Iraida E. Andreeva

Self-association of phosphorylase kinase from rabbit skeletal muscle in the presence of natural osmolyte, trimethylamine N-oxide

The effect of trimethylamine N-oxide (TMAO) on self-association of phosphorylase kinase (PhK) has been studied by analytical ultracentrifugation and turbidimetry in 40 mM N-(2-hydroxyethyl)piper-azine-N′-ethanesulfonic acid buffer, pH 6.8 and 8.2. PhK is a hexade-camer (αβγδ)4 with a molecular mass of 1,300 kDa. The oligomeric state of the native enzyme is dependent on the protein concentration and the concentrations of Ca2+ and Mg2+, which are essential for the enzymatic activity. In the absence of Ca2+ and Mg2+ the enzyme exists in the monomeric and dimeric forms (with s 20,w = 23 and 36.5 S); however, the addition of 0.1 mM Ca2+ and 10 mM Mg2+ results in the appearance of associates of higher order. TMAO (0.6–1.0 M) was found to favor greatly self-association of PhK. In the presence of TMAO, apart from the association products, consisting of a rather low number (n) of PhK molecules (n = 2, 3, 4,...), two distinct rapidly moving boundaries with S 20,w = 189 and 385 S are registered on the sedimentation profiles. These boundaries correspond to 24-mers and 70-mers (the molecular masses of associates were estimated using the Mark-Houwink—Kuhn—Sakurada equation, assuming a spherical form. The kinetics of TMAO-induced association of PhK was monitored by following the increase in the turbidity of the enzyme solution at varying concentrations of the protein and TMAO. The initial rate of the association is proportional to the enzyme concentration squared, suggesting that the initial step of PhK association is the stage of dimerization.

A tentative mechanism of the ternary complex formation between phosphorylase kinase, glycogen phosphorylase b and glycogen

The kinetics of rabbit skeletal muscle phosphorylase kinase interaction with glycogen has been studied. At pH 6.8 the binding of phosphorylase kinase to glycogen proceeds only in the presence of Mg2+, whereas at pH 8.2 formation of the complex occurs even in the absence of Mg2+. On the other hand, the interaction of phosphorylase kinase with glycogen requires Ca2+ at both pH values. The initial rate of the complex formation is proportional to the enzyme and glycogen concentrations, suggesting the formation of the complex with stoichiometry 1:1 at the initial step of phosphorylase kinase binding by glycogen. According to the kinetic and sedimentation data, the substrate of the phosphorylase kinase reaction, glycogen phosphorylase b, favors the binding of phosphorylase kinase with glycogen. We suggest a model for the ordered binding of phosphorylase b and phosphorylase kinase to the glycogen particle that explains the increase in the tightness of phosphorylase kinase binding with glycogen in the presence of phosphorylase b.

Effect of flavins on the rate of proteolytic digestion of muscle glycogen phosphorylase b

The kinetics of tryptic proteolysis of rabbit skeletal muscle phosphorylase b has been registered by the diminishing of protein fluorescence intensity at lambda = 335 nm (excitation at 290 nm) or by the disappearance of the enzyme activity (0.02 M Hepes buffer, pH 6.8, 37 degrees C). The first procedure showed that flavins (riboflavin, FMN, FAD) protected the enzyme against tryptic digestion. Microscopic dissociation constants for the complexes of phosphorylase b with riboflavin, FMN and FAD were calculated from dependences of the initial digestion rate on the flavin concentration. They where equal to 30 +/- 1, 15.8 +/- 0.2 and 36 +/- 1 microM, respectively. No influence of FMN on the rate of the tryptic hydrolysis of phosphorylase b was observed when using the second procedure (enzyme activity test). FMN completely prevents the formation of 69-, 81- and 85-kDa fragments during 20 min incubation of phosphorylase b with trypsin.

Studies on interaction of phosphorylase kinase from rabbit skeletal muscle with glycogen in the presence of ATP and ADP.

The influence of ATP on complex formation of phosphorylase kinase (PhK) with glycogen in the presence of Ca(2+) and Mg(2+) has been studied. The initial rate of complex formation decreases with increasing ATP concentration, the dependence of the initial rate on the concentration of ATP having a cooperative character. Formation of the complex of PhK with glycogen in the presence of ATP occurs after a lag period, which increases with increasing ATP concentration. The dependence of the initial rate of complex formation (v) on the concentration of non-hydrolyzed ATP analogue, beta,gamma-methylene-ATP, follows the hyperbolic law. A correlation between PhK-glycogen complex formation and (32)P incorporation catalyzed by PhK itself and by the catalytic subunit of cAMP-dependent protein kinase has been shown. For ADP (the product and allosteric effector of the PhK reaction) the dependence of v on ADP concentration has a complicated form, probably due to the sequential binding of ADP at two allosteric sites on the beta subunit and the active site on the gamma subunit.

Interaction of phosphorylase kinase from rabbit skeletal muscle with flavin adenine dinucleotide.

The interaction of flavin adenine dinucleotide (FAD) with rabbit skeletal muscle phosphorylase kinase has been studied. Direct evidence of binding of phosphorylase kinase with FAD has been obtained using analytical ultracentrifugation. It has been shown that FAD prevents the formation of the enzyme-glycogen complex, but exerts practically no effect on the phosphorylase kinase activity. The dependence of the relative rate of phosphorylase kinase-glycogen complex formation on the concentration of FAD has cooperative character (the Hill coefficient is 1.3). Under crowding conditions in the presence of 1 M trimethylamine-N-oxide (TMAO), FAD has an inhibitory effect on self-association of phosphorylase kinase. The data suggest that the complex of glycogen metabolism enzymes in protein-glycogen particles may function as a flavin depot in skeletal muscle.

Hysteretic properties of rabbit skeletal muscle phosphorylase kinase: Synergistic activation by phosphorylase b, Ca2, and Mg2

To study the hysteretic properties of rabbit skeletal muscle phosphorylase kinase the method of continuous registration of the kinetics of the kinase reaction developed by us earlier has been used. It was shown that duration of the lag period on the kinetic curves is independent of the phosphorylase kinase concentration and the simultaneous presence of phosphorylase b, Ca2+, and Mg2+ is required for the complete transition of the enzyme into the activated state.