Oxidative and Phosphorylative Activities of Tightly Coupled Mitochondria Isolated fromVicia fabaL.

Abstract

A procedure is described for the isolation of tightly coupled mitochondria from Vicia faba L. These mitochondria rapidly oxidized malate, succinate, and NADH and had good respiratory control (RC) with ADP together with high ADP/O ratios. The ADP/O ratios with malate as substrate varied between 2.33 and 3.0 with average RC of 2–4. With succinate as substrate the average ADP/O ratios observed were 1.75–2.0 and RC 2.0–4.0. Broad bean mitochondria oxidized externally added NADH with ADP/O ratios never more than 2.0. α-Oxoglutarate was oxidized slowly when TPP was incorporated in the reaction medium. Broad bean mitochondria were sensitive to classical uncouplers and inhibitors in the normal fashion but NADH oxidation was relatively insensitive to rotenone and amytal. The effect of some other parameters, viz. age of seedlings, stability of isolated mitochondria, pH of the reaction medium, presence and absence of EDTA, EGTA, BSA, and MgCl₂ is also described.