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.

The regulatory alpha subunit of phosphorylase kinase may directly participate in the binding of glycogen phosphorylase.

The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, alpha, of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One fragment, corresponding to residues 17-484 (PhbN'), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb. Truncation fragments of the alpha subunit were screened for interactions against these two substrate fragments. PhbC was not found to interact with any alpha constructs; however, PhbN' interacted with a region of alpha (residues 864-1014) that is near the phosphorylatable region of that subunit. PhbN' was also screened for interactions against a variety of fragments of the catalytic gamma subunit of PhK; however, no interactions were detected, even with full-length gamma. Our results support the idea that amino acid residues proximal to the convertible serine of Phb are important for its specific interaction with the catalytic subunit of PhK, but that regions distinct from the convertible serine residue of Phb and from the catalytic domain of PhK may also be involved in the interaction of these two proteins.

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 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.

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.

[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.

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.

[Molecular mechanisms of the regulation of phosphorylase kinase from skeletal muscles of mammals and birds]. / Molekuliarnye mekhanizmy reguliatsii aktivnosti kinazy fosforilazy iz myshts mlekopitaiushchikh i ptits.

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.

[Purification, quaternary structure and regulatory properties of phosphorylase kinase from pigeon skeletal muscle]. / Ochistka, chetvertichnaia struktura i reguliatornye svoistva kinazy fosforilazy iz skeletnykh myshts golubia.

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.

[Limited proteolysis and phosphorylation of phosphorylase kinase from chicken skeletal muscles]. / Ogranichennyi proteoliz i fosforilirovanie kinazy fosforilazy iz skeletnykh myshts kuritsy.

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.

Phosphorylase kinase from chicken skeletal muscle. Quaternary structure, regulatory properties and partial proteolysis.

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'.

[Regulatory properties of phosphorylase from chicken skeletal muscle]. / Reguliatornye svoistva fosforilazy iz skeletnykh myshts kuritsy.

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.

[Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle]. / Ochistka, chetvertichnaia struktura i nekotorye immunologicheskie svoistva kinazy fosforilazy iz skeletnykh myshts kuritsy.

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.

[Activation of phosphorylase kinase from rabbit muscle by actin and calmodulin]. / Aktivatsiia kinazy fosforilazy iz myshts krolika aktinom i kal'modulinom.

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.

Regulation of muscle phosphorylase kinase by actin and calmodulin.

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.