In situ compartmentation of creatine kinase in intact sarcomeric muscle: The acto-myosin overlap zone as a molecular sieve

Abstract

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.

Cytoarchitectural and metabolic adaptations in muscles with mitochondrial and cytosolic creatine kinase deficiencies

“…In muscle, MM-CK mediated ATP production is mainly coupled to the local activity of SR-and plasma-bound Ca 2+ -ATPases [6,8,9,32], the Na + /K + -ATPase [33,34] and the myosin ATPase involved in actin-myosin sliding during contraction in the sarcomeric M-band [35][36][37][38][39]. The MM-CK mediated reaction is also topologically coupled to the glycolytic metabolic reaction cascade in the I-band thinfilament region [35].…”

Biochemical characterization of the mouse muscle-specific enolase: developmental changes in electrophoretic variants and selective binding to other proteins

“…Previous observations suggest that glycolytic enzymes are involved in the formation of multiprotein complexes [18,19]. Thus in muscle cells, the structural characteristics of muscle-specific isoenzymes [20][21][22] would permit their accumulation near the muscle contractile apparatus, so that the ATP produced via glycolysis could be more efficiently delivered and used for contraction.…”

Impaired Intracellular Energetic Communication in Muscles from Creatine Kinase and Adenylate Kinase (M-CK/AK1) Double Knock-out Mice

“…cDNAs for five isoforms of AK (AK1-AK5) along with the variant of AK1 (AK1␤, a membranebound form with a presumed role in cell cycle regulation) have been cloned from metabolically active tissues (9 -12). Mammalian skeletal muscle is particularly rich in AK1, the major isoform of the family (9), present in the sarcoplasm, and clustered along the myofibrillar I-band or bound as AK1␤ to membranes (13)(14)(15). By donating the energy of the ␤-phosphoryl group of ATP/ADP to the cellular energetic pool, AK isoenzymes protect cells against energy deprivation in periods of high metabolic demand (6, 16 -20).…”

“…Creatine kinases are foremost found in cells with high peak demands in metabolic energy such as the brain, heart, or skeletal muscle (1,5). In skeletal muscle, the principal CK isoform is the cytosolic isoform, MM-CK, a homodimer mainly present as a soluble protein in the cytosol and bound to the myofibrillar M-and I-bands (13) as well as to the sarcoplasmic reticulum membranes (25). Skeletal muscle also contains an additional mitochondrial CK isoform (ScCKmit), which amounts to 1-10% of the total CK activity depending on the type of muscle fiber (26,27).…”