[Determination of glycogen phosphorylase B activity by the turbidimetric method in the presence of the forward and reverse reaction substrates]. / Opredelenie aktivnosti glikogenfosforilazy B turbidimetricheskim metodom pri odnovremennom prisutstvii substratov priamoi i obratnoi reaktsii.

The turbidimetric method based on monitoring changes in the turbidity of glycogen solution at wavelengths above 300 nm has been developed for the measurement of catalytic activity of rabbit skeletal muscle glycogen phosphorylase B. The dependences of the initial rate of enzymatic reaction on molar ratio of inorganic phosphate in the inorganic phosphate-glucose-1-phosphate mixture have been obtained at various concentrations of the allosteric activator, AMP, or in the presence of inhibitors (D-glucose, glucose 6-phosphate). It has been shown that the changes in glycogen phosphorylase B activity in the presence of these modifiers are not affected by variations in the molar ratios of the substrate. A theoretical expression for the dependence of the initial rate of the reversible reaction S in equilibrium P catalyzed by the enzyme has been deduced as a function of the substrate (S) molar ratio.

[Effect of glucose 1-phosphate and inorganic phosphate on binding of muscle phosphorylase b by glycogen]. / Vliianie gliukozo-1-fosfata na sviazyvanie myshechnoi glikogenfosforilazy b glikogenom.

Using analytical ultracentrifugation, the affinity of rabbit skeletal muscle phosphorylase b for glycogen was shown to increase 11.6-fold in the presence of saturating concentrations of glucose 1-phosphate and to decrease 3.7-fold in the presence of saturating concentrations of inorganic phosphate (20 degrees, 0.05 M glycyl-glycine buffer, pH 6.8, ionic strength 0.3).

[Kinetics of the reaction between muscle glycogen phosphorylase B and glycogen]. / Kinetika vzaimodeistviia myshechnoi glikogenfosforilazy B s glikogenom.

Kinetics of glycogen binding by glycogen phosphorylase b has been studied by stopped flow and temperature jump methods. This reaction is followed by increase in light scattering whose amplitude depends upon the enzyme binding sites concentration of glycogen particles occupied by the enzyme. It has been shown that the complex formation has the first order with respect to enzyme and glycogen concentrations. Relaxation kinetics is compatible with proposed bimolecular reaction scheme. Microscopic rate constants of the forward and reverse reactions of glycogen binding by glycogen phosphorylase b are determined in temperature range from 12,7 to 30 degrees C. The possibility of diffusional control of the binding rate is discussed.

[Effect of polyethylene glycol on association of muscle phosphorylase B]. / Vliianie poliétilenglikolia na assotsiatsiiu myshechnoi fosforilazy B.

It has been shown that a neutral polymer poly(ethyleneglycol) with molecular weight of 4000 and more shifts the equilibrium between the dimer and tetramer forms of phosphorylase B from rabbit skeletal muscles in the direction of formation of species with higher molecular weights. Poly(ethyleneglycol) with molecular weight below 600 has no effect on the association process. Phosphorylase B is precipitated in the form of crystals in the presence of poly(ethyleneglycol) mol. weight 40,000), when the concentration of the latter is higher than 50 mg/ml. AMP and sulfate ions are required for the crystallization process.

[Binding of muscle phosphorylase B by glycogen]. / Izuchenie sviazyvaniia myshechnoi fosforilazy b glikogenom.

The binding of phosphorylase B with glycogen has been studied by means of analytical ultracentrifuge with absorption optics and a photoelectric scanner. Adsorbtion capacity for pig liver glycogen with respect to phosphorylase B (aM) and microscopic dissociation constant of enzyme -- glycogen complex (K) have been determined (aM = 3.64 . 10(-6) moles of bound enzyme per 1 g of glycogen: K = 2.6 . 10(-7) M at 12.7 degrees, pH 6.8). For oyster glycogen the value of aM is equal to 3.92 . 10(-6) moles of the bound protein per 1g of glycogen and K = 6.8 . 10(-7) M. The method of determination of microscopic Michaelis constant with respect to glycogen using known value of aM has been demonstrated.