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Dynamic light scattering was used to study the interaction of phosphorylase kinase (PhK) and glycogen phosphorylase b (Phb) from rabbit skeletal muscle with glycogen under molecular crowding conditions arising from the presence of 1 M trimethylamine N-oxide and at physiological ionic strength. The mean value of hydrodynamic radius of the initial glycogen particles was 52 nm. Crowding stimulated Phb and PhK combined binding on glycogen particles. Two-stage character of PhK binding to glycogen particles containing adsorbed Phb was found in the presence of the crowding agent. At the initial stage, limited size particles with hydrodynamic radius of approximately 220 nm are formed, whereas the second stage is accompanied by linear growth of hydrodynamic radius. Flavin adenine dinucleotide (FAD) selectively inhibited PhK binding at the second stage. The data indicate that in the first stage Phb is involved in PhK binding by glycogen particles containing adsorbed Phb, whereas PhK binding in the second stage does not involve Phb.

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Thermal aggregation of rabbit skeletal muscle glycogen phosphorylase b (Phb) has been investigated using dynamic light scattering under conditions of a constant rate of temperature increase (1 K/min). The linear behavior of the dependence of the hydrodynamic radius on temperature for Phb aggregation is consistent with the idea that thermal aggregation of proteins proceeds in the kinetic regime wherein the rate of aggregation is limited by diffusion of the interacting particles (the regime of "diffusion-limited cluster-cluster aggregation"). In the presence of alpha-crystallin, a protein exhibiting chaperone-like activity, the dependence of the hydrodynamic radius on temperature follows the exponential law; this suggests that the aggregation process proceeds in the kinetic regime where the sticking probability for colliding particles becomes lower than unity (the regime of "reaction-limited cluster-cluster aggregation"). Based on analysis of the ratio between the light scattering intensity and the hydrodynamic radius of Phb aggregates, it has been concluded that the addition of alpha-crystallin results in formation of smaller size starting aggregates. The data on differential scanning calorimetry indicate that alpha-crystallin interacts with the intermediates of the unfolding process of the Phb molecule. The proposed scheme of thermal denaturation and aggregation of Phb includes the stage of reversible dissociation of dimers of Phb into monomers, the stage of the formation of the starting aggregates from the denatured monomers of Phb, and the stage of the sticking of the starting aggregates and higher order aggregates. Dissociation of Phb dimer into monomers at elevated temperatures has been confirmed by analytical ultracentrifugation.

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Kinetics of thermal aggregation of model protein substrates (glycogen phosphorylase b from rabbit skeletal muscle and yeast alcohol dehydrogenase) were investigated under heat stress conditions (41-48 degrees C) in the presence of macrophage migration inhibitory factor (MIF), a heat-stable hydrophobic protein (12.5 kD). Anti-chaperone MIF activity found by turbidimetry manifests itself in significantly accelerated protein aggregation and increased limiting value of apparent optical absorption at 360 nm and t --> infinity in the sub-stoichiometric range of MIF concentrations. The aggregation kinetics is shown to have cooperative character. Possible reversibility of aggregation after removal of denaturing conditions was demonstrated using alcohol dehydrogenase aggregation at a temperature close to the physiological level (41.5 degrees C). This reversibility is caused by solubility of aggregates and stabilization of oligomeric structure of the substrate as a result of MIF binding to the partially denatured protein. The data suggest that in spite of distinct anti-chaperone effect, the chaperone-like activity of MIF can be observed in the case of heat stress removal and restoration of the system to normal conditions.

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The effects of the osmolytes trimethylamine-N-oxide (TMAO), betaine, proline, and glycine on the kinetics of inactivation and aggregation of rabbit skeletal muscle glycogen phosphorylase b by guanidine hydrochloride (GuHCl) have been studied. It is shown that the osmolytes TMAO and betaine exhibit the highest protective efficacy against phosphorylase b inactivation. A test system for studying the effects of macromolecular crowding induced by osmolytes on aggregation of proteins is proposed. TMAO and glycine increase the rate of phosphorylase b aggregation induced by GuHCl.

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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.

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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.

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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).

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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.

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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.

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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.

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The effect of specific ligands on the initial rate of muscle glycogen phosphorylase b digestion by trypsin has been studied. The kinetics of tryptic proteolysis were followed by measuring the decrease in phosphorylase b fluorescence intensity at 335 nm (excitation at 290 nm). The kinetic curves were linear at least in the region 0-400 s (0.02 M HEPES, pH 6.8; 37 degrees C). An allosteric activator (AMP) and allosteric inhibitors (flavins) protected the enzyme from tryptic digestion when trypsin was added to the enzyme preincubated with the ligand. Differences were found between the kinetic curves of trypsinolysis initiated by addition of the trypsin-ligand mixture to phosphorylase b an by addition of trypsin to the enzyme preincubated with the ligand for 10 min. It is concluded that the specific ligands under study (AMP, flavins, and the substrate--glucose 1-phosphate) induce relatively slow conformational changes in the phosphorylase b molecule with the half-conversion time of several minutes.

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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.

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Using DEAE-Toyopearl column chromatography, a preparation of pigeon skeletal muscle phosphorylase kinase was obtained in a state approaching homogeneity. The molecular mass of the native enzyme (1320 kDa) and the subunit formula (alpha beta gamma delta)4 are similar to those of rabbit and chicken counterparts. Both red and white pigeon skeletal muscle isozymes contain the alpha'-subunit instead of alpha. Gradient SDS-PAGE electrophoresis revealed small but well-reproducible differences in the molecular masses of rabbit, chicken and pigeon muscle beta- and gamma-subunits. The activity ratio at pH 6.8/8.2 is 0.06-0.15 for different preparations of phosphorylase kinase b. The activity of pigeon muscle phosphorylase kinase b is Ca2+-dependent. The [Ca2+]0.5 value at pH 7.0 is 20 microM, which exceeds that for the chicken muscle enzyme by two orders of magnitude. In the presence of Ca2+, pigeon phosphorylase kinase b is activated 4-fold by saturating concentrations of calmodulin and troponin C. Pigeon muscle phosphorylase b is activated 3-5-fold during autophosphorylation or phosphorylation by the catalytic subunit of cAMP-dependent protein kinase.

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The changes in the quaternary structure of chicken skeletal muscle phosphorylase kinase during limited proteolysis by trypsin and chymotrypsin were studied. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the products of phosphorylase kinase limited proteolysis revealed a similarity in the structure of the alpha'- and beta-subunits and some differences in the structure of the gamma-subunits of the chicken and rabbit enzymes. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol of 32P/mol of alpha' beta gamma' sigma monomer) and autophosphorylation (up to 8 mol of 32P/mol alpha' beta gamma' delta monomer) increased the activity of chicken phosphorylase kinase 1.5-fold and 2.0-fold, respectively. The incorporation of phosphate into the alpha' and beta-subunits in the course of the protein kinase-catalyzed reaction was demonstrated.

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Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.

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The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.

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