Oxidative phosphorylation in rat liver mitochondria isolated by rate zonal centrifugation: Examination of Ficoll gradients and subpopulations of mitochondria

Abstract

In order to measure the parameters of oxidative phosphorylation it is necessary to isolate physiologically intact mitochondria. The isolation of rat liver mitochondria by rate zonal centrifugation utilizing isoosmotic Ficoll gradients resulted in the uncoupling of oxidative phosphorylation in these organelles. Analysis of the Ficoll solutions used to construct the gradients indicated that the Ca2+ content (200-400 nmole Ca2+/mg protein) was sufficiently high to cause an uncoupling of oxidative phosphorylation. Treatment of the Ficoll solutions with Amberlite MB-3 resin reduced the Ca2+ content to levels below the limit of determination of the assay procedure. This resulted in the retention of respiratory control (1.42) in rate-zonally centrifuged mitochondria. The addition of bovine serum albumin (100 mg %) to the Ficoll gradients increased the respiratory control index to 2.10. The increase is due to an elevation in state 3 respiration rather than any change in state 4 respiration. The addition of 200 mg % bovine serum albumin to the Ficoll gradient did not further enhance the respiratory control index. Examination of subpopulations of rat liver mitochondria revealed that they are heterogeneous with regard to states 3 and 4 respiration, respiratory control indices, and ADP : O ratios. In mitochondrial subpopulations respiratory control indices ranged from 1.00 to 4.13 and ADP : O ratios ranged from 1.22 to 1.83. This investigation defined a procedure for the isolation of physiologically intact mitochondria from rat liver homogenates.