Chronic hepatitis C: T-helper1/T-helper2 imbalance could cause virus persistence in peripheral blood.

Interferon gamma (IFN gamma) and interleukin 4, 10 and 12 (IL-4, -10, -12) production was measured in whole peripheral blood (WPB) and peripheral blood mononuclear cells (PBMC) of 10 chronic hepatitis C (CHC) patients. The level of IFN gamma in supernatants in mitogen-activated WPB was lower than in healthy donors. IL-10 served as a possible downregulative factor for IFN gamma, since its spontaneous IL-10 production was enhanced in CHC. Neutralization of IL-10 partly restored IFN gamma response in CHC patients. Recombinant IL-12 (rIL-12) also enhanced IFN gamma of CHC patients, but IL-12 production was decreased in CHC. Thus, IFN gamma production deficiency in CHC patients is secondary to blockage by high levels of IL-10-impaired IL-12 production.

[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'.

[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.

[Comparative study of the circular dichroism of rabbit liver and muscle glycogen phosphorylases a and b]. / Sravnitel'noe issledovanie krugovogo dikhroizma glikogenfosforilaz a i b iz myshts i pecheni krolika.

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

[Purification and kinetic properties of glycogen phosphorylase A from rabbit liver]. / Ochistka i kineticheskie svoistva glikogenfosforilazy a iz pecheni krolika.

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

[Isolation and properties of glycogen phosphorylase b from rabbit liver]. / Vydelenie glikogenfosforilazy b iz pecheni krolika i issledovanie nekotorykh svoistv fermenta

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