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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.

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.

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.

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).

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.

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.

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.

Phosphorylase kinase is the key enzyme in the control of glycogen metabolism in skeletal muscles, the heart and the liver. The quaternary structure of the enzyme, the primary structure of the enzyme subunits as well as the kinetic properties and regulation of the skeletal muscle enzyme activity by covalent modification, phosphorylation and some physiological effectors (Ca2+, calmodulin, troponin C) are reviewed.

Electron microscopy of negatively stained preparations of phosphorylase b with protamine added reveals three types of crystal formations: plane monolayers of particles, tubes and three-dimensional crystals. The methods of optical diffraction and filtering have been used to analyse the micrographs. The unit cell of the layer contains two packing units. Most of the tubes have one-and two-layer walls corresponding to monolayers of particles, but sometimes tubes occur with three and more layers in the wall. Optical diffraction shows that the packing units in tubes with a one-layer wall form a two-start helix in which 27 units fall on 3.5 turns. The inner tube in tubes with two walls has an analogous structure. The outer tube represents a three-start helix in which 19 packing units fall on 5 3 of a turn. The unit cell in the layers forming the wall contains one packing unit. The analysis of the image of particle packing-units shows that these are the same in the layer and tube walls. Particles of the same type were found in preparations of phosphorylase b tetramers from solution. A model for the particles is proposed; it consists of four elongated, bent subunits arranged with the point-group symmetry 222 at the vertices of a tetrahedron. One has good grounds for considering the observed particles to be the phosphorylase b molecules in the form of tetramers.

The structure, physical and kinetic properties as well as the regulatory mechanisms of skeletal muscle phosphorylase kinase have been studied extensively. In contrast to our current understanding of muscle phosphorylase kinase, the structure and regulatory properties of liver enzyme have not yet been fully clarified. Results obtained with crude and partially purified preparations of liver phosphorylase kinase suggest two main mechanisms of regulation: firstly, covalent modification through a phosphorylation reaction catalyzed by cyclic AMP-dependent protein kinase in response to glucagon and epinephrine (Vandenheede et al. 1979), and secondly, Ca2+ has been proposed as a possible candidate for an intracellular mediator in the hormonal control by cyclic AMP-independent hormones (e.g., vasopressin, angiotensin and α adrenergic agents) (Van de Werve et al. 1977); besides that, the control of kinase reaction can occur due to binding of effectors to the substrate, phosphorylase b, to the enzyme, phosphorylase kinase, or both.

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.

The activation of different forms of muscle phosphorylase kinase by actin has been studied. F-actin which is polymerized by 2 mM MgCl2 is a more effective activator of phosphorylase kinase than F-actin polymerized by 50 mM KCl. There is evidence suggesting that the activation of phosphorylase kinase b by actin is not due to the presence of trace amounts of calmodulin in actin preparations: (1) Troponin I and trifluoperazine inhibit the activation of phosphorylase kinase by calmodulin but do not inhibit the activation by actin. (2) The activation induced by saturating concentrations of calmodulin and actin is additive. (3) The activation of phosphorylase kinase by calmodulin and actin has different pH profiles. An addition of F-actin does not affect the apparent Km value for ATP but increases the sensitivity to phosphorylase b and the value of V. F-actin has no stimulating effect on the phosphorylated form (a) of phosphorylase kinase or on the form a previously activated by proteolysis.

The purification of phosphorylated form of rabbit liver glycogen phosphorylase (phosphorylase A) using the chromathography on a omega-amino-hexyl-Sepharose column has been carried out. The yield is about 90%, the specific activity is equal to 90 mkmol Pi/min. mg. The enzyme samples appeared essentially homogeneous when analyzed by polyacrylamide gel electrophoresis. The Km value of glucose-1-phosphate in the presence of AMP is 4--6 mM, which is higher than that found previously. When the glucose-1-phosphate concentration was varied, deviations from Michaelis-Menten kinetics were observed in the presence of glucose or ATP (Hill slopes of 1.6), but these deviations were virtually abolished by the presence of AMP. With glucose-1-phosphate as the substrate the Ki value of glucose is 57 mM in the absence of AMP and 150 mM in the presence of this effector. The Ki value of ATP is less dependent on the presence of AMP. The synergism in combined action of glucose and ATP has been revealed.

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.

The activation of muscle phosphorylase kinase b by actin has been studied. F-actin which is polymerized by 2 mM MgCl2 is a more effective activator of phosphorylase kinase than F-actin polymerized by 50 mM KCl. There is evidence suggesting that the activation of phosphorylase kinase by actin is not due to trace contamination of actin preparations with calmodulin: (1) Troponin I and trifluoperazine inhibit the activation of phosphorylase kinase by calmodulin but do not inhibit the activation of phosphorylase kinase by F-actin. (2) The activation induced by saturating concentrations of calmodulin and actin is additive both at pH 8.2 and at pH 6.8. (3) The activation of phosphorylase kinase by calmodulin and actin has different pH profiles. An addition of F-actin does not affect the apparent Km value for ATP but increases the sensitivity to phosphorylase b and the value of Vmax.

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.

Red and white avian skeletal muscles (chicken and pigeon) contain the same alpha'-isoenzyme of phosphorylase kinase. According to data from gradient polyacrylamide slab electrophoresis in the presence of SDS, the molecular masses of beta- and gamma-subunits of phosphorylase kinase from rabbit, chicken and pigeon muscles are not identical. Electron microscopy data suggest that the quaternary structure of chicken and pigeon phosphorylase kinase is of the same type. The alpha'-isozyme of chicken and pigeon phosphorylase kinase is strongly activated by calmodulin and troponin C. Avian phosphorylase kinase is activated 2--3-fold by phosphorylation with cAMP-dependent protein kinase and by autophosphorylation. This activation is associated with the phosphorylation of both alpha'- and beta-subunits. The affinity of pigeon phosphorylase kinase a for Ca2+ is 20 times as high as that of phosphorylase kinase b.

Circular dichroism (CD) spectra of glycogen phosphorylase a and b from rabbit liver have been measured in the presence of various ligands in the near- and far-ultraviolet regions. Positive circular dichroism was detected in the absorption band of protein-bound pyridoxal phosphate (333 nm). The mean residue ellipticity of this dichroic band (35 deg cm2dmol-1) is of the same order for muscle and liver phosphorylase a and b and does not change upon binding of glucose-1-phosphate and AMP. Only glucose induces small changes in the ellipticity in this region. The CD spectra of muscle and liver phosphorylase a and b in the 250-300 nm region have at least five positive dichroic bands namely at 259, 264, 273, 281 and 288 nm and have strong resemblances for all these forms of the enzyme in spite of the fundamental differences in their properties. The binding of AMP and glucose to phosphorylase from both sources induces distinct perturbations in CD spectra; the changes are much larger for muscle and liver phosphorylase a than for phosphorylase b which indicates that conformational perturbations induced by binding of activator and inhibitor to the inactive form of phosphorylase are probably more local than for the active form. The CD spectra in far-ultraviolet region are similar for all forms of phosphorylase. The percent of alpha-helices calculated according to Chen is about 50; this value coincides very well with the value 51% received for muscle phosphorylase a by X-ray crystallographic analysis at 2.5 A resolution.

Purification of dephosphorylated form of rabbit liver glycogen phosphorylase (phosphorylase b) has been carried out. The purification procedure included 3 steps:sedimentation of protein-glycogen pellet by centrifugation at 62 000 x g during 2 hours, chromathography on omega-amino-hexyl-Sepharose column and isoelectric focusing. The yield of the enzyme is about 60%, the specific activity at glucose-1-phosphate concentration 100mM is 35 mkmol of Pi/mg protein-min. The final preparation revealed apparent homogeneity during ultracentrifugation and polyacrylamide gel electrophoresis. The subunit molecular weight of 100 000 was determined by disc-electrophoresis in the presence of sodium dodecyl sulfate. The absorption spectrum of liver phosphorylase b shows a protein maximum at 280 nm and a second peak at 333 nm produced by bound PLP. A kinetic analysis of initial rates with variable concentrations of the substrate revealed the homotropic cooperativity of glucose-1-phosphate binding sites. This cooperative effect is more pronounced in the presence of inhibitors, glucose and ATP and less pronounced in the presence of AMP.

Muscle glycogen phosphorylase exists in two enzymatically interconvertible forms: phosphorylase a and phosphorylase b. It has been shown that phosphorylase a in solution is a tetramer of molecular weight 400000 (s=13,2S), and phosphorylase b is a dimer of molecular weight 200000 (1). In the presence of p-chlormercuribenzoate (2) phosphorylases a and b dissociate into four and two monomers, respectively, both having the same molecular weight. On conversion of phosphorylase b into a, in the presence of ATP, Mg2+ and phosphorylase b kinase, one phosphate group appears to be bonded to the serine residue per monomer of the enzyme (3). If phosphorylase a is fully active in the native state without 5′-AMP, phosphorylase b, the non-phosphorylated form of the enzyme, is active only in the presence of 5′-AMP or IMP with protamin (4,5). The ultracentrifuge studies have shown that the dimeric form of phosphorylase b is converted into the tetrameric form (s=13,2S) in the presence of a mixture of 0,001M−AMP+0,03M cysteine+0,01M Mg2+(6) or 0,001M 5′−IMP+ 0,0001M protamin or protamin only (7). Under the same conditions, at a rather high protein concentration and a low temperature, phosphorylase b forms crystals quite readily (6,8); phosphorylase a forms crystals at a low temperature and high protein concentration in 0,03M cysteine or 0,03M β -mercaptoethanol as well as in the presence of protamin.