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.

Biochemical effects of molecular crowding.

Cell cytoplasm contains high concentrations of high-molecular-weight components that occupy a substantial part of the volume of the medium (crowding conditions). The effect of crowding on biochemical processes proceeding in the cell (conformational transitions of biomacromolecules, assembling of macromolecular structures, protein folding, protein aggregation, etc.) is discussed in this review. The excluded volume concept, which allows the effects of crowding on biochemical reactions to be quantitatively described, is considered. Experimental data demonstrating the biochemical effects of crowding imitated by both low-molecular-weight and high-molecular-weight crowding agents are summarized.

Pyridoxal 5'-phosphate as a catalytic and conformational cofactor of muscle glycogen phosphorylase B.

This review summarizes data on structure of muscle glycogen phosphorylase b and the role of the cofactor pyridoxal 5'-phosphate in catalysis and stabilizing the native conformation of the enzyme. Specific attention is paid to the stabilizing role of pyridoxal 5'-phosphate upon denaturation of phosphorylase b. Stability of holoenzyme, apoenzyme, and enzyme reduced by sodium borohydride is compared.

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.

Kinetics of denaturation of rabbit skeletal muscle glycogen phosphorylase b by guanidine hydrochloride.

The kinetics of denaturation and aggregation of rabbit muscle glycogen phosphorylase b in the presence of guanidine hydrochloride (GuHCl) have been studied. The curve of inactivation of phosphorylase b in time includes a region of the fast decline in the enzymatic activity, an intermediate plateau, and a part with subsequent decrease in the enzymatic activity. The fact that the shape of the inactivation curves is dependent on the enzyme concentration testifies to the dissociative mechanism of inactivation. The dissociation of phosphorylase b dimers into monomers in the presence of GuHCl is supported by sedimentation data. The rate of phosphorylase b aggregation in the presence of GuHCl rises as the denaturant concentration increases to 1.12 M; at higher concentration of GuHCl, suppression of aggregation occurs. At rather low concentration of the protein (0.25 mg/ml), the terminal phase of aggregation follows the kinetics of a monomolecular reaction (the reaction rate constant is equal to 0.082 min(-1); 1 M GuHCl, 25 degrees C). At higher concentration of phosphorylase b (0.75 mg/ml), aggregation proceeds as a trimolecular reaction.

Dissociative mechanism of thermal denaturation of rabbit skeletal muscle glycogen phosphorylase b.

The thermal stability of rabbit skeletal muscle glycogen phosphorylase b was characterized using enzymological inactivation studies, differential scanning calorimetry, and analytical ultracentrifugation. The results suggest that denaturation proceeds by the dissociative mechanism, i.e., it includes the step of reversible dissociation of the active dimer into inactive monomers and the following step of irreversible denaturation of the monomer. It was shown that glucose 1-phosphate (substrate), glucose (competitive inhibitor), AMP (allosteric activator), FMN, and glucose 6-phosphate (allosteric inhibitors) had a protective effect. Calorimetric study demonstrates that the cofactor of glycogen phosphorylase-pyridoxal 5'-phosphate-stabilizes the enzyme molecule. Partial reactivation of glycogen phosphorylase b preheated at 53 degrees C occurs after cooling of the enzyme solution to 30 degrees C. The fact that the rate of reactivation decreases with dilution of the enzyme solution indicates association of inactive monomers into active dimers during renaturation. The allosteric inhibitor FMN enhances the rate of phosphorylase b reactivation.

Kinetics of the interaction of rabbit skeletal muscle phosphorylase kinase with glycogen.

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.

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.

Continuous enzymatic assay for phosphorylase kinase in a monocascade enzyme system.

A turbidimetric method for continuous monitoring of the enzymatic reaction catalyzed by rabbit skeletal muscle phosphorylase kinase has been developed. The reaction mixture contained the substrates of glycogen phosphorylase a, i.e., glycogen and glucose 1-phosphate (or P(i)), in addition to the usual components of the kinase reaction. The kinetics of the cascade enzyme system were followed by the change in glycogen concentration over time, as measured by the absorbance of the reaction medium at 360 nm. The reliability of this turbidimetric method for measuring phosphorylase kinase activity was proven by comparison with a commonly used radiochemical assay. We present here a newly developed method for calculating the initial rate of phosphorylase kinase reaction in our conjugated system. We demonstrate that our procedure is applicable for investigating the hysteretic properties of phosphorylase kinase.

[Structure, regulation, and denaturation of muscle glycogen phosphorylase b]. / Struktura, reguliatsiia i denaturatsiia myshechnoi glikogenfosforilazy b.

The review summarizes data on structure, allosteric regulation, and denaturation of muscle glycogen phosphorylase b. Specific attention is paid to correlations between the structure and function of phosphorylase b and molecular mechanism of its allosteric regulation. Chemical and thermal denaturation of phosphorylase b is reviewed.

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.

[Effect of specific ligands on heat inactivation of muscle glycogen phosphorylase b]. / Vliianie spetsificheskikh ligandov na teplovuiu inaktivatsiiu myshechnoi glikogenfosforilazy b.

It has been shown that the rate constant, k, for thermal inactivation of rabbit skeletal muscle glycogen phosphorylase b decreases as the enzyme concentration increases. This effect is interpreted within the framework of a kinetic model which includes two parallelly occurring processes, namely: phosphorylase b denaturation in solution and denaturation of the enzyme absorbed on test-tube walls. The contribution of the latter process increases with a decrease in the enzyme concentration. The protective effect of the allosteric activator (AMP), allosteric inhibitors (glucose 6-phosphate and FMN) and the competitive inhibitor (glucose) against heat denaturation of glycogen phosphorylase b has been demonstrated. Quantitative analysis of the dependence of the rate constant, k, on concentration of AMP, glucose 6-phosphate and FMN allows the calculation of microscopic constants for dissociation of phosphorylase b complexes with these ligands for the given experimental conditions as equal to 0.34, 0.50 and 0.30 mM, respectively. The S-shaped dependence of the rate constant of thermal inactivation on glucose concentration points to the existence of positive cooperative interactions between glucose-binding sites in the dimeric molecule of phosphorylase b (nH = 1.8).

Thermal inactivation of skeletal muscle phosphorylase b: protective effect of the specific ligands.

The kinetics of the thermal inactivation of rabbit skeletal muscle phosphorylase b have been studied. Under the condition used (0.08 M HEPES, pH 6.8, 75 mu g/ml phosphorylase b) the enzyme quickly loses its activity at the temperature higher then 49 degrees C. All the specific ligands tested (substrate -glucose 1-phosphate, competitive inhibitor - glucose, allosteric activator - AMP, and allosteric inhibitors - glucose 6-phosphate and FMN) have the pronounced protective effect. The strongest protective action have glucose 6-phosphate and FMN. Quantitative analysis of the inactivation rate constant versus ligand concentration curves allow the enzyme affinity for ligands to be characterized.

Deimination of glycogen phosphorylase b by peptidylarginine deiminase. Influence on the kinetical characteristics and dimer-tetramer transition.

The kinetics of the native glycogen phosphorylase b from rabbit skeletal muscle and of the enzyme specifically deiminated by peptidylarginine deiminase have been studied. One arginine residue per phosphorylase b monomer is transformed into citrulline after 3 h of incubation with peptidylarginine deiminase. The maximal velocity of the enzymatic reaction for the modified phosphorylase b is 7-20% higher than that for the native enzyme. Deiminated phosphorylase b, like the native enzyme, shows a positive kinetic cooperativity with respect to glucose-1-phosphate. The affinity of the modified phosphorylase b for the allosteric activator AMP is one order of magnitude higher than that of the native enzyme. Deimination caused a pronounced reduction of the values of [I]0.5 for FMN and glucose, but the sensitivity of the deiminated enzyme to glucose-6-phosphate is much lower than that of the native phosphorylase b. Deiminated phosphorylase b, unlike the native enzyme, shows the positive cooperativity for FMN binding. Deiminated phosphorylase b, unlike the native enzyme, shows less capability to form tetramers in the presence of AMP as compared to the native enzyme.

[Kinetics of action of phosphorylase kinase in a cascade enzymatic system. III. Hysteretic properties of phosphorylase kinase from skeletal muscles]. / Kinetika deistviia kinazy fosforilazy v kaskadnoi fermentnoi sisteme. III. Gisterezisnye svoistva kinazy fosforilazy iz skeletnykh myshts.

The kinetic behaviour of rabbit skeletal muscle phosphorylase kinase at variable concentrations of the enzyme and the substrate (glycogen phosphorylase b) has been studied. The kinetic curves reveal a lag period whose duration decreases with a rise in the phosphorylase kinase concentration (when the reaction is initiated by an addition of the ATP + MgCl2 mixture to the enzyme preincubated with phosphorylase b, CaCl2, glycogen and glucose-1-phosphate or inorganic phosphate). A decrease of the phosphorylase b concentration eliminates the lag period. Under these conditions the specific activity of phosphorylase kinase decreases with a rise in the enzyme concentration. The kinetic behaviour of phosphorylase kinase is interpreted in terms of a model of a linearly associating system, such as M reversible M2 reversible M3 reversible ...Mi, where M is the dexadecameric molecule of phosphorylase kinase. Acceleration of the phosphorylase kinase-catalyzed reaction in the course of the enzymatic process seems to be due to the breakdown of inactive enzyme associates (Mi) caused by phosphorylase b. The short gamma-subunit of phosphorylase kinase devoid of the calmodulin-binding domain does not display any hysteretic properties.

[Kinetics of action of phosphorylase kinase in an enzymatic cascade system. II. Assessment of the initial rate of the enzymatic reaction catalyzed by phosphorylase kinase]. / Kinetika deistviia kinazy fosforilazy v kaskadnoi fermentnoi sisteme. II. Otsenka nachal'noi skorosti ferementativnoi reaktsii, kataliziruemoi kinazoi fosforilazy.

The turbidimetric method for determining the phosphorylase kinase activity has been developed. The reaction mixture contained, alongside with other components of the kinase reaction, also the substrates of glycogen phosphorylase a, the final product of the kinase reaction-glycogen and glucose 1-phosphate (or inorganic phosphate). The kinetics of the cascade enzymatic system were followed by the increment (decrement) of absorbance of the glycogen solution at 360 nm (delta A). The initial rate of the phosphorylase kinase-catalyzed enzymatic reaction, nu 0, can be calculated according to the formula: nu 0 = 2tg alpha/a2, where tg alpha is the initial slope of the kinetic curve in the coordinates: delta A-(time)2 and a2 is the specific enzymatic activity of phosphorylase a. The latter was estimated from the initial rates of the phosphorylase reaction measured by the addition of glycogen to the reaction mixture after the completion of the kinase reaction. The reliability of the turbidimetric method for determining the phosphorylase kinase activity was proved by comparison with a direct method based on the measurement of the amount of incorporated 32P.

[The effect of specific deimination of glycogen phosphorylase b by peptidylarginine deiminase on the allosteric properties of the enzyme and dimer-tetramer transition]. / Vliianie spetsificheskogo deziminirovanniia glikogenfosforilazy b peptidilarginindeziminazoi na allostericheskie svoistva fermenta i dimer-tetramernye perekhody.

The kinetics of the native glycogen phosphorylase b from rabbit skeletal muscle and of the enzyme specifically deiminated by peptidylarginine deiminase have been studied. According to the data on amino acid composition one arginine residue per phosphorylase b monomer is transformed into citrulline after 3 hours of incubation with peptidylarginine deiminase. The kinetics of the phosphorylase reaction were studied in the direction of glycogen synthesis. The native and the deiminated forms of phosphorylase b showed similar affinity to glucose 1-phosphate. The maximal velocity of the enzymatic reaction for the modified phosphorylase b is 8-20% higher than that for the native enzyme. Deiminated phosphorylase b like the native enzyme shows a positive kinetic cooperatively with respect to glucose 1-phosphate in the presence of the allosteric inhibitors (FMN, glucose), S-shaped dependences of the velocity of the enzymatic reaction on glucose 1-phosphate concentration (in the presence of FMN) pronouncing more distinctly for deiminated phosphorylase b than for the native enzyme (Hill coefficient is equal to 1.7 +/- 0.2 and 1.3 +/- 0.1, respectively). The affinity of the modified phosphorylase b to the allosteric activator AMP is one order of magnitude higher than that to the native enzyme. The cooperativity of AMP binding doesn't change significantly after deimination. The kinetics of inhibition of the native and modified phosphorylase b by FMN, glucose and glucose 6-phosphate are cooperative (the value of Hill coefficient is higher than unity). The more pronounced distinctions between two forms of the enzyme concern with the value of the "semisaturation" concentration [I]0.5. The deimination causes a pronounced reduction of the values of [I]0.5 for FMN and glucose, but the sensitivity of the deiminated enzyme to glucose 6-phosphate is much lower than that of the native phosphorylase b. Deiminated phosphorylase b unlike the native enzyme shows the positive cooperativity of the FMN binding (the value of the Hill coefficient is equal to 1.37 +/- 0.05). Deiminated phosphorylase b shows less capability to form tetramer in the presence of AMP as compared to the native enzyme.