Mitochondrial creatine kinase from cardiac muscle and brain are two distinct isoenzymes but both form octameric molecules.

J Schlegel,M Wyss,U Schürch,T Schnyder,A Quest,G Wegmann,H M Eppenberger,T Wallimann

doi:10.1016/s0021-9258(18)37484-2

Abstract

Mitochondrial creatine kinase (Mi-CK) from chicken cardiac muscle and brain, recently shown to differ in their N-terminal amino acid sequences and to be encoded by multiple mRNAs (Hossle, H.P., Schlegel, J., Wegmann, G., Wyss, M., Böhlen, P., Eppenberger, H. M., Wallimann, T., and Perriard, J.C. (1988) Biochim. Biophys. Res. Commun. 151, 408-416) were separated on two-dimensional nonequilibrium pH-gradient electrophoresis gels and visualized as two distinct protein spots by immunoblotting. Analysis of the two proteins purified by specific elution from Blue-Sepharose with ADP (Wallimann, T., Zurbriggen, B., and Eppenberger, H. M. (1985) Enzyme 33, 226-231) followed by fast protein liquid chromatography cation exchange chromatography showed obvious differences in peptide maps, in immunological cross-reactivity with monoclonal antibodies, and in kinetic parameters. However, even though the two proteins were different, tissue-specific mitochondrial isoforms, both formed regularly-sized, perforated cube-like octameric structures with Mr of 364,000 +/- 25,000 and 352,000 +/- 20,000 for the cardiac and brain isoform, respectively. Electron microscopy of cardiac and brain Mi-CK octamers revealed cube-like molecules with a central cavity or transverse channel filled by negative stain. The octameric molecular structure of Mi-CK isoforms differs from the generally accepted dimeric arrangement of "cytosolic" muscle MM- and brain BB-CK.

Highlights

Mitochondrial creatine kinase (Mi-CK)from chicken cardiac muscle and brain, recently shown to differ in their N-terminal amino acid sequences and to be encoded by multiple mRNAs
The two mitochondrial CK isoforms were resolved as two distinct spots when phosphate extracts from cardiac andbrain mitochondria adjusted to contain equal amounts of CK activity were coelectrophoresed
They were blotted by electrophoretic transfer onto nitrocellulose andstained by monospecific polyclonal rabbit antichicken cardiac Mi-CK and (Mi)-CK antibodies which were characterized in this laboratory [37]

Summary

Introduction

Mitochondrial creatine kinase (Mi-CK)from chicken cardiac muscle and brain, recently shown to differ in their N-terminal amino acid sequences and to be encoded by multiple mRNAs Play a crucial role in the energy supply within tissues of high, sudden energy demand, e.g. cardiac and skeletal muscle [911], brain [12, 13], spermatozoa [14, 15], and photoreceptor cells of the retina [16] In these tissues or cells Mi-CK is always coexpressed with one of the cytosolic CK-isoforms, MM-CK or BB-CK. The computer-assisted analysis of sea urchin sperm-tail oscillation clearly revealed that existing diffusional restraints between the spermatozoan mid-piece,where energy (ATP/CP) is generated inthe mitochondria, andthetail region, where energy is utilized by the dynein ATPase, are overcome by a CP shuttle working in a similar way in spermatozoa [14] as in muscle [11].A progressive loss of spermtail oscillation starting at thedistal end of the sperm tails is observed if the functionally segregated CK isoenzyme populations are inactivatedstepwise by dinitrofluorobenzene [30]

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