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Creatine kinase regulation by reversible phosphorylation in frog muscle
Authors:Christopher A Dieni  Kenneth B Storey  
Institution:aDepartment of Chemistry, The Pennsylvania State University, University Park, PA 16802, United States;bInstitute of Biochemistry and Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6
Abstract:Creatine kinase (CK) was analyzed from skeletal muscle of wood frogs, Rana sylvatica, a species that survives natural whole body freezing during the winter months. Muscle CK activity increased by 35% and apparent Km creatine decreased by 29% when frogs froze. Immunoblotting analysis showed that this activity increase was not due to a change in total CK protein. Frog muscle CK was regulated by reversible protein phosphorylation; in vitro incubations with 32P-ATP under conditions that facilitated the actions of various protein kinases (PKA, PKG, PKC, CaMK or AMPK) resulted in immunoprecipitation of 32P-labeled CK. Furthermore, incubations that stimulated CaMK or AMPK altered CK kinetics. Incubation under conditions that facilitated protein phosphatases (PP2B or PP2C) reversed these effects. Phosphorylation of CK increased activity, whereas dephosphorylation decreased activity. Ion-exchange chromatography revealed that two forms of CK with different phosphorylation states were present in muscle; low versus high phosphate forms dominated in muscle of control versus frozen frogs, respectively. However, CK from control versus frozen frogs showed no differences in susceptibility to urea denaturation or sensitivity to limited proteolysis by thermolysin. The increased activity, increased substrate affinity and altered phosphorylation state of CK in skeletal muscle from frozen frogs argues for altered regulation of CK under energy stress in ischemic frozen muscle.
Keywords:Rana sylvatica  Vertebrate freeze tolerance  Muscle energy metabolism  Reversible protein phosphorylation  Phosphagen metabolism
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