scholarly journals Purification and comparative properties of isoenzymes of nicotinamide–adenine dinucleotide phosphate–isocitrate dehydrogenase from rat heart and liver

1972 ◽  
Vol 129 (5) ◽  
pp. 1003-1011 ◽  
Author(s):  
M. Islam ◽  
Joyce L. Bell ◽  
D. N. Baron
Keyword(s):  
Myocardial Infarction ◽  
Ion Exchange ◽  
Ammonium Sulphate ◽  
Isocitrate Dehydrogenase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Differential Sensitivity ◽  
Michaelis Constants ◽  
Ammonium Sulphate Fractionation ◽  
Cytoplasmic Location

1. Rat liver and heart major isoenzymes of NADP–isocitrate dehydrogenase have each been purified about 100-fold by a combination of ammonium sulphate fractionation and chromatography on ion-exchange cellulose and their properties compared. 2. The properties were similar in respect of pH, inhibition by Hg2+and Michaelis constants for isocitrate and NADP. 3. Some of the properties of the isoenzymes were different. 4. The heart isoenzyme was activated about 210% by 0.8m-ammonium sulphate whereas the liver isoenzyme was unaffected. The heart isoenzyme showed greater sensitivity to inactivation by heat (30°C for 30min), whereas the liver isoenzyme was more sensitive to inactivation by p-chloromercuribenzoate and by Cu2+. 5. The Michaelis constants with 3-acetylpyridine–adenine dinucleotide phosphate showed a twofold difference between liver and heart isoenzyme. 6. The differential sensitivity to heat and its mainly non-cytoplasmic location may be an explanation of the failure of plasma isocitrate dehydrogenase activity to increase after a myocardial infarction.

scholarly journals Properties of a halophil nicotinamide–adenine dinucleotide phosphate-specific isocitrate dehydrogenase. Preliminary studies of the salt relations and kinetics of the crude enzyme

1970 ◽  
Vol 116 (1) ◽  
pp. 125-134 ◽  
Author(s):  
D. M. Aitken ◽  
A. J. Wicken ◽  
A. D. Brown
Keyword(s):  
Sodium Chloride ◽  
Potassium Chloride ◽  
Isocitrate Dehydrogenase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Competitive Inhibitor ◽  
Michaelis Constants ◽  
The Hill ◽  
Kinetics Of ◽  
Pronounced Inhibition

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. Vmax. 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 Vmax. 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.

scholarly journals Purification and properties of the nicotinamide–adenine dinucleotide phosphate-dependent isocitrate dehydrogenase from pig liver cytoplasm

1970 ◽  
Vol 118 (2) ◽  
pp. 253-258 ◽  
Author(s):  
J. A. Illingworth ◽  
K. F. Tipton
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Isocitrate Dehydrogenase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Soluble Fraction ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Turnover Number ◽  
Pig Liver ◽  
Extinction Coefficients ◽  
Purification And Properties

The NADP-dependent isocitrate dehydrogenase from pig liver soluble fraction was purified over 500-fold with an overall yield of 25%. The purified enzyme, which is homogeneous by all the usual criteria, has a molecular weight of about 75000 and is composed of two identical subunits. This has been demonstrated by ultracentrifugation, fluorescence titration and peptide `fingerprinting'. The maximal turnover number, extinction coefficients at 280nm and 260nm and amino acid analysis are described.

scholarly journals 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

1970 ◽  
Vol 117 (1) ◽  
pp. 73-83 ◽  
Author(s):  
A. P. F. Flint ◽  
R. M. Denton
Keyword(s):  
Malate Dehydrogenase ◽  
Isocitrate Dehydrogenase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Nicotinamide Adenine ◽  
Tissue Homogenate ◽  
Kinetic Properties ◽  
Rat Ovary ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Effect Of Glucose

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 Km 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.

scholarly journals Properties of halophil nicotinamide–adenine dinucleotide phosphate-specific isocitrate dehydrogenase. True Michaelis constants, reaction mechanisms and molecular weights

1972 ◽  
Vol 130 (3) ◽  
pp. 645-662 ◽  
Author(s):  
D. M. Aitken ◽  
A. D. Brown
Keyword(s):  
Reaction Mechanism ◽  
Sodium Chloride ◽  
Potassium Chloride ◽  
Salt Concentration ◽  
Isocitrate Dehydrogenase ◽  
Reaction Mechanisms ◽  
Substrate Concentration ◽  
Gel Filtration ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Michaelis Constants

True values of Michaelis constants of the NADP+-specific isocitrate dehydrogenase from Halobacterium salinarium were not very different from those of the apparent constants reported by Aitken et al. (1970). The true constants were affected by salt in a similar manner to that of the apparent constants obtained with NADP+ at fixed concentrations of 1.0–0.2mm and threo-ds-(+)-isocitrate at fixed concentrations of 2.0–0.125mm. The response of apparent Vmax. to salt concentration was highly dependent on fixed substrate concentration in solutions of sodium chloride but much less so in solutions of potassium chloride. At several levels the results emphasize the difficulty of generalizing about the salt relations of a halophil enzyme without adequate attention to substrate concentration. The enzyme has at least two different reaction mechanisms depending on salt concentration. In its ‘physiological’ form (i.e. in 1.0m-potassium chloride), and also in 1.0m-sodium chloride, the reaction mechanism is ordered with NADP+ the first substrate added and NADPH the last product released. In 0.25m-sodium chloride, however, the mechanism is different and is probably non-sequential. In 4.0m-sodium chloride with low concentrations of either fixed substrate, there was evidence of a co-operative action of the variable substrate. The evidence suggests that salt participates in the reaction mechanism in two ways: one is the reversible addition to the enzyme in a manner analogous to that of a substrate; the other is dead-end complex-formation. The relative contributions of these two types of reaction determine whether salt activates or inhibits the enzyme. In addition, the inhibition caused by high concentrations of sodium chloride is more complex than the corresponding inhibition by potassium chloride. Gel-filtration experiments indicated that at very low salt concentrations the enzyme has an apparent molecular weight of about 70800. In ‘physiological’ concentrations of potassium chloride the enzyme appears to be a dimer (mol.wt. 122000–135000) and, in 1.0–4.0m-sodium chloride, it behaves as a trimer or tetramer (mol.wt. 224000–251000). A preliminary method of purifying the enzyme is described.

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