SEPARATION OF MITOCHONDRIAL MEMBRANES OF NEUROSPORA CRASSA II. Submitochondrial Localization of the Isoleucine-Valine Biosynthetic Pathway

Separation of Neurospora mitochondrial outer membranes from the inner membrane/ matrix fraction was effected by digitonin treatment and discontinuous density gradient centrifugation . The solubilization of four isoleucine-valine biosynthetic enzymes was studied as a function of digitonin concentration and time of incubation in the detergent . The kinetics of the appearance of valine biosynthetic function in fractions outside of the inner membrane/ matrix fraction, coupled with enzyme solubilization patterns similar to that for the matrix marker, mitochondrial malate dehydrogenase, indicate that the four isoleucine-valine pathway enzymes are localized in the mitochondrial matrix . mitochondria of Neurospora crassa and characterized the enzyme L-kynurenine-3-hydroxylase (KH) as an outer membrane marker, and succinatecytochrome c reductase (SCCR) as an inner membrane marker. In this study, digitonin subfractionation has been used to uncover evidence that a group of four enzymes catalyzing the over-all biosynthesis of two branched chain amino acids, isoleucine and valine, from pyruvate14 C in isolated Neurospora mitochondria (9), behave as soluble enzymes loosely associated in the mitochondrial matrix. These enzymes are an acetohydroxy acid synthetase (AAS), an acetohydroxy acid reductoisomerase (RI), a dihydroxy acid dehydratase (DH), and a branch chained amino acid amino