Studies on the stabilization of an oxidative phosphorylation system. I. Resistance of a phosphorylating system of submitochondrial particles to trypsin, due to phosphorylation of ADP

Abstract

The action of trypsin on the phosphorylating system of submitochondrial particles was studied under various conditions. The rate of proteolysis of the phosphorylating system did not decrease when the particles, acted upon by trypsin, simultaneously oxidized some substrate (NADH or succinate). The phosphate acceptor (ADP) itself did not stabilize the phosphorylating system either. The latter became definitely resistant to trypsin only under conditions favouring oxidative phosphorylation ( i.e. in the presence of a substrate, oxygen, ADP and phosphate), or after treatment by ATP. The protective effect was manifested when measuring the P/O ratio, the rate of the ATP- 32P i exchange reaction and the rate of the ATP-dependent NAD + reduction by succinate. This effect depended on the rate and efficiency of the functioning of the oxidative phosphorylation system. Stabilization was maximal when the rate of NADH oxidation was 30–50 nmoles/min per mg of protein, and the P/O ratio was not lower than 1.9–2.0. The stabilizing action of succinate and ADP vanishes when oleate is added in the concentration necessary for complete uncoupling of respiration and phosphorylation. These facts indicate that under definite conditions stabilization of a phosphorylating system is a result of its functioning. An assumption is advanced according to which the composition and structural organization of mitochondria are such as to ensure maximum resistance of the phosphorylating system to proteolysis.