Mitochondrial creatine kinase from chicken brain. Purification, biophysical characterization, and generation of heterodimeric and heterooctameric molecules with subunits of other creatine kinase isoenzymes.
In a recent study it has been shown that mitochondrial creatine kinase from chicken brain (Mia-CK) and heart (Mib-CK) are two distinct isoenzymes differing in ten out of the thirty N-terminal amino acids (Hossle, J.P., Schlegel, J., Wegmann, G., Wyss, M., Böhlen, P., Eppenberger, H.M., Wallimann, T., and Perriard J.C. (1988) Biochem. Biophys. Res. Commun. 151, 408-416). The present article describes the purification and biophysical characterization of the mitochondrial creatine kinase isoenzyme from chicken brain (Mia-CK). Gel permeation chromatography, direct mass measurements of individual molecules by scanning transmission electron microscopy, and analytical ultracentrifugation confirmed the existence of two different oligomeric forms, dimeric and octameric Mia-CK, with molecular masses of 85 kDa and 306-352 kDa and with sedimentation constants of 4.9-5.3 and 11.6-12.0 S, respectively. In addition, it was tested if Mia- and Mib-CK can form heterodimeric and heterooctameric molecules with subunits of other CK isoenzymes. By denaturation in urea or guanidine hydrochloride and subsequent renaturation, MiaMib-CK and surprisingly also MiaM-CK heterodimers could be generated. In contrast, no heterodimers were obtained between Mib- and M- or B-CK. Furthermore, reoctamerization of a mixture of Mia- and Mib-CK homodimers led to the formation of MiaMib-CK heterooctamers. In these heterooctamers, the Mia- and Mib-CK homodimers remained the fundamental building blocks. No subunit exchange between adjacent dimers within the heterooctamer could be observed even after storage for 3 months at 4 degrees C. The relevance of these data on the structural organization of the Mi-CK octamer and on the physiological aspects of tissue-specific isoenzyme expression are discussed.
Institute of Cell Biology, ETH Hönggerberg, Zürich, Switzerland.
M
M
Wyss
J
J
Schlegel
P
P
James
H M
HM
Eppenberger
T
T
Wallimann
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002645:Chickens; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003402:Creatine Kinase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012689:Sequence Homology, Nucleic Acid
D007527:Isoenzymes; D046911:Macromolecular Substances; D003402:Creatine Kinase