Identification and primary structure of the cardiolipin‐binding domain of mitochondrial creatine kinase

Abstract
It was recently shown that the mitochondrial isozmye of heart creatine kinase binds to cardiolipin on the outer half of the inner membrane [Müller, M., et al. (1985) J. Biol. Chem. 260, 3839–3843]. The enzyme has now been extracted and purified to homogeneity from rat heart mitochondria, and cleaved with CNBr. The fragments have been separated on an FPLC system using a Mono Q HR 5/5 column. Only one of these binds to cardiolipin‐containing liposomes and has thus been identified as the cardiolipin‐binding domain of the enzyme. Its amino acid sequence has been determined. The fragment contains 25 amino acids and corresponds to the N‐terminal region of the protein. The binding of the fragment of cardiolipin‐containing liposomes was inhibited by adriamycin. Another and larger CNBr fragment could be specifically labelled with periodate‐oxidized (di‐aldehyde) ATP and has thus been identified as the ATP‐binding domain. Chemical modification of the basic amino acids Lys and Arg of the enzyme abolished its binding to cardiolipin.

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