Abstract
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 millimolar. 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 number of the matrix enzyme was 2.5. Upon digitonin extraction the cooperativity number of the membrane enzyme rose to 3.5. The isocitrate K(m) for the membrane enzyme was calculated to be approximately 5.9 x 10(-4) molar, while the S(0.5) for the matrix was 6.9 x 10(-4) molar. The NAD K(m) 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 0.32. 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.
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