Compartmentation of enzymes in the rat liver mitochondrial matrix

Abstract

Rat liver mitochondria were treated with increasing concentrations of digitonin (0.5–5 mg/mg protein) and the loss of latency of enzymes was measured as the permeability of the outer and inner membranes increased. The outer membrane became completely permeable to external cytochrome c at 0.1 mg digitonin/mg protein while succinate cytochrome c reductase was unmasked at a higher concentration 0.2 mg digitonin/mg protein. Enzymes bound on the inside of the inner membrane, e.g., β-hydroxybutyrate dehydrogenase, NADH oxidase, rotenone-sensitive NADH-vitamin K 3 reductase were unmasked at 0.3 mg digitonin/mg protein when the inner membrane became permeable to pyridine nucleotides. Matrix enzymes such as aconitase, NAD-isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, fumarase, pyruvate dehydrogenase, palmitoyl-CoA dehydrogenase, and ornithine transcarbamylase were also unmasked at this concentration. Much higher digitonin concentrations were required to unmask malate dehydrogenase, citrate synthase NADP-isocitrate dehydrogenase, glutamate dehydrogenase, aspartate-glutamate transaminase, and DT-diaphorase. Breaks in the Arrhenius plots were found in the former group of enzymes while the latter group showed a uniform activation energy change except in the case of citrate synthase and DT-diaphorase. An organization of enzymes in mitochondrial matrix was proposed and the significance of this finding was discussed with reference to the efficient operation and regulation of the Krebs cycle.