[Delayed activation of muscle glycogen phosphorylase b on incubation of the enzyme with adenosine-5'-monophosphate]. / Medlennaia aktivatsiia myshechnoi glikogenfosforilazy b pri inkubatsii fermenta s adenozin-5'-monofosfatom.

When studying the enzyme activity of glycogen phosphorylase b from rabbit skeletal muscles by the turbidimetric method or by the method based on the determination of inorganic phosphate (the product of the enzymatic reaction) in the direction of glycogen synthesis we observed that 10-15 min preincubation of the enzyme with the allosteric activator (AMP) results in an increase in the initial rate of the enzymatic reaction (nu) in relation to the corresponding value of nu measured by the initiation of the enzymatic reaction by the addition of the mixture of glucose 1-phosphate and AMP (0.02 M Hepes, pH 6.8; 37 degrees C). Glycogen with molecular mass of (264-276).10(6) dalton was used in the kinetic experiments. For 1 mM AMP the time-dependent process of phosphorylase b activation under the action of AMP follows the exponential law with the apparent rate constant of the first order equal to 0.43 min-1 (turbidimetric method of measurement of enzyme activity). When AMP concentration increases, the degree of activation of phosphorylase b reaches a limiting value equal to 1.75 (6 mM glucose 1-phosphate, 0.2 mg/ml glycogen). The activation effect decreases with increasing glycogen concentration and disappears at saturating concentrations of high-molecular weight substrate. Incubation of phosphorylase b with AMP causes the lowering of Michaelis constant for glucose 1-phosphate. It is assumed that enhancement of the rate of the enzymatic reaction catalyzed by phosphorylase b during incubation with AMP is due to association of the enzyme molecules adsorbed to a glycogen particle resulting in an increase in the affinity of the enzyme for glycogen.

[Retarded conformational transitions in muscle glycogen phosphorylase b induced by specific ligands]. / Medlennye konformatsionnye perekhody v myshechnoi glikogenfosforilaze b, indutsiruemye spetsificheskimi ligandami.

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.

[Formation of adducts of pyridine nucleotides in the active center of swine lactate dehydrogenase (isoenzyme M4)]. / Obrazovanie adduktov piridinovykh nukleotidov v aktivnom tsentre laktatdegidrogenazy svin'i (izoferment M4)

Isozyme M4 of pig lactate dehydrogenase (LDH-M4) catalyzes reaction of NAD-adduct formation with a nucleophylic agent that is perhaps OH--ion. The T 1/2 of the reaction is 10-30 sec at concentration NAD 2,0-10(-3) M, LDH-M4 50 gamma/ml at pH greater than 8. Initial velocity and limit of the reaction increase at high LDH-M4, NAD and OH--ion concentrations. Pyridine-3-aldehyde and 3-acetyl pyridine analogs of NAD forms fluorescent adducts too, but at OH--ion concentration approximately 0,01 of that in the case of NAD reaction. Isoelectrical point of LDH-M4 determined by isoelectrofocusing method is 8,65 +/- 0,04 pH unit.