Kinetic properties of NADP-specific isocitrate dehydrogenase from bovine adrenals

At low concentrations of Mg2+ or Mn2+ the reaction catalyzed by isocitrate dehydrogenase from bovine adrenal cortex proceeds with a lag period which disappears as a result of the enzyme saturation with Mn2+ or Mg2+. The nu o versus D,L-isocitrate concentration curve is non-hyperbolic, which may be interpreted either by the presence of two active sites with different affinity for the substrate (K'mapp = 2.3 and 63 microM) within the enzyme molecule or by the "negative" cooperativity of these sites. The apparent Km value for NADP lies within the range of 3.6-9 microM. High concentrations of NADP inhibit isocitrate dehydrogenase (Ki = 1.3 mM). NADP.H inhibits the enzyme in a mixed manner with respect to NADP (Ki = 0.32 mM). In the presence of NADP.H the curve nu o dependence on NADP concentration shows a "negative" cooperativity between NADP binding sites. The reverse enzyme-catalyzed reaction of reductive carboxylation of 2-oxoglutarate does not exhibit any significant deviations from the Michaelis-Menten kinetics. The Km value for 2-oxoglutarate is 120 microM, while that for NADP.H is 10 microM.     

Purification and properties of NADP-specific malate dehydrogenase from bovine adrenal cortex mitochondria

An electrophoretically homogeneous preparation of mitochondrial NADP-dependent malate dehydrogenase with a specific activity of 155 u./mg and a 67% yield has been obtained, using ammonium sulfate fractionation, gel filtration through Toyopearl HW-55 F, ion-exchange chromatography on DEAE-Toyopearl 650 M and affinity chromatography on 2',5'-ADP-Sepharose 4B. The molecular mass of native malate dehydrogenase is 260 kD; Mr of the SDS-treated enzyme is 61 kD, which is suggestive of a tetrameric structure of the protein. Malate dehydrogenase is active only in the presence of Mg2+ or Mn2+, but not Ca2+ or Ba2+. The Km' values for Mn2+ and Mg2+ are 50 and 66 microM, respectively. At low malate concentrations and NADP saturation, the enzyme is characterized by a sigmoidal kinetics which changes to hyperbolic at low concentrations of NADP. The Lineweaver--Burk plots for the dependence of the initial reaction rate on the concentration of one substrate at several fixed concentrations of the other substrate intersect to the left of the B-axis. NADPH competes with NADP:pyruvate inhibits malate dehydrogenase ++noncompetitively with respect to the coenzyme. NADPH and pyruvate inhibit the malate dehydrogenase-catalyzed reaction via a mixed type mechanism with respect to malate. The data obtained are consistent with a consecutive mechanism of reaction, whose first substrate is NADP and the last product is NADPH.     

Purification and properties of NADP-dependent isocitrate dehydrogenase from the bovine adrenal cortex

NADP-dependent isocitrate dehydrogenase (EC 1.1.42) was isolated and 430 times purified from the hyaloplasm fraction of bull adrenal cortex using fractionation by ammonium sulphate and acetone, heat treatment, chromatography on DEAE-Sephadex A-50, gel-filtration on Sephadex G-200 and affinity chromatography on 2',5'-ADP-sepharose 4B. The specific activity of homogeneous enzyme is 60 units per 1 mg of protein at 30 degrees C, yield--34%, pH optimum--8.0, molecular weight, determined by gel filtration on Sephadex G-200, is 96 kDa. The preparation electrophoresis in PAAG in the presence of DS-Na reveals one protein fraction with the mobility corresponding to that of protein having molecular weight of 46 kDa. The data obtained evidence for a dimer structure of the isocitrate dehydrogenase molecule from bull adrenals.     

NADP-dependent malate dehydrogenase from bovine adrenal cortex cytoplasm. Isolation and properties

The NADP-dependent decarboxylating malate dehydrogenase was isolated from the cytoplasmic fraction of bovine adrenal cortex and purified 3530-fold by 3-fold ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl 650 M and DEAE-Sephadex A-50 with subsequent two-fold gel filtration through Toyopearl HW-55. The specific activity of homogeneous enzyme preparations was equal to 60 U/mg protein with a 30% yield. The enzyme molecular weight as determined by gel filtration on Sephadex G-20 was 155000. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate malate dehydrogenase dissociated into two subunits with Mr 77000. The Arrhenius plot for the reaction rate showed a break at 30 degrees C. The values of activation energy and temperature coefficient above and below the breakpoint were equal to 45049 and 147188 J X mol-1; 1.68 and 2.63, respectively. Within the temperature range of 26-40 degrees C, malate dehydrogenase exhibited hyperbolic kinetics with respect to the substrate. At 30 degrees C, Km for malate was equal to 250 microM, whereas at 40 degrees C it was 130 microM. The curve for the dependence of the initial reaction velocity versus NADP concentration was S-shaped. The Hill coefficient was 1.4, which testifies to positive cooperativity of NADP interaction with malate dehydrogenase.     

Various kinetic and spectral-fluorescent properties of NADP-dependent malate dehydrogenase from bovine adrenal cortex cytoplasm

Malate dehydrogenase from bovine adrenal cortex has been purified to homogeneity, using affinity chromatography on 2',5'-ADP-Sepharose 4B. The kinetic data do not contradict the consecutive mechanism of the reaction with the ordered addition of substrates: NADP binds first, then malate. The enzyme conformation initiated by NADP and malate binding is less thermostable. Malate dehydrogenase has intrinsic tryptophan fluorescence with the spectrum maximum at 335 +/- 1 nm, half-width of 50 +/- 1 nm and quantum yield of 0.08. The tryptophan residues belonging to class 1 (75%) and class 2 (25%) make the main contribution to the intrinsic fluorescence of malate. The binding of cofactors and substrates results in the quenching of enzyme fluorescence. The values of dissociation constants for malate dehydrogenase complexes with NADP (4 microM), with NADP . H (8 microM) and with pyruvate (2.5 mM) correlate with the corresponding values of Km. The shifts in pH of the medium induce changes in the fluorescence parameters which are probably related to conformational changes in the enzyme molecule. The changes in the fluorescence parameters correlate with the alterations of the malate dehydrogenase enzymatic activity.     

Effect of thiol reagents on the activity of NADP-dependent malate dehydrogenase isolated from bovine adrenal cortex cytoplasm

NADP-dependent malate dehydrogenase was rapidly inactivated in the presence of mercurous chloride. Titration of malate dehydrogenase by 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) in a solution of 8 M urea revealed 18 SH groups per molecule of the enzyme. Eight sulphydryl groups reacted with DTNB in native malate dehydrogenase and their modification was not accompanied by a loss of the enzyme activity. The interaction of p-chloromercury benzoate (PCMB) with malate dehydrogenase resulted in a 70% decrease in the enzyme activity. The binding of the thiol reagents by the malate dehydrogenase molecule appreciably increased the Michaelis constant value for the substrate. In the presence of magnesium ions, NADP and malate did not affect the process of malate dehydrogenase modification by DTNB and did not protect the enzyme from the inactivation by PCMB. It is suggested from the data obtained that the sulphyryl groups are involved in maintaining the active conformation of the enzyme.     

The role of Ca++ in regulation of oxoglutarate dehydrogenase complex activity from bovine adrenal cortex

Binding of Ca2+ ions by EGTA is established to increase the oxoglutarate dehydrogenase complex K'm for 2-oxoglutarate up to 25 mM in spite of the Mg2+ ions presence in the medium. The maximum reaction rate remains unchanged. Addition, besides EGTA of an equimolar Ca2+ amount to the medium induces a more than 100-fold decrease in K'm. A positive effect of Ca2+ ions is intensified by ADP. Ca2+ counteracts the inhibition of the oxoglutarate dehydrogenase complex activity by NADH. When chelating Ca2+ by EGTA with NADH available, a nonhyperbolic dependence of the reaction rate on the 2-oxoglutarate concentration is observed. When Ca2+ is absent, signs of a positive cooperative interaction of the enzyme with ADP and NADH are observed under conditions when 2-oxoglutarate concentration is much lower than the saturating one.     

Role of NADPH in the regulation of NADP-specific isocitrate dehydrogenase from pig heart.

The present results show that the NADP specific isocitrate dehydrogenase from pig heart exhibits a time lag before the reaction rate approaches a constant value at low metal ion concentrations. Addition of NADPH or EDTA to the assay mixture abolished the lag, and will under certain conditions activate the enzyme.The lag time increased with increasing concentrations of isocitrate and decreased with increasing enzyme concentration. The NADP and metal ion concentration affected the lag in a complex manner. At low NADP and isocitrate concentration, the lag was reduced 50% by an NADPH concentration of less than 2 μm. Stopped flow experiments showed that premixing of NADP or NADPH with the enzyme abolished the effect of NADPH on the lag time. NADPH activated the enzyme at high NADP concentrations. This activating effect could be accounted for by removal of substrate inhibition by NADP.Evidence was obtained to show that the effect of NADPH on the activity was caused by binding of the reduced coenzyme to a site separate from the normal coenzyme binding site. Binding of metal ions by the reduced coenzyme is probably of importance as EDTA affects the lag time and activity in a manner similar to NADPH. The NADPH effect seems to be a general property of NADP-linked isocitrate dehydrogenases.     

Purification and various properties of hyaloplasmic NADP-dependent isocitrate dehydrogenase from the rabbit adrenal gland

NADP-dependent isocitrate dehydrogenase was isolated from the hyaloplasmic fraction of rabbit adrenal glands and purified by ammonium sulfate and polyethylene glycol fractionation and chromatography on DEAE-Sephadex A-50 to a specific activity of 26.8 U/mg with a 53% yield. Polyacrylamide gel electrophoresis revealed one distinct protein band with mobility corresponding to Mr approximately 50 000 in the presence of SDS. Data from gel filtration suggest that the detergent-untreated isocitrate dehydrogenase has a twice as great molecular mass, which is indicative of its dimeric structure of identical subunits. The pH optimum for the adrenal isocitrate dehydrogenase-catalyzed reaction is 7.5-7.7; the apparent activation energy is 61.3 kJ X mol-1. Mn2+ activate the enzyme more effectively than Mg2+. The curve for the dependence of the isocitrate dehydrogenase reaction rate versus D-isocitrate and NADP concentrations is S-shaped. At low substrate or coenzyme concentrations the Hill coefficient is 2.0 and 1.9, respectively, which serves as a kinetic attribute of positive cooperativity of their interaction with isocitrate dehydrogenase. The concentrations of D-isocitrate and NADP providing for the half-maximal rate of the reaction are 3.8 and 6.6 microM, respectively.     

Kinetic model of the functioning of pyruvate kinase from bovine adrenal cortex

The dependence of pyruvate kinase reaction rate on the concentration of one of the ligands--ADP or MgCl2--at constant concentrations of the other ligand was studied. The enzyme activity vs ligand concentration curves have fairly symmetrical peaks which correspond to the range of approximately equal ligand concentrations. The S-shaped dependence is observed only over the range of concentrations close to the dissociation constant for the Mg-ADP- complex (0.7 mM) under the given experimental conditions. The data obtained are consistent with the results of the first model kinetics within the framework of the London-Steck theory. The substrate for pyruvate kinase is the Mg-ADP- complex, while free Mg2+ and ADP3- competitively inhibit the enzyme. The inhibition constants are equal to 44 and 1 mM, respectively. The inhibiting effects of the metal and dinucleotide may be due to the competition with the substrate for the enzyme active site. Taking into consideration the fact that the binding of one of the ligands to the enzyme depends on the presence of the other ligand, a conclusion is drawn that Mg2+ forms a bridge with ADP3- and pyruvate kinase from adrenal cortex.     

Alpha-ketoglutarate supply for amino Acid synthesis in higher plant chloroplasts: intrachloroplastic localization of NADP-specific isocitrate dehydrogenase

Isocitrate dehydrogenase was found in Pisum sativum chloroplasts purified on sucrose density gradients. A chloroplast-enriched pellet obtained by differential centrifugation formed two chlorophyll-containing bands. The lower one containing intact chloroplasts had NADP-specific isocitrate dehydrogenase and triose-phosphate isomerase activities. Mitochondria and peroxisomes were observed to band well away from the intact chloroplast region, as indicated by peak activities of fumarase and catalase, respectively. The presence of isocitrate dehydrogenase in chloroplasts suggests that chloroplasts may generate at least some of the α-ketoglutarate required for glutamate synthesis.     

The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. The tryptic peptides.

The NADP-specific glutamate dehydrogenase of Neurospora crassa was digested with trypsin, and peptides accounting for 441 out of the 452 residues of the polypeptide chain were isolated and substantially sequenced. Additional experimental detail has been deposited as Supplementary Publication SUP 50052 (11 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem J. (1975) 145, 5.     

NADP-specific isocitrate dehydrogenase in regulation of urea synthesis in rat hepatocytes.

The effect of inhibition of NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) by DL-threo-alpha-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylase) on urea synthesis was studied in isolated rat hepatocytes. alpha-Methylisocitrate substantially inhibited the rate of urea synthesis (35--84%) with substrates requiring net reductive amination of 2-oxoglutarate to glutamate for aspartate synthesis (i.e., L-serine, D-alanine, or NH4Cl + L-lactate). alpha-Methylisocitrate did not inhibit synthesis of urea from substrates not requiring reductive formation of glutamate (i.e. L-alanine, L-glutamine, L-asparagine, or NH4Cl + L-ornithine). The rate-limiting role of NADPH in urea synthesis was correlated with the decrease in NADPH content that occurred upon addition of NH4Cl or of alpha-methylisocitrate to hepatocytes incubated with lactate and pyruvate, indicating utilization of NADPH for reductive amination of 2-oxoglutarate and inhibition of NADPH generation via NADP-isocitrate dehydrogenase, respectively. Similar results were obtained with D-alanine and L-serine; however, alpha-methylisocitrate or NH4Cl did not substantially decrease NADPH content when L-alanine was the substrate. Inhibitors or ornithine--2-oxo acid transaminase (L-canaline or gabaculine) decreased the uptake of ornithine by hepatocytes and inhibited the alpha-methylisocitrate insensitive urea synthesis from ornithine and NH4Cl. Canaline did not inhibit urea synthesis from lactate, ornithine, and NH4Cl but the inhibition by alpha-methylisocitrate of urea formation from this combination was appreciably larger with canaline (approx. 82%) than without canaline (approx. 48%). Inhibition of urea synthesis from NH4Cl + lactate by alpha-methylisocitrate was partially prevented by oleate, octanoate, or 3-hydroxybutyrate. When the NADH content of hepatocytes was increased by 3-hydroxybutyrate, the addition of NH4Cl and/or alpha-methylisocitrate caused a decline in NADH (and NADPH) content, suggesting that reducing equivalents from NADH as well as from NADPH can support net reductive amination of 2-oxoglutarate when required for urea synthesis.     

The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptides from digestion with a staphylococcal proteinase.

The extracellular proteinase of Staphylococcus aureus strain V8 was used to digest the NADP-specific glutamate dehydrogenase of Neurospora crassa. Of 35 non-overlapping peptides expected from the glutamate content of the polypeptide chain, 29 were isolated and substantially sequenced. The sequences obtained were valuable in providing overlaps for the alignment of about two-thirds of the sequences found in tryptic peptides [Wootton, J. C., Taylor, J, G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 739-748]. The blocked N-terminal peptide of the protein was isolated. This peptide was sequenced by mass spectrometry, and found to have N-terminal N-acetylserine by Howard R. Morris and Anne Dell, whose results are presented as an Appendix to the main paper. The staphylococcal proteinase showed very high specificity for glutamyl bonds in the NH4HCO3 buffer used. Partial splits of two aspartyl bonds, both Asp-Ile, were probably attributable to the proteinase. No cleavage of glutaminyl or S-carboxymethylcysteinyl bonds was found. Additional experimental detail has been deposited as Supplementary Publication SUP 50053 (5 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K, from whom copies may be obtained under the terms given in Biochem. J. (1975) 1458 5.     

NADP-specific isocitrate dehydrogenase from the simian malaria parasite Plasmodium knowlesi: partial purification and characterization.

Cell-free schizonts of Plasmodium knowlesi, a simian malaria parasite, possess significant isocitrate dehydrogenase (IDH) activity, about 90% of which is contributed by the NADP-specific enzyme that is localized in the cytosolic fraction. The enzyme has been partially purified by affinity chromatography using Blue sepharose CL-6B. Although unstable in nature, it is stabilized by citrate and glycerol. Kinetic studies with DL-isocitrate and NADP yielded hyperbolic curves with Michaelis constants of 0.210 and 0.038 mM, respectively. Manganous or magnesium ions are essential for activity. The enzyme is thermosensitive, shows maximum activity at pH 8.0, and has a molecular mass of about 48.5 kDa. It is strongly inhibited by thiol-blocking agents but protected against them by thiol-providing agents. Cupric and argentic ions also have a marked inhibitory effect on its activity. The enzyme is significantly inhibited by chloroquine and oxytetracycline in vitro, but to a lesser degree by tetracycline.     

Identification of essential amino acid residues in valine dehydrogenase from Streptomyces albus

Cys-29 and Cys-251 of Streptomyces albus valine dehydrogenase (ValDH) were highly conserved in the corresponding region of NAD(P)(+)-dependent amino acid dehydroganase sequences. To ascertain the functional role of these cysteine residues in S. albus ValDH, site-directed mutagenesis was performed to change each of the two residues to serine. Kinetic analyses of the enzymes mutated at Cys-29 and Cys-251 revealed that these residues are involved in catalysis. We also constructed mutant ValDH by substituting valine for leucine at 305 by site-directed mutagenesis. This residue was chosen, because it has been proposed to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). Kinetic analysis of the V305L mutant enzyme revealed that it is involved in the substrate binding site. However it displayed less activity than the wild type enzyme toward all aliphatic and aromatic amino acids tested.     

Insulin stimulatory action on amino acid uptake in bovine adrenal cortex or glomerulosa zone.

1. Insulin stimulated the [1-14C] methylaminoisobutyric acid and [1-14C] aminoisobutyric acid uptake in the bovine adrenal cortex or in the glomerulosa zone through the A system. 2. Verapamil nullified the insulin stimulatory action indicating that this hormonal action is probably related to the voltage-dependent Ca2+ channels.