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

ABSTRACT: 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.

SUBMITTER: Aitken DM

PROVIDER: S-EPMC1185331 | biostudies-other | 1970 Jan

REPOSITORIES: biostudies-other

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Project description: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.

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

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