Abstract
Creatine kinase isoenzymes from Torpedo californica electric organ, skeletal muscle, and brain were purified and characterized. Torpedo electric organ and skeletal muscle creatine kinase have identical apparent Mr, electrophoretic mobility, and cyanogen bromide fragments. The electrophoretic mobility of the Torpedo creatine kinase was anodal as compared to mammalian MM creatine kinase. No creatine kinase isoenzyme with an electrophoretic mobility similar to mammalian BB creatine kinase was seen in any of the Torpedo tissues examined. Hybridization studies demonstrate the Torpedo electric organ creatine kinase to be composed of identical subunits and capable of producing an enzymatically active heterodimer when combined with canine BB creatine kinase. Creatine kinase from sucrose gradient-purified Torpedo electric organ acetylcholine receptor-rich membranes has an electrophoretic mobility identical with the cytoplasmic isoenzyme and an apparent Mr identical with mammalian MM creatine kinase. Western blot analysis showed Torpedo electric organ skeletal muscle creatine kinase and acetylcholine receptor-enriched membrane creatine kinase reacted with antiserum specific for canine MM creatine kinase. NH2-terminal amino acid sequence determinations show considerable sequence homology between human MM, Torpedo electric organ, chicken MM, and porcine MM creatine kinase. The acetylcholine receptor-associated creatine kinase is, therefore, identical with the cytoplasmic form from the electric organ and is composed of M-subunits.
Highlights
Creatine kinase was purified from Torpedo electric organ, skeletal muscle, andbrain forcomparison tocanineand human creatinekinase
Molecularweight standards were obtained fromPharmacia Fine Chemicals (Piscataway, NJ). was obtained fromAmersham.Creatine kinase was assayed with Calbiochem Single Vial Reagent, Calbiochem-Behring Corp., and CK-NAC Reagent, Boehringer Mann-Acetylcholine receptor-rich membranes from Torpedo are enriched for three 43-kDa proteins referred to as the peripheral or Y proteins aswell as the five structural polypeptides of the acetylcholine receptor
Electrophoresis-Creatine kinase purifiedfrom Torpedo californicu electric organ and skeletal muscle migrated as a single specieswith an apparenmt olecular weightof 43,000on
Summary
$ T o whom correspondence and requests for reprints should be addressed. The abbreviationsused are:AcChoR,acetylcholine receptor;SDS, sodium dodecyl sulfate. The preparation was dialyzed overnight against 50 mM Tris-HC1, pH7.5,5 mM 2-mercaptoethanol.The preparation was centrifuged and concentrated to a volume of 30 ml in an Amicon chamber using a PM-30 membrane. The sample was applied to a chromatofocusing column (1 X 60 cm) packed with Polybuffer Exchanger-94 equilibrated with 25 mM Tris/acetate, pH 8.3,5 mM 2mercaptoethanol. All other creatine kinases were purified as previously described [7]. Isolation of AChoR-rich Membranes-Acetylcholine receptor-rich membranes were purified and analyzed by the procedure of Burden et al [12].The creatine kinase specific activity was 16-22 units/mg. Hybridization Experiments-To determine whether Torpedo electric organ creatine kinase is a heterodimer or composed of identical subunits, the isoenzymewas denatured by exposure to 6 M guanidine HCI, 50 mM Tris-HC1, pH 7.5, 5 mM 2-mercaptoethanol for 30 min. Protein SequenceDetermination-The amino-terminal amino acid sequence of Torpedo and human M creatine kinase was determined as described by Hewick et al [18]
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