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Identification and primary structure of the cardiolipin-binding domain of mitochondrial creatine kinase
1Laboratory of Biochemistry, Swiss Federal Institute of Technology, Zürich.
Abstract:
It was recently shown that the mitochondrial isozyme 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.
Insights
Researchers identified the cardiolipin-binding domain of heart creatine kinase, a crucial mitochondrial enzyme. This N-terminal fragment
Area of Science:
- Biochemistry
- Molecular Biology
- Cardiology
Background:
- Mitochondrial heart creatine kinase (CK) is known to bind cardiolipin.
- Cardiolipin is a key phospholipid in the inner mitochondrial membrane.
Purpose of the Study:
- To identify and characterize the cardiolipin-binding domain of heart creatine kinase.
- To investigate the role of specific amino acids in cardiolipin binding.
Main Methods:
- Purification of heart creatine kinase from rat mitochondria.
- Cleavage of the enzyme with CNBr and separation of fragments using FPLC.
- Liposome binding assays to identify the cardiolipin-binding domain.
- Amino acid sequencing of the identified fragment.
- Chemical modification of basic amino acids (Lysine and Arginine).
Main Results:
- A 25-amino acid N-terminal fragment was identified as the cardiolipin-binding domain.
- Adriamycin inhibited the binding of this fragment to cardiolipin-containing liposomes.
- A separate CNBr fragment was identified as the ATP-binding domain.
- Chemical modification of Lysine and Arginine residues abolished cardiolipin binding.
Conclusions:
- The N-terminal region of heart creatine kinase contains the cardiolipin-binding domain.
- Specific basic amino acids (Lysine and Arginine) are essential for cardiolipin interaction.
- Adriamycin may interact with or affect the cardiolipin-binding site.