Isolation and Characterization of Inner Membrane-Associated and Matrix

The isotherm for isocitrate oxidation by potato (Solanum tuberosum L. var. Russet Burbank) mitochondria in the presence of exogenous NAD is characterized by a hyperbolic phase at isocitrate concentrations below 3 millimolar, and a sigmoid, or positively cooperative phase from approximately 3 to 30 mllmlar. The two forms of isocitrate dehydrogenase were separated and characterized following the sonication of mitochondria in 15% glycerol in the absence of buffer, followed by fractionation in a density step gradient to yield inner membrane and matrix components. The membrane-associated isocitrate dehydrogenase was found to have a Hill, or cooperativity, number of 1, while the Hill nmber of the matrix enzyme was 2.5. Upon digitonin extraction the cooperativity number of the membrane enzyme rose to 3.5. The isocitrate Km for the membrane enzyme was calculated to be approximately 5.9 x 10' molar, while the SO.5 for the matrix was 6.9 x 10' molar. The NAD Km for both enzymes was 150 micromolar. Whereas the membrane enzyme proved indifferent to adenine nucleotides, the matrix enzyme was arguably inhibited by AMP and ADP, and inhibited some 25% by 5 millimolar ATP. Both enzymes were negatively responsive to the mole fraction of NADH, the membrane enzyme being 50% inhibited at a mole fraction of 0.26, and the matrix enzyme by a mole fraction of 032. The suggestion is offered that the enzymes in question constitute two forms of a single enzyme, one peripherally associated with the inner membrane, and one soluble in the matrix. It Is proposed that a degree of regulation may be achieved by the apportionment of the enzyme between the bound and free forms. NAD (12). Whereas tricarboxylic acid cycle dehydrogenases have most often been assigned to the matrix, Srere (24) and Matlib and O'Brien (15) have made a persuasive case for the association to varying degrees ofputative matrix enzymes to the inner membrane ofmammalian mitochondria. Castor bean mitochondrial pyruvate dehydrogenase has been shown to be tightly bound to the inner membrane (19). In the presence of exogenous NAD, potato mitochondria show a dual, or dromedary, ICDH isotherm (12). The latter has been taken to reflect the superimposition of a positively cooperative isotherm with an S0.s of roughly 7 mm, representing matrix ICDH, on a hyperbolic isotherm with a Km of from 0.1 to 0.2 mM, representing membrane-associated ICDH. Herein we compare the kinetic characteristics of the two enzymes following their separation, and reaffirm the view that two forms of NAD-linked ICDH exist in potato mitochondria. MATERIALS AND METHODS Source of Enzyme. Potato tubers (Solanum tuberosum L. var. Russet Burbank) were cultivated by the Department of Vegetable Crops, University of California, Davis, and provided by courtesy of Professor Herman Timm. Tubers were stored at 7°C till use. Potato mitochondria were fractionated as described below, and the fractions were assayed for NAD-specific isocitrate dehydrogenase. Mitochondria Preparation. Four hundred g of potato tubers were homogenized as described previously (10), except that the The inability of fresh potato slices to oxidize citrate (9), together with the known regulatory characteristics of NAD-linked isocitrate dehydrogenase (1, 22), caused us to examine the behavior of ICDH4 in potato mitochondria from fresh slices. In the preceding paper (12), we were led to the view that potato mitochondria contain either two species of ICDH, or a single species in two states, one free in the matrix and another associated with the inner mitochondrial membrane on the matrix side. This deduction stemmed primarily from the disparate behavior ofICDH in intact potato mitochondria in the presence and absence of exogenous ' Supported by Research Grant GM 19807 from the United States Public Health Service. 2Cuffent address: Laboratory of Horticultural Science, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464, Japan. To whom reprint requests should be addressed. Abbreviations: ICDH, threo-Ds-isocitrate:NAD+ oxidoreductase (decarboxylating), EC 1.1.1.41. Z