Cytochemical Studies of Mitochondria

Abstract

1. Mitochondria isolated from rat liver were disrupted with 0.3 per cent deoxycholate and a number of subfractions were isolated from this preparation by differential centrifugation. 2. The protein N, RNA and phospholipide content, as well as the succinoxidase, cytochrome c oxidase, adenylate kinase, and DPNH-cytochrome c reductase of these fractions were determined. 3. Two of these subfractions, found to consist of mitochondrial membranes (2), contained ∼ 12 per cent of the protein N and ∼ 35 per cent of the phospholipide of the whole mitochondria and accounted for ∼ 70 per cent of the succinoxidase and cytochrome c oxidase activity of the original mitochondrial preparation. There was no discernible adenylate kinase, DPNH-cytochrome c reductase, or phosphorylating activities in these fractions, nor could they oxidize other substrates of the Krebs's cycle. 4. The most active fraction (60 minutes at 105,000 g pellet) had a higher phospholipide/protein value than the whole mitochondria and showed a seven-to elevenfold concentration of succinoxidase and cytochrome c oxidase activities. 5. Evidence has been given to indicate that the various components of the succinoxidase complex are present in this membrane fraction in the same relative proportions as in the whole mitochondria. 6. The implications of these findings are discussed.

Enzyme-Structure Relationships in the Endoplasmic Reticulum of Rat Liver

“…A possible explanation of this parallelism is an extensive contamination of each of these two fractions by whole units or subunits that properly belong to the other. This possibility is ruled out or rendered negligible, however, by the fact that certain activities are not shared by the two fractions: glucose-6-phosphatase is restricted to the microsomes, and suecinoxidase activity is found only in mitochondria, more precisely in mitochondrial membranes (62). Mitochondrial subunits poor in succinoxidase but rich in NADH oxidase activity have been isolated (63,64) by a rather drastic and elaborate procedure, but it is hardly conceivable that such subunits could form at the expense of mitochondria during tissue homogenization (to be available for the contamination of the microsomes).…”

Studies on the Content and Organization of the Respiratory Enzymes of Mitochondria

“…The details of this figure are discussed in the text. (2) and Siekevitz and Watson (22). These pigments are presumed to be bound with lipids (30) and are, according to present speculation, of rather high molecular weight (cytochrome c, the only member of the respiratory chain to be isolated in relatively pure form is, however, of rather low molecular weight, i.e., 12,000).…”

Studies on the Cornea

“…Siekevitz and Watson (65,66) have demonstrated, in combined electron microscopic and cytochemical studies of isolated membrane fractions from rat liver mitochondria, that better than 70 per cent recovery of the succinic dehydrogenase and cytochrome c oxidase activity of the intact mitochondria is found associated with one of the membrane fractions.…”