 相似文献
11.
The purification and properties of the respiratory-chain reduced nicotinamide–adenine dinucleotide dehydrogenase of Torulopsis utilis
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1. An NADH-ferricyanide reductase activity has been isolated from the respiratory chain of Torulopsis utilis by using detergents. The isolated enzyme contains non-haem iron, acid-labile sulphide and FMN in the molar proportions 27.5:28.4:1. The preparation is free of FAD and largely free of cytochrome. 2. The enzyme catalyses ferricyanide reduction by NADPH at about 1% of the rate with NADH, and reacts poorly with acceptors other than ferricyanide. The rates of reduction of some acceptors are, as percentages of the rate with ferricyanide: menadione, 0.35%; lipoate, 0.01%; cytochrome c, 0.065%; dichlorophenolindophenol, 0.35%; ubiquinone-1, 0.08%. 3. Several properties of submitochondrial particles of T. utilis (non-haem iron, acid-labile sulphide, FMN and an NADH-reducible electron-paramagnetic-resonance signal) were found to co-purify with the NADH-ferricyanide reductase activity. Thus about 70% of the FMN and, within the limits of accuracy of the experiments, 100% of the non-haem iron and acid-labile sulphide of submitochondrial particles derived from T. utilis cells grown under conditions of glycerol limitation (but relatively low iron availability) can be attributed to the NADH-ferricyanide reductase. 4. It was also shown that the component of submitochondrial particles specifically bleached at 460nm by NADH [species 1 of Ragan & Garland (1971)] co-purifies with the NADH-ferricyanide reductase. 5. This successful purification of an NADH dehydrogenase from T. utilis forms a starting point for investigating the molecular properties of phenotypically modified mitochondrial NADH oxidation pathways that lack energy conservation between NADH and the cytochromes. 相似文献
12.
The synthesis of nicotinamide–adenine dinucleotide and poly(adenosine diphosphate ribose) in various classes of rat liver nuclei
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1. The activities of NMN adenylyltransferase and an enzyme that synthesizes poly (ADP-ribose) from NAD were investigated in the various classes of rat liver nuclei fractionated by zonal centrifugation. 2. The highest specific activities of these two nuclear enzymes occur in different classes of nuclei. In very young and in mature rats it was shown that a correlation exists between DNA synthesis and NMN adenylyltransferase activity, but in rats of intermediate age this correlation is less evident. The highest activities of the enzyme that catalyses formation of poly (ADP-ribose) are in the nuclei involved in the synthesis of RNA. 3. The significance of these results in relation to NAD metabolism is discussed. 相似文献
13.
Of the two NADP-linked isocitrate dehydrogenases in Acinetobacter lwoffi the higher-molecular-weight form, isoenzyme-II, is reversibly stimulated sixfold by low concentrations of glyoxylate or pyruvate. Kinetic results indicate that this stimulation of activity involves both an increase in V(max.) and a decrease in the apparent K(m) values for substrates, most markedly that for NADP(+). Other changes brought about by glyoxylate or pyruvate include a shift in the pH optimum for activity and an increased stability to inactivation by heat or urea. Mixtures of glyoxylate plus oxaloacetate, known to inhibit isocitrate dehydrogenases from other organisms, produce inhibition of both A. lowffi isoenzymes, and do not reflect the stimulatory specificity of glyoxylate for isoenzyme-II. Isoenzyme-II is also stimulated by AMP and ADP, but the activation by glyoxylate or pyruvate is shown to be quite independent of the adenylate activation. Differential desensitization of the enzyme by urea to the two types of activator further supports the view that the enzyme possesses two distinct allosteric regulatory sites. The metabolic significance of the activations is discussed. 相似文献
14.
The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide–adenine dinucleotide phosphate-linked isoenzymes 总被引:1,自引:0,他引:1
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Two isoenzymes of NADP-linked isocitrate dehydrogenase have been identified in Acinetobacter lwoffi and have been termed isoenzyme-I and isoenzyme-II. The isoenzymes may be separated by ion-exchange chromatography on DEAE-cellulose, by gel filtration on Sephadex G-200, or by zonal ultracentrifugation in a sucrose gradient. Low concentrations of glyoxylate or pyruvate effect considerable stimulation of the activity of isoenzyme-II. The isoenzymes also differ in pH-dependence of activity, kinetic parameters, stability to heat or urea and molecular size. Whereas isoenzyme-I resembles the NADP-linked isocitrate dehydrogenases from other organisms in having a molecular weight under 100000, isoenzyme-II is a much larger enzyme (molecular weight around 300000) resembling the NAD-linked isocitrate dehydrogenases of higher organisms. 相似文献
15.
Guanosine 3′:5′-monophosphate has a slight hydroosmotic effect on toad urinary bladder. Furthermore, this nucleotide strongly inhibits the responses to 3′:5′-adenosine monophosphate and oxytocin. The response to an increase in medium tonicity is not modified by the guanosine nucleotide. A role for guanosine 3′:5′-monophosphate in the regulation of water permeability in toad urinary bladder is proposed. 相似文献
16.
Properties of a halophil nicotinamide–adenine dinucleotide phosphate-specific isocitrate dehydrogenase. Preliminary studies of the salt relations and kinetics of the crude enzyme 总被引:5,自引:0,他引:5
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The effects of chlorides on NADP-specific isocitrate dehydrogenase from Halobacterium salinarium were investigated. The enzyme is stabilized by potassium chloride and sodium chloride and this effect is discussed in relation to the Hill (1913) equation. Kinetics of the enzyme were studied within a range of concentrations of potassium chloride and sodium chloride. Apparent Michaelis constants for both substrates were affected by salt concentration, the effect being greater in sodium chloride than in potassium chloride. Minimal apparent Michaelis constants for both substrates were similar to the corresponding constants reported for yeast isocitrate dehydrogenase. V(max.) was maximal in each salt at a concentration of about 1m. The maximum was higher in sodium chloride than in potassium chloride. At salt concentrations above about 2.3m, the apparent V(max.) was lower in sodium chloride than in potassium chloride, and at salt concentrations below 0.75-1.0m, each salt behaved as a linear activator of the enzyme. Within this concentration range salt and NADP(+) acted competitively; the activation by salt was overcome at finite concentrations of NADP(+). At concentrations above about 1m, potassium chloride was a linear non-competitive inhibitor of the enzyme. Within the range 1.0-2.5m, sodium chloride was also a linear non-competitive inhibitor, but above 2.5m it caused more pronounced inhibition. 相似文献
17.
The hormonal regulation of enzymes in prenatal and postnatal rat liver. Effects of adenosine 3′,5′-(cyclic)-monophosphate
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Olga Greengard 《The Biochemical journal》1969,115(1):19-24
1. The administration of glucagon, cAMP [adenosine 3',5'-(cyclic)-monophosphate], BcAMP [6-N-2'-O-dibutyryladenosine 3',5'-(cyclic)-monophosphate] or adrenaline to foetal rats during the last 2 days of gestation evoked the appearance of tyrosine aminotransferase and enhanced the accumulation of glucose 6-phosphatase in the liver. In foetuses 1-2 days younger only BcAMP was effective. After birth liver glucose 6-phosphatase no longer responds to glucagon or BcAMP. Tyrosine aminotransferase is still inducible by these agents in 2-day-old rats, but not in 50-day-old rats. After adrenalectomy of adults glucagon or BcAMP can enhance the induction of the enzyme by hydrocortisone. The results indicate that the ability to synthesize tyrosine aminotransferase and glucose 6-phosphatase when exposed to cAMP develops sooner than the ability to respond to glucagon with an increase in the concentration of cAMP; the responsiveness of enzymes to different hormones changes with age. A scheme illustrating the sequential development of competence in regulating the level of an enzyme is presented. 2. Actinomycin inhibited the effects of glucagon and BcAMP on liver tyrosine aminotransferase and glucose 6-phosphatase in foetal rats. Growth hormone, insulin and hydrocortisone did not enhance the formation of these enzymes. 3. The time-course of accumulation of glucose 6-phosphatase in the kidney is different from that in the liver. Hormones that increase the accumulation in foetal liver do not do so in the kidney of the same foetus or in the livers of postnatal rats. 相似文献
18.
Malate dehydrogenase of the cytosol. Preparation and reduced nicotinamide–adenine dinucleotide-binding studies
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1. Two methods of preparing pig heart soluble malate dehydrogenase are described. A slow method yields an enzyme composed of three electrophoretically separable subforms. The more rapid method reproducibly gives a high yield of an enzyme that consists predominantly of the least acid subform. 2. The A(1%) (1cm) of the protein was redetermined as 15 at 280nm. By using this value the enzyme molecule was found to contain two independent and indistinguishable NADH-binding sites in titrations with NADH. 3. No evidence was found for the dissociation of the enzyme in the concentration range 0.02-7.2mum. 4. l-Malate (0.1m) tightened the binding of NADH to both pig and ox heart enzyme (2-fold), but, in contrast with the report by Mueggler, Dahlquist & Wolfe [(1975) Biochemistry14, 3490-3497], did not cause co-operative interactions between the binding sites. 5. Fructose 1,6-bisphosphate had no effect on the binding of NADH to the pig heart enzyme, but with the ox heart enzyme the NADH is slowly oxidized. This slow oxidation explains the ;sigmoidal' binding curves obtained when NADH was added to ox heart soluble malate dehydrogenase in the presence of fructose 1,6-bisphosphate [Cassman (1973) Biochem. Biophys. Res. Commun.53, 666-672] without the postulate of site-site interactions. 6. It is concluded that neither l-malate nor fructose 1,6-bisphosphate could in vivo modulate the activity of soluble malate dehydrogenase and alter the rates of transport of NADH between the cytosol and the mitochondrion. 7. Details of the preparation of soluble malate dehydrogenase have been deposited as Supplementary Publication SUP 50080 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1978) 169, 5. 相似文献
19.
Proton translocation coupled to quinone reduction by reduced nicotinamide–adenine dinucleotide in rat liver and ox heart mitochondria
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Measurements were made of the stoicheiometry of proton-translocation coupled to NAD(P)H oxidation by several quinones (duroquinone, ubiquinone(0), ubiquinone(1), ubiquinone(2)) in mitochondria from rat liver and ox heart. Observed stoicheiometries of protons translocated per mol of NADH oxidized (-->H(+)/2e(-) ratios; Mitchell, 1966) ranged from 0.75 (rat liver mitochondria with ubiquinone(1)) to 1.55 (ox heart mitochondria with ubiquinone(1) or ubiquinone(2)). Only the rotenone-sensitive pathway of NADH oxidation by quinone was able to support proton translocation. Correction of the observed -->H(+)/2e(-) ratios for the loss of reducing equivalents to the rotenone-insensitive pathway increased their value to approx. 2.0. It is concluded that the rotenone-sensitive NADH- ubiquinone reductase activity of the respiratory chain may be organized in the mitochondrial membrane as a proton-translocating oxidoreduction loop. The number of such loops between NADH and ubiquinone is one, and not two, as initially proposed by Mitchell (1966). 相似文献