Isolation and characterization of acetylcholine receptor membrane-associated (nonreceptor v2-protein) and soluble electrocyte creatine kinases.
Creatine kinase has been identified as a most prominent component of Torpedo electric organ and a minority constituent of the acetylcholine receptor (AChR) membranes obtained therefrom. Purification by low temperature ethanol extraction, precipitation of the Mg2+-enzyme complex, and mercurial-agarose chromatography yield preparations of soluble kinase with specific activities greater than 550 units/mg protein. Retention times in ion-exchange high performance liquid chromatography, electrophoretic behavior, immunochemical properties, tryptic mapping, and amino acid composition enable the comparison of creatine kinase isoenzymes. The denatured subunits of the predominant species have pI values of 6.3-6.8 and Mr = 40,000-42,000 characteristic of the so-called v2 proteins and show cross-reactivity with antibodies against the BB ("brain" type) creatine kinase. The MM ("muscle" type) antigens could be detected in the total electrocyte, but not in the AChR membranes; they have a slightly lower molecular weight and higher pI. The in situ membrane association of the BB isoenzyme is confirmed by immunocytochemistry. The apparent Km values for the substrate creatine phosphate are 2.2 mM for the AChR membrane-associated enzyme and 2.5 mM for the muscle form. The apparent Km values for Mg2+-ADP are 0.54 and 0.22 mM, respectively. Thus, a 2-fold higher affinity in the binding of ADP to the binary enzyme-creatine-P complex results from membrane association.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003402:Creatine Kinase / Q000032:analysis*; D004557:Electric Organ / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D007527:Isoenzymes; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D009132:Muscles / Q000201:enzymology; D011950:Receptors, Cholinergic / Q000302:isolation & purification*; D012995:Solubility; D014101:Torpedo
D000596:Amino Acids; D007527:Isoenzymes; D046911:Macromolecular Substances; D011950:Receptors, Cholinergic; D003402:Creatine Kinase; D008274:Magnesium