Kinetic regime of Ca2+ and Mg2+-induced aggregation of phosphorylase kinase at 40 °C

Abstract

Many functions of phosphorylase kinase (PhK) are regulated by Ca²⁺ and Mg²⁺ ions. Ca²⁺ and Mg²⁺ ions stimulate activity of PhK, induce the changes in the tertiary and quaternary structure of the hexadecameric enzyme molecule, provoke association/aggregation of PhK molecules, enhance PhK binding to glycogen. To establish the kinetic regime of Ca²⁺ and Mg²⁺-induced aggregation of PhK from rabbit skeletal muscles at 40 °C, in the present work the kinetics of aggregation was studied at various protein concentrations using the dynamic light scattering. The proposed mechanism of aggregation involves the stage of unfolding of the protein molecule with retention of the integrity of its oligomeric structure, the nucleation stage and stages of the growth of protein aggregates. The initial rate of the aggregation process at the stage of aggregate growth depends linearly on the protein concentration. This means that the order of aggregation with respect to the protein is equal to unity and the aggregation rate is limited by the rate of protein unfolding. The rate constant of the first order characterizing the stage of protein unfolding was found to be equal to 0.071 min^-1 (40 mM Hepes, pH 6.8, 100 mM NaCl, 0.1 mM Ca²⁺, 10 mM Mg²⁺).

Keywords: Aggregation; Analytical ultracentrifugation; Dynamic light scattering; Kinetic regime; Phosphorylase kinase.