[The allosteric nature of substrate inhibition of rabbit skeletal muscle fructose-1,6-diphosphatase]. / Allostericheskaia priroda substratnogo ingibirovaniia fruktozo-1,6-bisfosfatazy iz skeletnykh myshts krolika.

The mechanism of substrate inhibition of rabbit skeletal muscle fructose-1.6-bisphosphatase was examined. Analysis of substrate saturation curves obtained at different concentrations of Mg2+ revealed that the inhibiting effect of the substrate is manifested only within the complex with Mg2+, whereas the free form of the substrate causes no inhibition. Evidence for the allosteric nature of substrate inhibition was obtained by partial desensitization of the enzyme in the presence of salicylates. It was shown that fructose-1.6-bisphosphatase inhibition by the substrate obeys positively cooperative kinetics and is noncompetitive with respect to the substrate involved in the catalytic process. Studies on enzyme modification in the presence of DTNB and pyridoxal-5'-phosphate demonstrated that the inhibiting concentrations of the substrate are bound to the center which differs from the allosteric site for AMP. It is suggested that the antagonism of simultaneous action of AMP and high substrate concentrations may be due to the competition of the phosphate groups of these ligands for binding to the common lysine residue located in the overlapping region of two allosteric sites.

[The mechanism of rat liver fructose-1,6-bisphosphate inhibition by fructose-2,6-bisphosphate]. / O mekhanizme ingibirovaniia fruktozo-1,6-bisfosfatazy iz pecheni krysy fruktozo-2,6-bisfosfatom.

It was found that a decrease in the activating cation (Mg2+) concentration below [A]0.5 causes the disappearance of cooperativity of the fructose 1.6-bisphosphatase substrate binding sites induced by high fructose 2.6-bisphosphate concentrations without any significant alteration in the extent of the enzyme inhibition. Under these conditions, a competitive type of inhibition (with respect to the substrate) is transformed into a non-competitive type with an increase in the fructose 2.6-bisphosphate concentration. The data obtained confirm the viewpoint that fructose 2.6-bisphosphate binds to the enzyme at two distinct sites, a catalytic and an allosteric ones, differing in their affinity for the inhibitor. It is supposed that the interaction between the allosteric fructose 2.6-bisphosphate binding site and the activator site occupied by Mg2+ is necessary for the cooperative response of the enzyme to the substrate.

[Effect of exogenous ribose-5-phosphate on the ATP level in a cardiomyocyte culture during "metabolic ischemia" and anoxia]. / Vliianie ékzogennogo ribozo-5-fosfata na uroven' ATF v kul'ture kardiomiotsitov v usloviiakh "metabolicheskoi ishemii" i anoksii.

Addition of ribose-5-phosphate into the incubation medium of primary culture of cardiomyocytes was shown to prevent a decrease in the ATP content under conditions of "metabolic ischemia" and anoxia. Possible mechanisms of ribose-5-phosphate participation in maintaining of the energetic status of myocardial cell in pathological conditions are discussed.

[Relation between Mg2+ activation and AMP inhibition of fructose-1,6-diphosphatase from rabbit skeletal muscles]. / Vzaimosviaz' mezhdu aktivatsiei Mg2+ i ingibirovaniem AMP fruktozo-1,6-bifosfatazy iz skeletnykh myshts krolika.

It was shown that AMP, an allosteric inhibitor of fructose-1.6-bisphosphatase, decreases the apparent affinity of the enzyme for the activating cation, Mg2+, which is accompanied by a decrease of the kinetic cooperativity between the Mg2+-binding sites. In its turn, the Mg2+ increase diminishes the enzyme sensitivity to the inhibiting effect of AMP and decreases the cooperativity of the inhibitor binding. The heterotropic interactions between the allosteric inhibitor and activator binding centers are consistent with the predictions of the Monod-Wyman-Changeux model which involves two conformational states of the enzyme (of which one is catalytically inactive) differing in their affinity for the ligands. An increase in pH from 7.4 to 9.0 increases the enzyme affinity for Mg2+ and causes an equilibrium shift towards the catalytically active state of the enzyme.

[Desensitization with acetylsalicylate and salicylate of fructose-1,6-diphosphatase from the rabbit liver and skeletal muscles to allosteric inhibition of AMP]. / Desensibilizatsiia atsetilsalitsilatom i salitsilatom fruktozo-1,6-bifosfatazy iz pecheni i skeletnykh myshts krolika k allostericheskomu ingibirovaniiu AMF.

Acetylsalicylate and salicylate partially desensitized fructose 1,6-biphosphatase from rabbit liver and skeletal muscle to allosteric AMP inhibition. The desensitization was accompanied by a decrease in cooperativity between allosteric sites especially distinct for the liver isoenzyme. The effect of salicylate on both isoenzymes was more pronounced than that of acetylsalicylate. No essential differences in the effect of the compounds studied on these isoenzymes were found.

Purification of commercial preparations of NADP+ from AMP contamination.

A method for purification of commercial preparations of NADP+ from AMP contamination is described. The purification procedure includes one-step anion-exchange chromatography on Dowex-1 (formate) and results in a highly purified salt-free coenzyme with a yield of 70-80%. The chromatography conditions have been selected allowing for complete separation of AMP from NADP+ in a HCOOH concentration gradient. This is followed by NADP+ elution with 1.5 M HCOOH containing HCOOK at a concentration at which the salt remains in solution during subsequent precipitation and washing of NADP+ with acetone. An addition of HCOOK is necessary to reduce the coenzyme elution volume that is important for further precipitation of NADP+ with acetone.

[Purification of commercial preparations of NADP from AMP]. / Ochistka kommercheskikh preparatov NADF ot AMF.

A method in described for elimination of AMP from commercially available preparations of NADP. The method enables to obtain desalted NADP preparations of 98-99.5% purity with a yield of 70-80% using only one chromatographic step. The procedure included anion exchange chromatography on Dowex 1, HCOO- -form. This chromatographic step completely separated AMP from NADP in a gradient of HCOOH concentrations from 0 to 2 N, and NADP was eluted by 1.5 N HCOOH containing 0.15 N HCOOK; during subsequent precipitation and washing of NADP by means of acetone HCOOK remained in a solution. Addition of HCOOK was required for a decrease in NADP elution volume which was important for the subsequent precipitation with acetone.