Biochemical studies on sub-fractions of rat liver mitochondria

Abstract

Highly purified mitochondria from rat liver were separated into six sub-fractions by differential centrifugation. The subfractions represent a spectrum from “heavy” to “very light” mitochondria. Enzymes representative of mitochondrial compartments were assayed to see whether functional differences occurred among the various mitochondrial sub-fractions. Respiratory control and NADH oxidase activity, both of which are indicators of mitochondrial structural integrity, were also measured. An enzyme marker for endoplasmic reticulum (glucose-6-phosphatase, G-6-Pase) was also assayed. Specific activities for monoamine oxidase (outer membrane marker), cytochrome oxidase (inner membrane marker) and malate-cytochrome c reductase did not vary within experimental error in all sub-fractions; similarly, for respiratory control and NADH oxidase activity. Malate dehydrogenase, a component of malatecytochrome c reductase is located within the matrix surrounded by the inner membrane. Specific activity of adenylate kinase (located between the outer and inner membrane) decreased markedly from the “heavy” mitochondria to the “very light” fractions. Specific activity for G-6-Pase, very low in the “heavy” fractions, increased markedly in the “light” to “very light” fractions. Isopycnic density centrifugation on a linear sucrose density gradient of each of the fractions indicated that the correlation coefficient for the sucrose concentrations at which cytochrome oxidase and G-6-Pase activities peaked was 0.995. Thus the “light” to “very light” mitochondria may represent mitochondria whose outer membrane is still contiguous with the endoplasmic reticulum. Microsomes containing the endoplasmic reticulum peaked on the gradient at a significantly lower sucrose concentration than any of the mitochondrial sub-fractions. A buoyant effect of endoplasmic reticulum still attached to any of the mitochondrial sub-fractions would be expected to lower the density of attached mitochondria and thus give rise to “light” and “very light” mitochondria.