[Association of rabbit skeletal muscle phosphorylase kinase with sarcoplasmic reticulum membranes]. / Assotsiatsiia kinazy fosforilazy skeletnykh myshts krolika s membranami sarkoplazmaticheskogo retikuluma.

The binding of phosphorylase kinase to sarcoplasmic reticulum has been studied using gel chromatography. The presence of Ca2+, Mg2+ and glycogen was found to be necessary for the maximal binding. The phosphorylase kinase adsorbed on sarcoplasmic reticulum is capable of phosphorylating exogenous phosphorylase b. Phosphorylase kinase was shown to take no part in the phosphorylation of sarcoplasmic reticulum proteins. Exogenous calmodulin initiates the incorporation of [gamma-32P] of ATP into sarcoplasmic reticulum proteins. The data obtained point to a possibility that another Ca(2+)-calmodulin-dependent protein kinase may participate in the phosphorylation of sarcoplasmic proteins.

[Isolation and study of certain properties of caldesmon from cattle aorta]. / Vydenenie i izuchenie nekorotykh svoistv kal'desmona iz aorty byka.

A modified method for bovine aorta caldesmon isolation has been developed. When isolated from large vessels, caldesmon is copurified with connective tissue proteins whose molecular weight is similar to that of caldesmon. Separation of these proteins can be achieved by stepwise anion- (Q-Sepharose) and cation (phosphocellulose)-exchange chromatography. Bovine aorta and duck gizzard caldesmons have similar apparent molecular weights, absorption at 280 nm and one-dimensional peptide maps. Casein kinase II transfers about one mol of phosphate per mol of bovine aorta or duck gizzard caldesmon. In both cases, the sites of phosphorylation are located in the N-terminal peptides of apparent molecular weights of 26-28 kDa. It is concluded that there are no substantial differences between the structures and properties of avian gizzard and mammalian vessel caldesmons.

[Interaction between glycogen phosphorylase b and creatinine kinase from rabbit skeletal muscle]. / Vzaimodeistvie mezhdu glikogenfosforilazoi b i kreatinkinazoi skeletnykh myshts krolika.

Phosphorylase b association with creatine kinase has been studied by frontal elution affinity chromatography, using CNBr-Sepharose 4B immobilized creatine kinase as the affinity matrix. The quantitative parameters of this interaction were estimated from the volumes of phosphorylase b elution at various concentrations of the enzyme. The dissociation constants for phosphorylase b complexes with immobilized creatine kinase and of the phosphorylase b complex with free creatine kinase were found to be equal to 0.49 and 0.191 microM, respectively. In the presence of AMP the interaction between the proteins became weaker. With a rise in AMP concentration from 0.02 to 0.15 mM the value of the dissociation constants increased from 1.59 up to 9.66 microM. One molecule of AMP was shown to bind on the phosphorylase b-immobilized creatine kinase complex.

[Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]. / Vydelenie i svoistva trekh form fosforilazy i kinazy fosforilazy iz skeletnykh myshts cheloveka.

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.

[Interaction of phosphorylase kinase with thin filament proteins of rabbit skeletal muscles]. / Vzaimodeistvie kinazy fosforilazy s belkami tonkikh filamentov skeletnykh myshts krolika.

The binding of phosphorylase kinase to thin filaments and their effects on the enzyme activity as well as the contribution of the enzyme to contractile protein phosphorylation have been studied. The data obtained suggest that the kinase binding to thin filaments is controlled by the regulatory proteins, troponin and tropomyosin. The bulk of the enzyme is bound to the F-actin-tropomyosin-troponin complex which activates the enzyme in a far greater degree than each of its constituent components. Ca2+ and ATP control the kinase binding to F-actin. ATP increases the enzyme binding 6-fold; Ca2+ decrease the S0.5 value for F-actin 5-fold. In acetone powder extracts phosphorylase kinase phosphorylates thin filament-bound phosphorylase b, troponin T and troponin I as well as 51-58 kDa and 114 kDa proteins. These results suggest that phosphorylase kinase plays a role in the mechanism of synchronization of glycogenolysis and muscle contraction rates.

The pH-dependent conformational transition in glycogen phosphorylase b. The effect of carnosine and anserine on its activity.

The conformational transition of phosphorylase b which occurs upon pH change from 6.0 to 7.0 is linked to the pH-dependent activity regulation. Skeletal muscle dipeptides, carnosine and anserine, activate rabbit and bovine phosphorylase b at pH 6.5-6.0 and inhibit it at pH 6.5-7.0. The enzyme activation constant was found to be equal to 20 mM, and the inhibition constant corresponded to 22 mM for both carnosine and anserine. The data obtained suggest that glycogen phosphorylase b adopts different conformational states at pH 6.0 and 7.0. Evidence for the pH-induced conformational transition of the enzyme was obtained using chemical modification of histidine residues. The change in the phosphorylase activity under effects of the dipeptides in the pH range of 7.0-6.0 may be due to their physiological role in muscle contraction.

[The role of phosphorylase kinase subunits in interaction with glycogen]. / Rol' sub''edinits kinazy fosforilazy vo vzaimodeistii s glikogenom.

The interaction of rabbit skeletal muscle phosphorylase kinase with CNBr-activated glycogen results in the formation of a covalent complex. The non-bound kinase was removed by chromatography on DEAE-cellulose and phenyl-Sepharose. The amount of the bound protein increased with an increase in the number of activated groups in the glycogen molecule; the enzyme activity was thereby decreased. The kinase covalently and non-covalently bound to glycogen exhibited a higher affinity for the protein substrate (phosphorylase b) as well as for Mg2+ and Ca2+ than did the kinase in the absence of glycogen. Electrophoresis performed under denaturating conditions showed that the gamma-subunit of phosphorylase kinase is responsible for the enzyme binding to CNBr-glycogen. The effect of cross-linking reagents (glutaric aldehyde, 1.5-difluoro-2.4-dinitrobenzene) on the binding of phosphorylase kinase subunits was studied. Glycogen afforded protection of the gamma-subunit from the cross-linking to other enzyme subunits. An analysis of the subunit composition of phosphorylase kinase covalently bound to CNBr-glycogen and of the enzyme treated with cross-linking reagents in the presence of glycogen-revealed that the gamma-subunit is involved in the specific binding of phosphorylase kinase to glycogen.

[Regulation of the activity of rabbit skeletal muscle phosphorylase kinase by glycogen]. / Reguliatsiia aktivnosti kinazy fosforilazy iz skeletnykh myshts krolika glikogenom.

The effects of glycogen on the non-activated and activated forms of phosphorylase kinase were studied. It was found that in the presence of glycogen the activity of non-activated kinase at pH 6.8 and 8.2 and that of the activated (in the course of phosphorylation) form are enhanced. The degree of activation depends on glycogen concentration. At saturating concentrations, this enzyme activity increases 2-3-fold; the enzyme affinity for the protein substrate, phosphorylase b, also shows an increase. The polysaccharide has no effect on the activity of phosphorylase kinase stimulated by limited proteolysis. In the presence of glycogen, the rate of autocatalytic phosphorylation of the enzyme is increased. Glycogen stabilizes the enzyme activity upon dilution. The experimental results suggest that the polysaccharide directly affects the phosphorylase kinase molecule. The maximal binding was shown to occur at the enzyme/polysaccharide ratio of 1:10 (w/w) in the presence of Ca2+ and Mg2+.

[Effect of pH on conformation and kinetic properties of phosphorylase from bovine skeletal muscles]. / Vliianie pH na konformatsionnye i kineticheskie svoistva fosforilazy iz skeletnykh myshts byka.

Interaction of phosphorylase with 8-anilino-1-naphthalene-sulfonate (ANS) results in the formation of an ANS-protein complex. The microenvironment of the protein-bound dye changes depending on pH. Using fluorimetric titration, the dissociation constants for the complex (Kd = 23 and 57 microM for pH 6.2 and 6.8, respectively) were determined. The mode of the enzyme inhibition by ANS also changes depending on pH. At pH 6.8, ANS competitively inhibits the enzyme with respect to AMP, but does not compete with the nucleotide at pH 6.2; the corresponding Ki values are equal to 160 and 26 microM. The protective effect of ligands from the inhibiting effect of ANS was studied. It was shown that at pH 6.2, the enzyme is protected from the inhibition only by the substrate, glucose-1-phosphate, whereas at pH 6.8--by the allosteric inhibitor, glucose-6-phosphate. These findings suggest that at pH 6.2 the conformation of the enzyme molecule is induced by the substrate, while at pH 6.8--by the allosteric inhibitor. ANS binding in the vicinity of the active or allosteric centers is due to the pH-dependent conformational transition. The data obtained suggest that the pH changes within the range of 6.2-6.8 are essential for the regulation of enzyme activity.

[Purification and properties of glycogen phosphorylase isolated from bovine skeletal muscles]. / Ochistka i svoistva glikogenfosforilazy, vydelennoi iz skeletnykh myshts byka.

Glycogen phosphorylase isolated from bovine skeletal muscles was found to be homogeneous during polyacrylamide gel electrophoresis. The enzyme phosphorylation by phosphorylase kinase is accompanied by the incorporation of one mole of labeled phosphate per protein dimer; therefore the enzyme is represented by a partly phosphorylated form. The presence of a phosphate group prevents the removal of the protein-bound pyridoxal phosphate. The partly phosphorylated bovine phosphorylase possesses a low affinity for AMP and is inactive in the presence of IMP. Bovine phosphorylase a obtained from the partly phosphorylated enzyme has a molecular mass corresponding to a dimer. Both forms of bovine phosphorylase exhibit high cooperativity towards the substrate. The mechanism of phosphorylase a activation by AMP and IMP is identical: the nucleotides increase the enzyme affinity for the substrate as well as the maximal rate of the enzymatic reaction. Study of the enzyme inhibition by caffeine revealed the cooperativity of caffeine-binding centers. The equilibrium between the active and inactive enzyme conformations in the presence of caffeine is markedly shifted towards the inactive (T) form of glycogen phosphorylase.

[Allosteric properties of phosphorylase b]. / Allostericheskie svoistva fosforilazy b.

Rabbit skeletal muscle phosphorylase b was separated into two fractions by column chromatography on AMP-Sepharose. The first fraction protein was eluted by glucose-6-phosphate while the second fraction protein was eluted in an AMP concentration gradient. The bulk of the protein eluate was represented by the first fraction protein. Chromatography of phosphorylase b from bovine skeletal muscle under identical conditions also resulted in two fractions, however, with a reverse correlation: the bulk protein of this fraction was eluted by AMP. It was shown that the two phosphorylase b forms eluted by glucose-6-phosphate and AMP differ by their kinetic and physico-chemical properties as well as by the SH-group reactivity. The phosphorylase b forms eluted by the nucleotide were practically uninhibited by glucose-6-phosphate. It can thus be assumed that the equilibrium between the "active" (R) and "inactive" (T) conformations of the protein changes depending on metabolic peculiarities of a given tissue used as a source for enzyme isolation.

[The presence of glycogen synthase in phosphorylase kinase preparations isolated from rabbit skeletal muscles]. / Prisutstvie glikogensintazy v preparatakh kinazy fosforilazy, vydelennykh iz skeletnykh myshts krolika.

Phosphorylase kinase isolated from rabbit skeletal muscle contains a protein whose molecular mass as determined by polyacrylamide gel electrophoresis is 571 000 Da. The protein was found to possess a higher affinity for glycogen as compared to phosphorylase kinase and phosphorylase. The protein separated from kinase by chromatography on a DEAE-cellulose column produced during SDS electrophoresis one protein band corresponding to Mr of 95 200 Da. The above properties of the protein and the glycogen synthetase activity revealed in the presence of glucose-6-phosphate suggest that phosphorylase kinase preparations contain a hexameric form of glycogen synthetase.

[Role of the sulfhydryl groups of phosphorylase kinase]. / Izuchenie roli sul'fgidril'nykh grupp kinazy fosforilazy.

The cysteinyl residues of rabbit skeletal muscle non-activated phosphorylase kinase were titrated by 5,5'-dithiobis (2-nitrobenzoic acid). According to their reactivity toward SH-reagent the accessible cysteinyl residues may be classified into three groups. Modification of the first group residues is accompanied by the enzyme activation. The inactivation is characterized by a biphasic kinetic profile. The fast and slow phases of inactivation correlate in time with modification of the second and third groups of cysteinyl residues. The apparent order of the reaction with respect to inhibitor concentrations for the slow and fast reactions gives a value of about 1, thus indicating that the enzyme contains two essential cysteinyl residues per protomer (alpha beta gamma delta). Mg-ATP and Mg-ADP protect the enzyme against inactivation and modification of sulfhydryl groups. It may be assumed that the two essential cysteinyl residues are located near the nucleotide-binding sites.

[Properties of the catalytically active fragment obtained by limited proteolysis of phosphorylase kinase]. / Svoistva kataliticheski aktivnogo fragmenta, poluchennogo putem ogranichennogo proteoliza kinazy fosforilazy.

The activation of phosphorylase kinase by limited proteolysis with subtilisin results in a formation of new enzyme forms differing in their molecular weights. Using gradient electrophoresis in polyacrylamide gel, it was shown that the high molecular weight fraction is made up of active fragments having different molecular weights. The low molecular weight fraction was found to contain only one active fragment with molecular weight of 80 000. Disc electrophoresis in polyacrylamide gel demonstrated that the active fragments of the high and low molecular weight fractions are not homogenous. The kinetic properties of the low molecular weight fragment were investigated. It was found that at pH 8.2 the native non-activated kinase and the catalytically active fragment have identical Km values for the substrates (phosphorylase B and MgATP); however, unlike the non-activated kinase, this fragment possesses a decreased sensitivity to Ca2+ and effectors (glycogen and glucose 6-phosphate) and has no optimum of activity within the pH range of 6.0-9.0.

[Interaction of phosphorylase B with SH-reagents and properties of the modified enzyme]. / Vzaimodeistvie fosforilazy v C SH-PEAgentami i izuchenie svoistv modifitsirovannogo fermenta.

The interaction of phosphorylase B with the SH-reagents, i.e. 2-chloromercuri-4-nitrophenol and ethylmercurichloride was studied. It was shown that phosphorylase B inhibition obeys the pseudo-first-order kinetics, the inactivation rate constants being equal to 11 M-1 s-1 and 17,5 M-1 s-1, respectively. Data from the SH-group titration with 2-chloromercuri-4-nitrophenol and p-chloromercuri benzoate suggest that the number of modified cysteine residues and the amount of bound 2-chloromercuri-4-nitrophenol in the phosphorylase B dimer is equal to 2. In the modified phosphorylase B the absorption maximum of pyridoxal phosphate is decreased at 330 nm and is increased at 410 nm. The binding of 2-chloromercuri-4-nitrophenol is accompanied by quenching of the protein and coenzyme fluorescence. Upon interaction with ethylmercurichloride only the pyridoxalphosphate fluorescence is quenched. The increase of the spin label mobility in the modified enzyme calculated from the EPR spectra of the spin-labelled preparations is indicative of the changes in the protein conformation coupled with the blocking of one SH-group in the enzyme monomer. The rate of enzyme inactivation under effects of the SH-reagents is a function of pH and is considerably increased within the pH range of 5.7-6.7. The pH-optimum of activity of partly modified enzyme remains practically unchanged; however, at the pH shift towards the acidic values the activity is drastically decreased as compared to that of the native enzyme. The data obtained suggest that the enzyme inactivation is due to modification of one SH-group in the phosphorylase B monomer vicinal to the pyridoxal phosphate binding site and probably involved in the enzymatic reaction.

[Conformational changes of spin-labeled native and modified phosphorylase B]. / Izuchenie konformatsionnykh izmenenii spin-mechenykh nativnoi i modifitsirovannoi fosforilazy B.

The phosphorylase B labelled with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-iodacetamide (phosphorylase I) and with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-ethylmaleinimide (phosphorylase II) was studied. It was shown that label I is characterized by a greater mobility with respect to the protein as compared to label II. In spin-labelled preparations of phosphorylase B the 1,5--2,0 SH-groups of the enzyme monomer having no effect on the enzyme activity were modified. The effects of AMP, glucose-1-phosphate and glucose-6-phosphate on the EPR spectrum of phosphorylase I were studied. The greatest changes in the spectrum (especially in the high field line) were found to occur in the presence of glucose-6-phosphate. These changes are due to the increase in the degree of anisotropic spin rotation. The experimental and theoretical spectra allowing to determine the correlation time for the protein moiety (tau b = 160 ns) were shown to be similar. The local conformation changes were found to occur in the vicinity of one of the two label-bound SH-groups of phosphorylase I. The EPR spectra demonstrate the S-shaped dependence of mobility of phosphorylase I label on concentration of glucose-6-phosphate (0,1--10 mM). In the presence of AMP no S-shaped dependence is observed. Reduced NaBH4 phosphorylase I does not reveal the S-shaped dependence of the label mobility on concentration of glucose-6-phosphate. The degree of the label immobilization in the apo-phosphorylase I--pyridoxal-5-chloromethylphosphonate complex in the presence of glucose-6-phosphate and AMP is the same as in cholophosphorylase I; however, in contrast to the choloenzyme it does not depend on glucose-6-phosphate (0,1--10,0 mM). The changes in the mobility of the spin label of apophosphorylase I and its complex with the AMP analog--adenosine-5'-chloromethylphosphonate--during the choloenzyme reconstruction by pyridoxalphosphate are indicative of participation of AMP and the phosphate group of AMP in the formation of the enzyme active center.

[Structural-functional analysis of the complex between phosphorylase B and adenosine-5'-chloromethylphosphonate]. / Strukturno-funktsional'nyi analiz kompleksa fosforilazy B s adenozin-5'-khlormentilfosfonatom.

A complex of phosphorylase B with a tritium-containing AMP analogue, adenosine-5'-chloromethylphosphonate, was obtained. It is found on the basis of the results of the determination of N- and C-terminal amino acids, amino acid composition and sequence, that the peptide 1, modified by adenosine-5'-chloromethylphosphonate, corresponds to the fragment 185-191 in the primary structure of phosphorylase B, and is probably located in the allosteric center of the enzyme. The peptide 2, which is bound with the AMP analogue and corresponds to the fragment 795-798, is suggested to be located at the site of binding the second AMP molecule. The arginine residue 184 is discussed as a possible functional amino acid protein interacting with 5'-phosphate AMP group.