In situ compartmentation of creatine kinase in intact sarcomeric muscle: the acto-myosin overlap zone as a molecular sieve.
Creatine kinase isoenzymes (CK = ATP: creatine N-phosphoryl transferase, EC 2.7.3.2) were localized in situ in cryosections of intact sarcomeric muscle by immunocytochemical staining. Similar to cardiac muscle, spermatozoa and photoreceptor cells, mitochondrial-type CK (Mi-CK) localization in skeletal muscle was also restricted to mitochondria. Besides the well-documented localization of muscle-type (M-CK) at the M-line and at the sarcoplasmic reticulum, surprisingly, most of the sarcoplasmic M-CK was also highly compartmentalized and was mainly confined to the I-band. The localization of M-CK at the I-band coincided with that of adenylate kinase and aldolase. In intact muscle, the diffusion equilibrium decisively favours occupancy by all three enzymes of the I-band, with the acto-myosin overlap region of the A-band acting as a molecular sieve, excluding to a large extent all three enzymes from the acto-myosin overlap region. This indicates that in intact muscle, this region of the A-band may be less accessible in vivo to soluble, sarcoplasmic enzymes than thought before. If muscle were permeabilized by chemical skinning before fixation, I-band CK, as well as aldolase and adenylate kinase, were solubilized and disappeared from the myofibrils, but the fraction of M-CK which was specifically associated with the M-line remained bound to the myofibrils. Implications of these findings are discussed with respect to the functional coupling of I-band-CK with glycolysis, to the formation of large multienzyme complexes of glycolytic enzymes with CK and to the supply of energy for muscle contraction in general.
Wegmann
G
G
Institute for Cell Biology, Swiss Federal Institute of Technology, Zürich.
Zanolla
E
E
Eppenberger
H M
HM
Wallimann
T
T
D000199:Actins / Q000502:physiology; D000818:Animals; D002451:Cell Compartmentation; D002645:Chickens; D003402:Creatine Kinase / Q000032:analysis* / Q000378:metabolism; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000648:ultrastructure; D008954:Models, Biological; D009218:Myosins / Q000502:physiology; D012518:Sarcomeres / Q000201:enzymology* / Q000648:ultrastructure; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000648:ultrastructure
D000199:Actins; D007527:Isoenzymes; D003402:Creatine Kinase; D009218:Myosins