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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 experimental data on the kinetics of irreversible aggregation of proteins caused by exposure to elevated temperatures or the action of denaturing agents (guanidine hydrochloride, urea) have been analyzed. It was shown that the terminal phase of aggregation followed, as a rule, first order kinetics. For the kinetic curves registered by an increase in the apparent absorbance (A) in time (t) the methods of estimation of the corresponding kinetic parameters A(lim) and kI (A(lim) is the limiting value of A at t --infinity and kI is the rate constant of the first order) have been proposed. Cases are revealed when the reaction rate constant kI calculated from the kinetic curve of aggregation of the enzymes coincides with the rate constant for enzyme inactivation. Such a situation is interpreted as a case when the rate of aggregation is limited by the stage of denaturation of the enzyme. A conclusion has been made that, in order to establish the mechanism of protein aggregation, the kinetic investigations of aggregation should be carried out over a wide range of protein concentrations. The refolding experiments after denaturation of proteins by guanidine hydrochloride or urea have been also analyzed. It was shown that aggregation accompanying refolding follows first order kinetics at the final phase of the process. The model of protein refolding explaining such a kinetic regularity has been proposed. When aggregation of protein substrate follows first order kinetics, parameters A(lim) and kI may be used for the quantitative characterization of the chaperone-like activity in the test-systems based on suppression of protein aggregation.

Kinetic analysis of the glycogen chain growth reaction catalyzed by glycogen phosphorylase b from rabbit skeletal muscle has been carried out over a wide range of AMP concentration under the saturation of the enzyme by glycogen. Applicability of some variants of the kinetic model involving the interaction of AMP- and glucose 1-phosphate-binding sites in the dimeric enzyme molecule is considered. A kinetic model of the enzymatic reaction describing adequately the activation of the enzyme by AMP and inhibition at sufficiently high concentrations of AMP is proposed.

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 oligomeric state and formation of supramolecular structures of glycogen phosphorylase b from rabbit skeletal muscle was studied in the system of aerosol OT (AOT) reversed micelles in octane. The sedimentation experiments have shown that the enzyme oligomeric state depends on the degree of micelle hydration. The enzyme monomer, dimer, trimer, tetramer, hexamer, and octamer were observed, depending on the degree of hydration.

Large-scale functionally significant changes in the intramolecular dynamics of muscle glycogen phosphorylase b (EC 2.4.1.1) in solution upon ligand binding, transition from dimeric to tetrameric form, temperature denaturation and aggregation were registered at room temperature using the tryptophan phosphorescence technique. It was shown that binding of glucose-1-phosphate (substrate, 0.25-4 mM) and glucose (competitive inhibitor, 0.5-8 mM) to the active site and temperature-induced protein aggregation decrease large-scale structural fluctuations of the protein matrix at the level of domains and subunits; whereas the transition of glycogen phosphorylase b to tetrameric form (R-conformation) leads to a dramatic increase in the structural flexibility of the peripheral parts of the protein globule.

Hydrolysis of N-trans-cynnamoylimidazole catalyzed by conjugates and complexes of alpha-chymotrypsin (ChT) with poly(ethylene glycol) (PEG) of different molecular mass (from 300 to 5000 daltons) was studied in the system of the hydrated reversed micelles of aerosol OT (AOT) in octane at 25 degrees C. The plot of the deacylation constant k3 for PEG--ChT conjugates and complexes versus the degree of hydration of reversed micelles (w0 = [H2O]/[AOT]) was studied. These plots are bell-shaped with maxima shifted to higher degrees of micelle hydration compared to the corresponding value of the shift for ChT. As for PEG--ChT conjugates, the value of the shift of w0 increases with increasing of molecular mass of the attached PEG and/or with the number of polymer chains per ChT molecule. Another picture was observed for PEG--ChT complexes for which the position of the maximum on k3 versusw0 curves was practically the same for all compounds. The values of the thickness of the polymer layer for PEG--ChT conjugates and complexes were calculated. Thus, polymer chains in conjugates placed in hydrated micelles are highly packed, whereas in the case of complexes they form a flat layer on the surface of the protein.

The mechanism of heat aggregation of proteins proposed by the author involves the stage of irreversible denaturation, the stage of nucleation, and the stage of growth of aggregates. It was shown that the initial parts of the kinetic curves of aggregation followed by monitoring the increase in absorbance (A) or intensity of light scattering (I) are linearized in coordinates (dA/dt; t) and (A; t2) (or, respectively, in coordinates (dI/dt; t) and (I; t2)). The slope of these linear anamorphoses is proportional to the product of the rate constant of irreversible denaturation and the rate constant of growth of aggregates. The mechanism of heat aggregation proposed is fulfilled for pig heart citrate synthase. The dI/dt versus t curves for heat aggregation of glycogen phosphorylase b from rabbit skeletal muscles display a lag period whose appearance is caused by intramolecular predenaturational changes in the enzyme molecule.

The thermostability of conjugates, non-covalent complexes and mixtures of alpha-chymotrypsin (alpha-ChT) with poly(alkylene oxides)--poly(ethylene glycol) (PEG) with molecular mass of 1.9 kD and diblock copolymers of ethylene and propylene oxides (proxanols)--has been investigated. It was shown that the addition of PEG in concentration up to 2 wt. % to the solution of alpha-ChT did not affect the rate of the enzyme thermoinactivation. Meanwhile the addition of proxanol in the same concentration resulted in twofold decrease in the rate constant for the slow inactivation step, k2. Even more pronounced decrease in the thermoinactivation rate was observed for alpha-ChT--proxanol complexes obtained by heating or under the action of high pressure. The general tendency in the behavior of complexes of both types was the decrease in the k2 constant as the temperature or pressure used for complex preparation increased. The highest stabilizing effect was observed for complex obtained by heating up to 52 degreesC and containing maximal number of polymer chains (molar ratio proxanol/alpha-ChT was 10). For this complex fourfold decrease in the k2 value was observed. Covalent attachment of PEG or proxanol to enzyme gives maximal stabilizing effect with up to tenfold decrease in the k2 value. The investigation of the thermal denaturation kinetics of alpha-ChT and its adducts with poly(alkylene oxides) by means of fluorescence spectroscopy has shown that the presence of polymer chains practically does not affect the rate of protein denaturation registered by the decrease in the intensity of protein fluorescence. The polymer chains, probably, diminish the rate of melting of the active site-containing region of the protein molecule. At the same time, the overall denaturation rate is independent of the presence of polymer chains in the vicinity of the protein globule.

A new two-step procedure of protein refolding in vitro, proposed by Rozema and Gellman and named artificial chaperone-assisted refolding, is discussed. The new approach has been inspired by the two-step mechanism of the GroE system. In the first step, the protein is captured by a detergent under conditions that would normally lead to irreversible protein aggregation (heating or denaturant removal). In the second step, removal of detergent from the protein--detergent complex is triggered by addition of a cyclodextrin which is capable of forming "inclusion complexes" with detergent, allowing the protein to refold. The protein refolded with artificial chaperones (detergent and cyclodextrin) may be purified via a two-step protocol. After refolding was complete, the solution was passed through a 0. 22-micro(m) filter, to remove aggregated protein, and then through a M = 10 kD cutoff filter. The second filtration was intended to allow the low-molecular-weight artificial chaperones to pass, but to retain the refolded enzyme. The application of the above procedure for refolding of carbonic anhydrase B from human erythrocytes, hen egg white lysozyme, pig heart citrate synthase, and creatine kinase from rabbit skeletal muscles (MM isoenzyme) is discussed.

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 inhibition of glycogen phosphorylase b from rabbit skeletal muscles by the derivatives of riboflavin, FMN, FAD, and 2', 3', 4', 5'-tetraacetylriboflavin substituted in positions 6 and 8 of the isoalloxazine part of the flavin molecule is found to be cooperative (the Hill coefficient, h, exceeds 1.0). The modification of the flavin molecule slightly changes the value of the Hill coefficient, but results in the increase of the "half-saturation" concentration [I]0.5.

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 interaction of the myosin subfragment-1 (S-1) isoforms containing different alkali light chains A1 and A2 with F-actin has been studied using the stopped-flow technique and sedimentation in an analytical ultracentrifuge. The data obtained suggest a different mode of attachment of the S-1 isoforms to F-actin filaments.

Based on a theoretical analysis of functioning of a monocascade enzymatic system, a method for continuous monitoring of the phosphorylase kinase-catalyzed enzymatic reaction has been developed. The method is based on the ability of the kinase reaction product--the phosphorylated form of glycogen phosphorylase (form a)-to catalyze glycogen phosphorolysis (with inorganic phosphate as the low molecular weight substrate) or synthesis (with glucose 1-phosphate) in the absence of AMP. A turbidimetric method may be used for the monitoring of the reaction of glycogen degradation (or synthesis) by phosphorylase a formed in the course of the kinase reaction. A method to calculate the initial rate of the kinase reaction from an absorbance versus (time)2 plot has been theoretically substantiated.

The kinetics of glycogen chains growth catalyzed by glycogen phosphorylase b from rabbit skeletal muscles at saturating glycogen concentrations have been studied. The applicability of twelve different variants of the Monod-Wyman-Changeux model and six kinetic schemes assuming the interaction of AMP- and glucose 1-phosphate binding sites in the dimeric enzyme molecule is discussed. The kinetic model of the reaction is proposed which suggests: (1) independent binding of AMP and a molecule of glucose 1-phosphate to the dimeric enzyme molecule saturated by glycogen; (2) exclusive binding of the second glucose 1-phosphate molecule by the dimeric enzyme molecule containing two molecules of bound AMP and one molecule of bound glucose 1-phosphate; (3) exclusive ability of the enzyme complex with glycogen, two molecules of AMP and two molecules of glucose 1-phosphate in catalytic transformation. The parameters of the rate equation were calculated by nonlinear regression analysis. The proposed kinetic scheme satisfies the following criteria: (1) convergence in the regressional analysis; (2) reliability of the parameter values; (3) the minimal sum of the weighed squares of residuals. This model was found to fit adequately the kinetics of muscle phosphorylase b reaction at saturating glycogen concentrations.

The effect of specific ligands (the substrate glucose 1-phosphate, AMP, and flavins) on the initial rate of tryptic digestion of muscle glycogen phosphorylase b has been studied (0.02 M HEPES, pH 6.8; 37 degrees C). Differences were observed between the kinetic curves of the enzyme trypsinolysis initiated by the addition of a trypsin/ligand mixture and once trypsin was added to the enzyme preincubated with a ligand for 10 min. It was suggested that the specific ligands studied induce relatively slow conformational changes in the phosphorylase b molecule.

A fluorescent study of some structural and functional properties of conjugates of a number of proteins (bovine serum albumin, pyruvate kinase, alpha-chymotrypsin, and the two toxic proteins of plant origin--ricin and viscumin) with polyalkylene oxides (polyethylene glycol and pluronic) has been carried out. Analysis of the intrinsic protein fluorescence showed that the structure and stability of various protein conjugates to denaturing agents change only slightly: the conformational mobility of tryptophan residues accessible to the solvent decreases, whereas that of tryptophan residues localized in the protein regions of low polarity remains unchanged. Besides, the conjugates display a higher thermal stability in comparison with their native proteins. The fluorescence of 1-anilinonaphthalene-8-sulfonic acid and water insoluble 2',3',4',5'-tetrabenzoylriboflavin bound to the native and modified proteins indicated that modification of the proteins with polyalkylene oxides decreased the polarity and increased the viscosity of the microenvironment. Hence, this modification makes it possible to change some functional characteristics of the protein without causing any significant changes in its structure.

The effect of polyalkylene oxides and pluronics (block copolymers of polyethylene oxide and polypropylene oxide) and their conjugates with bovine serum albumin (BSA) on respiration and oxidative phosphorylation in rat heart mitochondria has been studied. The pluronics and their conjugates with BSA stimulate state 4 respiration in mitochondria, inhibit state 3 respiration and 2,4-dinitrophenol-uncoupled state respiration, decrease the respiratory control, ADP/O ratio values and display weak uncoupling properties. The pluronics inhibit respiration and oxidative phosphorylation more effectively than their conjugates with BSA. The effect of the conjugate on the mitochondrial membrane is reversible, in contrast with that of the pluronics. The compounds under study act preferentially on the NADH-dehydrogenase complex of the respiratory chain. Inhibition of respiration in thymocytes by these compounds confirms their ability to penetrate into the cell membrane. The dependence of membranotropic properties of polyalkylene oxides and their conjugates with the protein on their structure is discussed.

Solubilization of 2',3',4',5'-tetrabenzoyl-5-acetyl-1,5-dihydroriboflavin (benzaflavin) by conjugates of BSA with poly(alkylene oxide)s [poly(ethylene glycol) and block co-polymers of ethylene oxide and propylene oxide (pluronics)] was investigated. Conjugates of BSA with pluronics were shown to have more solubilizing efficiency towards benzaflavin than BSA itself. Solubilized forms of benzaflavin are able to inhibit NADPH-dependent peroxidation of lipids in rat liver microsomes. A study of solubilized riboflavin ester transfer to mitochondria demonstrated that conjugates of BSA with pluronics may be advantageously employed for this purpose. Inhibitory properties of solubilized forms of benzaflavin were demonstrated by the study of their influence on the enzyme activity of D-amino-acid oxidase from pig kidney. The results show that solubilized forms of biologically active compounds based on conjugates of proteins with poly(alkylene oxide)s may be used for testing of chemical substances in biochemical systems.

A fraction of coarse mitochondria from the rat brain was deposited after short-term effect of supersound to obtain "metabolones". Activity of dehydrogenases of alpha-ketoacids, succinate dehydrogenase, aspartate-, alanine- and GABA-alpha-ketoglutarate amino-transferases has been determined in the supernatant liquid and in "metabolones". It is shown that dehydrogenase activity is mainly (93-100%) localized in "metabolones", while the level of aminotransferase activity in the latter is lower (72-94%). Nonproportionally high activity of aminotransferases in the supernatant liquid is found to considerably suprpass a decrease in activity of these enzymes in "metabolones" against a background of extremely scanty losses of protein (within 5%) induced by the supersound effect. A hypothetic model of a "metabolone" containing the enzymes of the Krebbs cycle and GABA-shunt is suggested.

The two-phase character of essential histidine residues modification of pyruvate dehydrogenase component of pyruvate dehydrogenase complex from pigeon breast muscle by diethylpyrocarbonate has been demonstrated. The relative amplitude of the fast phase increases with increasing the modificator concentration. The model of chemical modification of dimeric enzyme where the modification of the residue in one subunit leads to the change of reactivity of corresponding residue in the other subunit is used for the description of inactivation kinetics. The expression for the diminishing of enzyme activity in the course of chemical modification and the methods of kinetic parameters estimation have been proposed. The following values of kinetic parameters for the modification of pyruvate dehydrogenase component by diethylpyrocarbonate were obtained (pH 6.0; 20 degrees C): k1 = 6400 +/- 400 M-1 min-1 (the microscopic rate constant for the modification of histidine residue in the intact dimer), k2 = 890 +/- 200 M-1 min-1 (the rate constant for the modification of histidine residue in the intact subunit in the dimer which contains one modified subunit) and kt = 0.9 +/- 0.2 min-1 (the rate constant for conformational transition of the dimer induced by modification of histidine residue in one of the subunits in the dimeric molecule).

Inhibition of rabbit skeletal muscle glycogen phosphorylase b by biotin, pyridoxine, lipoic acid, as well as by thiamine and cobalamine vitamins and coenzymes has been found. The values of "half-saturation" concentration and Hill coefficients are determined for biotin (27 mM, 1.3), pyridoxine (19 mM, 1.7), 5'-deoxyadenosyl-cobalamine (2.5 mM, 1.5), lipoic acid (3.4 mM, 1.1), thiamine (11 mM, 1.3), thiamine diphosphate (11 mM, 1.0). Effectiveness of the enzyme inhibition by vitamins and coenzymes containing different heterocyclic groups is analysed; riboflavin and its coenzymic forms are suggested to be the most effective inhibitors.

The notion of the "primary blocks" of cellular metabolism (designated as "metabolic system") has been introduced. Metabolic system is defined as a metabolic pathway which corresponds to the really existing multienzyme complex. The complex of glycolytic enzymes which catalyzes the anaerobic reduction of glucose-6-phosphate with production of ATP may serve as an example of metabolic system (this complex does not contain hexokinase). The complex is formed on thin filaments of I-band of the muscle fibers or on dimers of band 3 protein embedded in the erythrocyte membranes. The fixation of the multienzyme complex to the support of biological nature provides the material basis for regulation of the metabolic system by chemical signals produced by the higher levels of metabolic control. Owing to interaction with anchor protein of the support the chemical signals exert the general control of functioning the multienzyme complex (switching on--switching-off of the metabolic system). It is assumed that the glycolytic system in skeletal muscles is stimulated by Ca2+ ions which interact with the anchor protein of the support (troponin C).

The vitamin B2 and its coenzyme forms binding to glycogen phosphorylase b from rabbit skeletal muscle has been studied by the spectrophotometric method. The spectral properties of riboflavin, FMN and FAD bound to muscle glycogen phosphorylase b were found to be identical at the wavelengths of 300 to 500 nm. According to data on spectrophotometric titration of muscle glycogen phosphorylase b by FMN, each subunit of the enzyme contains one flavin-binding site.

A theoretical analysis has been made of dependencies of specific enzymatic activity (a) on the concentration of the enzyme for associating enzyme systems, in which the association of protein molecules leads to the formation of linear associates of an unlimited length (M in equilibrium M2 in equilibrium M3 in equilibrium ...) and is accompanied by steric shielding of active centers, and also for systems of the type 2 M in equilibrium D in equilibrium D2 in equilibrium D3 in equilibrium ... (M is an inactive monomer and D is an active dimer), in which the specific enzymatic activity of the dimer does not depend on the degree of association. For both models an analysis has been made of the S-shape of the curves of the dependence of a on the concentration of the substrate. Experimental data for glutamate dehydrogenase from ox liver and phosphofructokinase from rabbit skeletal muscles have been used as illustrations.

Kinetic studies have demonstrated that vitamin B2 and its coenzyme forms FMN and FAD are potent inhibitors of glycogen phosphorylase b from rabbit skeletal muscle. The inhibition of the enzyme by flavins has a co-operative character (Hill coefficients exceed unity). Glycogen phosphorylase b bound to FMN or FAD does not reveal catalytic activity, whereas the enzyme bound to riboflavin retains about 16% of the initial catalytic activity.

The rate constants and equilibrium constants for four stages of process of the abortive ternary complex lactate dehydrogenase (porcine isoenzyme H4)-NAD-pyruvate formation are determined. These stages are 1) the enolization of pyruvate, 2) the formation of binary complex enzyme-NAD, 3) the formation of "intermediate" ternary complex enzyme-NAD-enol and 4) the transformation of "intermediate" complex into "final" ternary one, accumulation of the latter being followed by spectrophotometric and fluorometric methods. The constants obtained were compared with the corresponding ones for porcine isoenzyme M4 determined in earlier work. It was shown that the greater stability of ternary complex and the greater initial rate of ternary complex formation in the case of H4 is due to greater affinity of isoenzyme H4 with respect to NAD, greater magnitude of reaction rate constant for transformation of "intermediate" ternary complex into "final" one and lesser magnitude of reaction rate constant for the reverse transition.

Inhibition of rabbit skeletal muscle glycogen phosphorylase b by 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates has been studied. The inhibition is reversible and characterized by positive kinetic cooperativity (Hill coefficient exceeds 1). The values of pterin concentration causing two-fold diminishing of the enzymatic reaction rate increased in the order: 3',5'-dichloromethotrexate, 3'-chloromethotrexate, 5-methyl-5,6,7,8-tetrahydrofolic acid (0.24, 0.40 and 1.87 mM, respectively). Comparison of "half-saturation" concentrations for the above compounds and for methotrexate and folinic acid shows that pterin affinity to glycogen phosphorylase b is affected by substituents both in pteridine and in p-aminobenzoic moieties of the pterin molecule. The antagonism between 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates, on the one hand, and AMP and FMN, on the other, is revealed for combined action of modifiers on glycogen phosphorylase b.

General properties of enzymes and structurally ordered multienzyme complexes as controllable systems are discussed: the spatial isolation of working sites and sites of control and the realization of control mechanisms with the participation of "external" factors which provide the optimal functioning of the controllable system in the biological system of higher level of complexity. The basic mechanisms of the control of soluble enzymes are isosteric and allosteric mechanisms which directed to the maintenance of cellular homeostasis. The mechanism of functioning of a multienzyme complex as a whole which is realized with the participation of second messengers is classified as a mechanism for tracing of the signals from higher levels of the control of metabolism (from nervous, hormonal and immune systems). When discussing the control of functioning of the multienzyme complexes, special attention was paid to the complex of glycolytic enzymes formed on the structural proteins of skeletal muscles and on the membranes. An order of assembly of the complex of glycolytic enzymes is proposed. The possible localization of this complex in myofibrils is discussed.

It has been demonstrated that phenylhydrazine poisoning results in a significant activation of lipid peroxidation, reduction of dimethylaniline N-demethylation and NADPH-2.6-dichlorophenolindophenol and NADPH-cytochrome c-reductase activities as well as in the stimulation of the NADPH-nitrotetrazolium reductase activity in rabbit liver microsomes. In addition, phenylhydrazine administration causes disturbances in vitamin B12 metabolism which are manifested as changes in total cobalamin pool and in the spectrum of vitamin B12 individual physiologically active forms in the blood.

The reactions of NADPH- or dithionite-dependent reduction of cytochrome P-450 were studied using a stopped flow technique. It was found that the kinetic curves for both reactions may be fitted by a sum of the two exponents. The arrhenius plots for the fast phase rate constants are linear for both reactions. On the contrary, the breaks on the corresponding plots for the slow phase rate constants are observed at 22 and 33 degrees C for cytochrome P-450 reduction by dithionite and at 31 degrees C for NADPH-dependent reduction of cytochrome P-450. The coincidence of the values of the rate constants and activation energy (56 +/- 5 kJ/mol) for the fast phase of NADPH-dependent reduction of cytochrome P-450 with values of catalytic constants and activation energy for demethylation of tertiary amines suggests that the first electron transfer process from NADPH-cytochrome P-450 reductase to cytochrome P-450 may be the rate-limiting step. A diverse character of the kinetic parameters for the two cytochrome P-450 reduction reactions is indicative of different nature of biphasity of these processes.

Distinct activation of lipid peroxidation, reduction in N-demethylation of dimethyl aniline and in NADPH-dependent electron transport chain were observed in liver microsomes of rabbits poisoned with phenyl hydrazine. Methylcobalamine and adenosyl-cobalamine, two coenzyme forms of vitamin B12, were firstly shown to serve as modulators of the monooxygenase system, whereas methyl-cobalamine proved to be inductor and adenosylcobalamine-repressor of the system. Administration of methylcobalamine into the poisoned rabbits stimulated much higher the activities of dimethyl aniline N-demethylase, aniline p-hydroxylase, NADPH-cytochrome P-450- and NODH-cytochrome b5 reductases as compared with normal state, while adenosylcobalamine inhibited the reduction of all the monooxygenase system patterns studied. At the same time, both these coenzymes contributed to normalization of lipid peroxidation in liver microsomes of poisoned rabbits.

Interaction of muscle glycogen phosphorylase b (EC 2.4.1.1) with glycogen was studied by sedimentation, stopped-flow and temperature-jump methods. The equilibrium enzyme concentration was determined by sedimentation in an analytical ultracentrifuge equipped with absorption optics and a photoelectric scanning system. The maximum adsorption capacity of pig liver glycogen is 3.64 μmol dimeric glycogen phosphorylase b per g glycogen, which corresponds to 20 dimeric enzyme molecules per average glycogen molecule of M r 5.5 · 10 6 . Microscopic dissociation constants were determined for the enzyme-glycogen complex within the temperature range from 12.7 to 30.0°C. Enzyme-glycogen complexing is accompanied by increasing light scattering and its increment depends linearly on the concentration of the binding sites on a glycogen particle that are occupied by the enzyme. Complex formation and relaxation kinetics are in accordance with the proposed bimolecular reaction scheme. The monomolecular dissociation rate constant of the complex increases as the temperature increases from 12.7 to 30.0°C, whereas the bimolecular rate constant changes sligtly and is about 10 8 M −1 · s −1 . These data point to the possibility of diffusional control of the complex formation.

Kinetics of glycogen binding by glycogen phosphorylase b has been studied by stopped flow and temperature jump methods. This reaction is followed by increase in light scattering whose amplitude depends upon the enzyme binding sites concentration of glycogen particles occupied by the enzyme. It has been shown that the complex formation has the first order with respect to enzyme and glycogen concentrations. Relaxation kinetics is compatible with proposed bimolecular reaction scheme. Microscopic rate constants of the forward and reverse reactions of glycogen binding by glycogen phosphorylase b are determined in temperature range from 12,7 to 30 degrees C. The possibility of diffusional control of the binding rate is discussed.