Compartmented coupling of chicken heart mitochondrial creatine kinase to the nucleotide translocase requires the outer mitochondrial membrane

Abstract

The kinetic coupling of mitochondrial creatine kinase (MiMi-CK) to ADP ATP translocase in chicken heart mitochondrial preparations is demonstrated. Measuring the MiMi-CK apparent K m value for MgATP 2− (at saturating creatine) gives a value of 36 μ m when MiMi-CK is coupled to oxidative phosphorylation. This K m value is threefold lower than the K m for enzyme bound to mitoplasts or free in solution. The nucleotide translocase K m value for ADP decreases from 20 to 10 μ m in the presence of 50 m m creatine only with intact mitochondria. Similar experiments with mitoplasts do not give decreased K m values. The observed K m differences can be used to calculate the concentration of ATP and ADP under steady-state conditions showing that the observed differences in the kinetic constants accurately reflect the enzyme activities of MiMi-CK under the different conditions. The behavior of the K m values provides evidence for what we term compartmented coupling. Therefore, like the rabbit heart system (S. Erickson-Viitanen, P. Viitanen, P. J. Geiger, W. C. T. Yang, and S. P. Bessman (1982) J. Biol. Chem. 257, 14395–14404) compartmented coupling requires an intact outer mitochondrial membrane. The apparent K m values for normal or compartmentally coupled systems can be used to calculate steady-state values of ATP and ADP by coupling enzyme theory. Hence, the overall kinetic parameters accurately reflect the behavior of the enzymes whether free in solution or in the intermembrane space.