Nicotinamide Adenine Dinucleotide Phosphate - specific Isocitrate

Nicotiniamide adenine dintucleotide plhosphate-specific isocitrate dehydrogenase was extracted from etiolated pea (Pisurn satirum L.) seedlings and was purified 65-fold. The puirified enzyme exhibits one predoniinant protein band by pol acrylamide gel electrophoresis, w-hich corresponds to tiie dehy-drogenase activity as imieasuired by the nitro bluie tetrazolium technique. The reaction is readily reversible, the pH optima for the forward (nicotinamide adenine dinuticleotide phosphate reductiotn) and reverse reactions being 8.4 and 6.0, respectively. T'he enzyme has different cofactor and inhibitor characteristics in the two directions. iManganese ions can be used as a cofactor for the reaction inl each direction but magnesium ions only act as a cofactor inl the forward reaction. Zinc ions, and to a lesser extent calcium ions, inhibit the enzyme at low concentrations wlhen magnesiunm but niot manganese is the metal activator. It is suggested that there is a fundamental difference between magnesiumli and nmanganese in the activation of the enzmnie. The enzvme slhows normal kinetics anid the MNichaelis contant for eachi suibstrate was deternmined. The inhibition by nucleotides, inujcleosides, reactionproducts, and related compounds was stuidied. The enzy-nle slhows a linear response to the nmole fraction of reduced nicotiniamide adenine dintucleotide phosphlate wlhen total niicotinamide adenine dinucleotide plhosphate (nicotinaniide adenine dinucleotide phosphate plus reduced nicotintamllide adenine dinucleotide phosph-ate) is kept constant. Isocitrate in the presence of divalent metal ions will protect thte enzyme from inactivationi by p-chloromercuribenzoate. P'rotection is also afforded by mllanganese ions alone but not by magnesiuim ions alone There is a con-certed inhibition of the enzyme by oxalacetate anld glyoxylate. The NAD+-specific isocitrate dehydrogenase (threo-D,-isocitrate:NAD+ oxidoreductase [decarboxylating] EC 1 .1 .1 .41) from higher plant mitochondria has been studied (4-7, 9-11) and has properties consistent with a role in regulating the tricarboxylic acid cycle. The enzyme shows complex kinetics with respect to the substrate isocitrate and appears to require both free and magnesium complexed isocitrate (11). It has been sug1 This work was supported by grant no. A5051 from the National Research Council of Canada. gested that the principal regulation of the enzyme is by the ratio ofNADH to NAD+ (5,9, 10). In contrast, the NADP+-specific isocitrate dehydrogenase (threo-D,-isocitrate: NADP+ oxidoreductase [decarboxylating], EC 1 .1 .1.42) from higher plants has received little attention although it has been reported to be present in swede storage tissue (23). The enzyme from bacterial (14, 18, 19), protozoan (15-17) and pig heart sources (2, 3) has recently been studied and in all cases has shown to have normal kinetics with respect to the substrates. In contrast to the NAD+-specific enzyme it readily catalyzes the reactions both in the forward direction (NADP+ reduction) and in the reverse direction. The enzyme also shows activity with respect to oxalosuccinic acid (20, 21), a postulated intermediate in the reaction. It has been suggested that the NADP+-specific enzyme is regulated by the concentration of ATP in the cell (14, 16). There are also indications that there is a concerted inhibition when glyoxylate and oxalacetate are added together (15, 17-19). The importance of these findings when considering metabolic regulation is difficult to assess since the function of the enzyme in the cell of eucaryotic organisms which also possess the NAD+-specific enzyme is not known. It is possible that the NADP+-specific enzyme functions to supply NADPH for biosynthetic purposes or to provide NADH via a transhydrogenase reaction (22). In these cases it might be expected that the enzyme would be regulated by the NADPH:NADP+ ratio rather than by the ATP concentration. In view of these results it was decided to study the NADP+specific enzyme from etiolated pea stems and compare its properties with the properties of the NAD+-specific enzyme from the same source which have already been presented (4-7, 9-11). As with the NAD+-specific enzyme (4) there appears to be a considerable difference between the activation of the enzyme by Mn2+ and Mg2+, the latter only acting as cofactor in the forward direction. In the forward reaction the enzyme has a much lower Km for Mn2+ than Mg2+ and the maximal velocity is also considerably higher. In the reverse reaction, only Mn2+ will activate. Unlike the NAD+-specific enzyme the NADP+-specific enzyme is not greatly affected by the mole fraction of reduced pyridine nucleotide when total pyridine nucleotide (NADP+ + NADPH) is kept constant. It is also much more sensitive to Zn2+ inhibition than the NAD+-specific enzyme when Mg2+ but not Mn2+ is the metal activator. Ca + behaves in a similar manner to Zn2+. The enzyme is inhibited by certain nucleosides and nucleotides; part of the inhibition is due to metal complexing. The enzyme is sensitive to p-chloromercuribenzoate but can be protected by isocitrate in the presence of metal ions or by Mn + alone but not by Mg2+ alone. There is also an apparently concerted inhibition by glyoxylate and oxalacetate.