Compartmentation of mitochondrial creatine phosphokinase. II. The importance of the outer mitochondrial membrane for mitochondrial compartmentation.

In order to define further the nature of the apparent close interaction between mitochondrial creatine phosphokinase and oxidative phosphorylation (Erickson-Viitanen, S., Viitanen, P., Geiger, P. J., Yang, W. C. T., and Bessman, S. P. (1982) J. Biol. Chem. 257, 14395-14404), rabbit heart and rat skeletal muscle mitochondria prepared by gentle mechanical homogenization were compared with preparations isolated after tryptic digestion of tissues, a method which has been reported to yield superior mitochondria (Reichert, M., Schaller, H., Kung, W., and Gerber, G. (1978) Acta Biol. Med. Germ. 37, 1167-1176). The ability of de novo synthesized and exported mitochondrial ATP to interact with creatine phosphokinase prior to total mixing of the ATP pool, which we consider to be evidence of compartmentation, could not be demonstrated with mitochondria prepared via the trypsin procedure. Mitochondria from rabbit cardiac muscle treated with digitonin synthesized ATP and creatine phosphate, but failed to show apparent compartmentation of creatine phosphokinase. Km values for ATP were compared for four conditions: 1) respiring, digitonin-treated rabbit heart mitochondria, 2) atractyloside-inhibited, digitonin-treated rabbit heart mitochondria supplied with a pyruvate kinase-phosphoenolpyruvate regenerating system, 3) respiring rabbit heart mitochondria, 4) atractyloside-inhibited rabbit heart mitochondria supplied with an ATP regenerating system. The observed Km values for ATP for conditions 1, 2, and 3 were similar but lower than that for condition 4. These findings suggest that an outer membrane diffusion barrier influences or controls mitochondrial creatine phosphokinase compartmentation.