This project will be a study of structure and regulation of expression of mitochondrial creatine kinase (Mt-CK) gene, with emphasis of studies in the mouse heart. For this purpose, I propose to clone and characterize by sequence analysis mouse sarcomeric specific (sMt-CK) and ubiquitous (uMt-CK) Mt-CK cDNAs. I will also isolate and partially characterize both mouse Mt-CK genes to compare both exonic structure and potential 5-regulatory sequences of the human and mouse genes. I then propose to begin experiments designed to 'knock out' or ablate the mouse sMt CK gene using mouse embryonic stem (ES) cells with subsequent impregnation of the altere cells into mice. During the second year, I intend to study the structural basis of Mt-CK interaction with the mitochondrial oxidative phosphorylation system. It is proposed to study the influence of different CK isoenzymes artificially incorporated into human hepatoma cell mitochondria on the kinetics of oxidative phosphorylation in isolated mitochondria and permeabilized hepatocytes.