Kinetics of the Interaction of Rabbit Skeletal Muscle Phosphorylase Kinase with Glycogen

I. E. Andreeva, V. F. Makeeva, B. I. Kurganov^*, N. A. Chebotareva, and N. B. Livanova

Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia; fax: (095) 954-2732; E-mail: inbio@glas.apc.org

^* To whom correspondence should be addressed.

Received June 16, 1998; Revision received September 21, 1998

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The kinetics of the interaction of rabbit skeletal muscle phosphorylase kinase with glycogen was studied by the turbidimetric method at pH 6.8 and 8.2. Binding of phosphorylase kinase by glycogen occurs only in the presence of Ca²⁺ and Mg²⁺. The initial rate of complex formation is proportional to the enzyme and polysaccharide concentration; this suggests the formation of a complex with 1:1 stoichiometry in the initial step of phosphorylase kinase binding by glycogen. The kinetic data suggest that phosphorylase kinase substrate--glycogen phosphorylase b--favors the binding of phosphorylase kinase with glycogen. This conclusion is supported by direct experiments on the influence of phosphorylase b on the interaction of phosphorylase kinase with glycogen using analytical sedimentation analysis. The kinetic curves of the formation of the complex of phosphorylase kinase with glycogen obtained in the presence of ATP are characterized by a lag period. Preincubation of phosphorylase kinase with ATP in the presence of Ca²⁺ and Mg²⁺ causes the complete disappearance of the lag period. On changing the pH from 6.8 to 8.2, the rate of phosphorylase kinase binding by glycogen is appreciably increased, and complex formation becomes possible even in the absence of Mg²⁺. A model of phosphorylase kinase and phosphorylase b adsorption on the surface of the glycogen particle explaining the increase in the strength of phosphorylase kinase binding with glycogen in the presence of phosphorylase b is proposed.

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KEY WORDS:phosphorylase kinase, glycogen, turbidimetric method, glycogen phosphorylase, analytical ultracentrifugation

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