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

Highlights

• Many functions of phosphorylase kinase are regulated by Ca²⁺ and Mg²⁺ ions.

• Kinetic regime of aggregation of highly complexly regulated protein is established.

• The rate-limiting stage of the aggregation process is the stage of protein unfolding.

• Aggregation pathway has been analyzed at various protein concentrations.

• The mechanism of aggregation of phosphorylase kinase at 40 °C has been proposed.

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⁺).

Introduction

Phosphorylase kinase (PhK) plays a key role in the neural and hormonal regulation of glycogenolysis in skeletal muscle [[1], [2], [3]]. PhK with molecular mass of 1320 kDa [4] has a complex molecular organization and consists of four subunits that form a hexadecamer (αβγδ)₄, where γ-subunit possesses the catalytic activity and other subunits regulate its activity [[4], [5], [6]]. The regulation of PhK activity can be complex. In the absence of Ca²⁺, α- and β-subunits inhibit the activity of the γ-subunit. Ca²⁺ being an obligatory allosteric activator of PhK binds with the δ-subunit, a non-dissociable molecule of calmodulin, and thus discovers the activity of the γ-subunit. Ca²⁺-induced conformational changes in the δ-subunit are converted into conformational changes of α- and β-subunits. Another cationic activator of PhK, Mg²⁺ binds to the γ-subunit and stimulates its activity [7]. Ca²⁺ and Mg²⁺ ions can act synergistically on nonactivated PhK at pH 6.8. These cations stimulate PhK activity by inducing changes in the tertiary and quaternary structure of the molecule [[5], [6], [7], [8], [9]], also enhance binding to glycogen [[10], [11], [12]] and stimulate association/aggregation of PhK hexadecamer molecules [13,14]. It was shown that the oligomeric state of PhK strongly depends on the protein concentration, Ca²⁺ and Mg²⁺ ions concentrations, ionic strength and presence of crowding agents [[14], [15], [16], [17], [18]]. Upon binding Ca²⁺, Mg²⁺ ions PhK becomes more sensitive to thermal perturbation, suggesting that an active conformation of kinase is less stable [[6], [7], [8],17]. It should be stressed, that during last six decades the activity and properties of PhK from rabbit muscle were studied at ≤30 °C, despite the fact that the rabbit physiological temperature is 40 °C. Carlson and co-workers showed that when the temperature changed from 30 to 40 °C, there was a sharp increase in the enzyme activity and a change in many PhK functions [9]. However, there are no investigations of PhK association/aggregation induced by Ca²⁺ and Mg²⁺ cations at 40 °C. The goal of our work was to study a kinetic regime of Ca²⁺ and Mg²⁺-induced aggregation of PhK from rabbit skeletal muscle at 40 °C.