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N B Livanova

Publications and source records attributed to N B Livanova.

At least 37 records · Page 2Linked to original sources
PubMed1994

[Retarded conformational transitions in muscle glycogen phosphorylase b induced by specific ligands].

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

Adenosine Monophosphate↗
PubMed1993

Expression, purification, characterization, and deletion mutations of phosphorylase kinase gamma subunit: identification of an inhibitory domain in the gamma subunit.

A catalytic fragment, gamma 1-298, derived from limited chymotryptic digestion of phosphorylase b kinase (Harris, W.R. et al., J. Biol. Chem., 265: 11740-11745, 1990), is reported to have about six-fold greater specific activity than does the gamma subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the gamma subunit, full-length wild-type and seven truncated forms of gamma were expressed in E. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar (gamma 1-353 and gamma 1-341) or less (gamma 1-331 and gamma 1-276) specific activity than does the full-length wild-type gamma, gamma 1-386. Three truncated forms, gamma 1-316, gamma 1-300, and gamma 1-290 have molar specific activities approximately twice as great as those of the full-length wild-type gamma and the nonactivated holoenzyme. All recombinant gamma s exhibit similar Km values for both substrates, i.e., about 18 microM for phosphorylase b and about 75 microM for MgATP. Three truncated gamma s, gamma 1-316, gamma 1-300, and gamma 1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (Vmax/Km) than that of the full-length wild-type gamma and a 3.5- to 4.5-fold greater efficiency than that of the truncated gamma 1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of gamma, which is located at gamma 301-331. gamma 1-290, but not gamma 1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the gamma subunit to exhibit phosphotransferase activity. Both gamma 1-290 and gamma 1-300 have several properties similar to full-length wild-type gamma, including metal ion responses (activation by free Mg2+ and inhibition by free Mn2+), pH dependency, and substrate specificities.

Amino Acid Sequence↗
PubMed1993

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.

Animals↗
PubMed1993

[Structure and regulation of the activity of muscle phosphorylase kinase].

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.

Animals↗
PubMed1989

[Molecular mechanisms of the regulation of phosphorylase kinase from skeletal muscles of mammals and birds].

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.

Animals↗
PubMed1987

[Limited proteolysis and phosphorylation of phosphorylase kinase from chicken skeletal muscles].

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.

Animals↗
PubMed1986

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'.

Adenosine Diphosphate↗
PubMed1986

Interaction of actin with the enzymes of carbohydrate metabolism.

As was shown earlier, actin is present not only in muscles, but in other animal tissues as well. Now it is firmly established that actin is built into cytoskeleton structures and is a necessary component of all kinds of eukaryotic cells. As is known, the main functions of the cytoskeleton are maintenance of the cell form and participation in the motile processes. At the same time a lot of information is available which indicates the regulatory role of actin in carbohydrate metabolism. The interaction of F-actin with most of the muscle glycolytic enzymes which are located in the region of actin filaments was shown in vitro. In particular, it was established that F-actin forms a complex with glyceraldehyde phosphate dehydrogenase and increases its activity. F-actin interacts with lactate dehydrogenase, forming a complex in which one molecule of the enzyme binds four actin monomers. The formation of the complex results in inhibition of lactate dehydrogenase activity. And, finally, F-actin has a strong activating effect on cellulase which belongs to the other enzyme system. Now the information is available on the induction of cellulase gene expression accompanied by an increase in actin synthesis. These results prove the opinion that actin is a coordinator of carbohydrate metabolism in the cell.

Actins↗
PubMed1986

[Interaction of muscle glycogen phosphorylase B with F-actin].

The binding of rabbit muscle glycogen phosphorylase b to F-actin has been studied by sedimentation in analytical centrifuge in 10 mM Tris-acetate buffer pH 6.8 at 20 degrees C. The adsorption capacity of F-actin is equal to (7.8 +/- 0.9) X 10(-7) mole of glycogen phosphorylase b per 1 g of F-actin; the microscopic dissociation constant for the glycogen phosphorylase-F-actin complex is (5.4 +/- 0.5) X 10(-7) M. It was found that the allosteric activator, AMP, facilitates the adsorption of glycogen phosphorylase b on F-actin, whereas the substrate, Pi, and the inhibitor, ATP, cause an opposite effect.

Actins↗
PubMed1985

[Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle].

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.

Animals↗
PubMed1985

[Regulatory properties of phosphorylase from chicken skeletal muscle].

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.

Adenosine Diphosphate↗
PubMed1984

[Activation of phosphorylase kinase from rabbit muscle by actin and calmodulin].

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.

Actins↗
PubMed1983

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.

Actins↗
PubMed1982

Some comparative aspects of regulation of muscle and liver phosphorylase kinase.

The interaction of muscle and liver phosphorylase kinase with some proteins has been studied. It was shown that muscle G-actin has a visible stimulating effect on the dephosphorylated form of muscle phosphorylase kinase. The effect of F-actin on this enzyme is very low. The interaction of phosphorylase kinase with G-actin probably is one of the additional links between glycogenolysis and muscle contraction. To answer the question what subunit(s) of phosphorylase kinase is involved in the interaction with G-actin we studied the influence of actin on the kinase preparations previously activated to a different degree by partial proteolysis with endogenous protease(s) or with trypsin. G-actin has almost no stimulating effect on the preparations of phosphorylase kinase deeply activated by proteolysis (pH 6.8-8.2 activity ratio more than 0.2). The experiments with partial proteolysis allow us to suppose that alpha-subunit is involved in the interaction of phosphorylase kinase with G-actin. Skeletal muscle G-actin activates purified preparations of liver phosphorylase kinase but to a lower degree than muscle enzyme. Brain and liver calmodulin has a low activating effect on liver phosphorylase kinase in the presence of calcium. Calcium-independent action of calmodulin on the preparations of liver phosphorylase kinase is stronger; probably it is connected with a nonspecific effect of this small acidic protein on the liver enzyme. The basic protein protamine has a strong inhibitory effect on liver phosphorylase kinase.

Actins↗