The role of lipides in electron transport. III. Purification and identification of a cytochrome c reductase lipide cofactor.

Recent investigations have implicated vitamin E as a possible cofactor for the oxidized diphosphopyridine nucleotide (DPN)and succinate-cytochrome c reductases of rat skeletal muscle and bovine heart muscle. It was demonstrated that added vitamin E increases the activity of the above cytochrome c reductases. Moreover, the almost complete loss of enzymatic activity resulting from isooctane extraction can be completely restored by the addition of tocopherol, or even more effectively by addition of the lipide residue obtained from vacuum distillation of the isooctane after extraction (l-3). Morrison, Crawford, and Stotz (4) have recently confirmed these reactivation effects by using isooctane-extracted succinate-cytochrome c reductase from pig heart. The present paper elaborates on preliminary reports (5, 6) from this laboratory with regard to the purification and identification of the active component (designated as the lipide “cofactor”) in the crude lipide residue obtained by isooctane or n-hexane extraction of bovine heart muscle homogenate. Evidence is presented to show that the lipide “cofactor” is a mixed triglyceride with stearate, palmitate and oleate components. The subsequent paper of this series (7) describes experiments demonstrating that the crude lipide residue obtained by isooctane extraction of the cytochrome c reductases from rat skeletal muscle and bovine heart muscle contains vitamin E in amounts too small, however, to account for enzyme reactivation. Further evidence is given suggesting that the added lipide “cofactor” acts indirectly by releasing endogenous vitamin E of the preparation to the “active sites” of the enzyme.