[Physical-mechanical and functional characteristics of xenograft during various methods of stabilization and treatment]. / Fiziko-mekhanicheskie i funktsional'nye kharakteristiki ksenotkani pri razlichnykh vidakh stabilizatsii i obrabotki.

The paper gives the results of experimental studies of the elastic-strength and functional characteristics of xenotissue samples (the pericardium of the calf, pig and Glisson's capsule) stabilized with glutaric aldehyde and polyepoxy compounds. The paper discusses various treatments of biological materials (including Glisson's capsule) anticalcified with additives (sodium dodecyl sulfate, complexes of copper and zinc) and shows that their use improves proteolytic and calcinotic resistance without deteriorating the mechanical parameters of xenotissues. Comparative analysis of the physical-mechanic and functional characteristics of xenotissues treated in different ways allows one to make anticalcifying treatment of biological tissues with copper and zinc complexes physical-mechanic and functional characteristics of xenotissues (Glisson's capsule) for clinical application.

Oxygen exchange reactions catalyzed by vacuolar H(+)-translocating pyrophosphatase. Evidence for reversible formation of enzyme-bound pyrophosphate.

Vacuolar membrane-derived vesicles isolated from Vigna radiata catalyze oxygen exchange between medium phosphate and water. On the basis of the inhibitor sensitivity and cation requirements of the exchange activity, it is almost exclusively attributable to the vacuolar H(+)-pyrophosphatase (V-PPase). The invariance of the partition coefficient and the results of kinetic modeling indicate that exchange proceeds via a single reaction pathway and results from the reversal of enzyme-bound pyrophosphate synthesis. Comparison of the exchange reactions catalyzed by V-PPase and soluble PPases suggests that the two classes of enzyme mediate P(i)-HOH exchange by the same mechanism and that the intrinsic reversibility of the V-PPase is no greater than that of soluble PPases.

Aminomethylenediphosphonate: A Potent Type-Specific Inhibitor of Both Plant and Phototrophic Bacterial H+-Pyrophosphatases.

The suitability of different pyrophosphate (PPi) analogs as inhibitors of the vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) of tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata was investigated. Five 1,1-diphosphonates and imidodiphosphate were tested for their effects on substrate hydrolysis by the V-PPase at a substrate concentration corresponding to the Km of the enzyme. The order of inhibitory potency (apparent inhibition constants, Kiapp values, [mu]M, in parentheses) of the compounds examined was aminomethylenediphosphonate (1.8) > hydroxymethylenediphosphonate (5.7) [almost equal to] ethane-1-hydroxy-1,1-diphosphonate (6.5) > imidodiphosphate (12) > methylenediphosphonate (68) >> dichloromethylenediphosphonate (>500). The specificity of three of these compounds, aminomethylenediphosphonate, imidodiphosphate, and methylenediphosphonate, was determined by comparing their effects on the V-PPase and vacuolar H+-ATPase from Vigna, plasma membrane H+-ATPase from Beta vulgaris, H+-PPi synthase of chromatophores prepared from Rhodospirillum rubrum, soluble PPase from Saccharomyces cerevisiae, alkaline phosphatase from bovine intestinal mucosa, and nonspecific monophosphoesterase from Vigna at a PPi concentration equivalent to 10 times the Km of the V-PPase. Although all three PPi analogs inhibited the plant V-PPase and bacterial H+-PPi synthase with qualitatively similar kinetics, whether substrate hydrolysis or PPi-dependent H+-translocation was measured, neither the vacuolar H+-ATPase nor plasma membrane H+-ATPase nor any of the non-V-PPase-related PPi hydrolases were markedly inhibited under these conditions. It is concluded that 1, 1-diphosphonates, in general, and aminomethylenediphosphonate, in particular, are potent type-specific inhibitors of the V-PPase and its putative bacterial homolog, the H+-PPi synthase of Rhodospirillum.

[Effect of exogenously administered ATP on heart function and energy status]. / Vliianie ékzogenno vvedennoi ATF na funktsiiu i énergeticheskoe sostoianie serdtsa.

It has been shown that ATP (24.10(-6) M) administered once into the circulating solution improved strength and speed characteristics of aerobically perfused hearts. The increasing demands of the hearts in oxygen and energy substrates were satisfied due to coronary vessels dilatation without harming the energy status of the myocardium. When the functional parameters of the hearts in the test and control groups did not differ, an exogenously administered ATP was included into the energy metabolism and increased considerably ATP and CP tissue levels, the sum of high-energy phosphates and ATP/ADP ratio. Artificial supplementation of tissue energy resources was accompanied by a decrease in energy-dependent end-diastolic pressure and diastolic left ventricular elasticity and by increased extensibility of the heart muscle, which may improve functional parameters.

Steady-state kinetics of substrate hydrolysis by vacuolar H(+)-pyrophosphatase. A simple three-state model.

The results of analyses of the steady-state kinetics of the vacuolar H(+)-translocating pyrophosphatase (V-PPase) of native tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata (mung bean) and purified enzyme from the same source under a wide range of Mg2+, pyrophosphate (PPi) and K+ concentrations are consistent with a minimal reaction scheme in which dimagnesium pyrophosphate is the active substrate species and catalysis is mediated by preformed enzyme-Mg2+ complex. When account is taken of the sensitivity of the V-PPase to ionic strength, additional kinetic interactions are not required to describe the behavior of the enzyme. N-Ethylmaleimide-protection assays show that the dissociation constant for Mg2+ binding in the absence of PPi is an order of magnitude smaller than that estimated from the steady-state kinetics of PPi hydrolysis. Two distinct Mg(2+)-binding sites are therefore invoked. Since the protective action of Mg2+ is independent of the nature of the monovalent cations and Mg2+ and K+ do not compete during substrate hydrolysis, divalent and monovalent cations are concluded to bind at separate sites. The pH dependencies of the kinetic parameters are consistent with the participation of groups of pKa 5.7 and 8.6 in substrate binding and groups of pKa 6.1 and 9.0 in the substrate-conversion step, indicating that at least four ionizable groups are essential for catalysis. These findings are discussed with respect to the reaction mechanism of the V-PPase and the potential regulatory significance of cytosolic free Mg2+ and K+ in vivo.

Differential sensitivity of membrane-associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme.

1,1-Diphosphonate analogs of pyrophosphate, containing an amino or a hydroxyl group on the bridge carbon atom, are potent inhibitors of the H(+)-translocating pyrophosphatases of chromatophores prepared from the bacterium Rhodospirillum rubrum and vacuolar membrane vesicles prepared from the plant Vigna radiata. The inhibition constant for aminomethylenediphosphonate, which binds competitively with respect to substrate, is below 2 microM. Rat liver mitochondrial pyrophosphatase is two orders of magnitude less sensitive to this compound but extremely sensitive to imidodiphosphate. By contrast, fluoride is highly effective only against the mitochondrial pyrophosphatase. It is concluded that the mitochondrial pyrophosphatase and the H(+)-pyrophosphatases of chromatophores and vacuolar membranes belong to two different classes of enzyme.

[The effect of droperidol on heart function]. / Vliianie droperidola na funktsiiu serdtsa.

The effect of droperidol (3.3 x 10(-6) M) on the functional capacities of the isolated heart has been studied. The drug inhibited myocardial contractility. The effect manifested directly after the introduction of droperidol into perfusate (prompt phase). Afterwards the progress of the drug inhibitory effect was slow (slow phase). It is assumed that the difficulties in the use of droperidol in the critical cardiosurgical patients are associated with the attenuation in the realization of the regulatory effects of the central nervous system.

A comparison of the protective effect of a modified StTH solution and HTK-B on the energy and functional status of the isolated rat heart.

Using a model of the isolated beating rat heart, the authors compared the protective effect of St. Thomas Hospital cardioplegic solution enriched with glucose and mannitol (StTH-M) and Bretschneider solution (HTK-B). Results showed that, during 120-minute global ischaemia in cardioplegia, StTH-M was able to maintain levels of high-energy phosphates comparable with those found in a group of hearts perfused with HTK-B at 20 degrees C only when the temperature had been decreased to 12-15 degrees C. Under these conditions, repair of metabolic and functional parameters during post-ischaemic perfusion was also similar in both groups.

[Effect of hypothermia on the metabolism and post-ischemia restoration of myocardial function]. / Vliianie gipotermii na metabolizm i postishemicheskoe vosstanovlenie funktsii miokarda.

Our studies have revealed a prognostically poor course in postinfarction angina pectoris patients on conservative medicamentous treatment. The mortality rate within the first year of this syndrome onset was 19.8 per cent among conservatively treated patients. Surgical management of these patients had a significantly better prognosis. Despite the fact that there was a great difference in survival rates between the studied groups at the hospital stage, the survival rate was significantly higher at subsequent stages in surgical patients compared to that in the conservatively treated group, and correlated with that in a larger population of matched age. In addition, surgical treatment was found to substantially improve the quality of life in patients with postinfarction angina pectoris which implies lowered frequency of attacks, increased exercise tolerance, and improved working capacity.

[Effectiveness of protecting the myocardium against ischemia with a normothermic cardioplegic solution and creatine phosphate]. / Effektivnost' zashchity miokarda ot ishemii normotermicheskim kardioplegicheskim rastvorom s kreatinfosfatom.

The protective effects of cardioplegic solutions (CS) containing creatine phosphate (CP) were studied in a rat heart model of cardiopulmonary bypass and ischemic cardiac arrest. Isolated rat hearts were subjected to a 3-minute coronary infusion with CS containing CP in normothermic (37 degrees C) and hypothermic (4-6 degrees C) regimes. In the normothermia group, the postischemic functional recovery was 70-75% of the preischemic control value, while the cellular ATP and CP content was reduced but insignificantly. By contrast, in the hypothermia group, the postischemic functional recovery was markedly depressed, with the tissue high-energy phosphate content being appreciably lowered. The data obtained confirm high efficacy of CP-containing cardioplegic solutions administered under normothermia conditions.

The flip-flop mechanism of the phosphorylation of yeast inorganic pyrophosphatase.

1. An active monomeric form of inorganic pyrophosphatase from baker's yeast was prepared by maleylation of the protein at pH 10.5. 2. The dimeric and monomeric pyrophosphatase bound at non-catalytic sites 0.5 and 1.0 mol of slowly dissociating Pi per mol subunit, respectively. This stoichiometry was not affected on active site blockage with PPi. 3. Added Pi accelerated the dissociation of Pi from the dimeric but not monomeric enzyme. 4. Our results indicate a strong interaction to occur between the non-catalytic sites of two subunits of native pyrophosphatase which results in diminished stability of Pi binding to one of them.

[Cooperative mechanism of phosphorylation of the monomeric and dimeric forms of inorganic pyrophosphatase from baker's yeast]. / Kooperativnyi mekhanizm fosforilirovaniia monomernoi i dimernoi form neorganicheskoi pirofosfatazy iz pekarskikh drozhzhei.

A comparative study of phosphorylation of native dimeric and artificial monomeric forms of inorganic pyrophosphatase and its fluoride-stabilized complex with PPi has been carried out. The maximal incorporation of Pi for the dimeric and monomeric proteins is 0.5 and 1 mole per mole of subunit, respectively. The saturation kinetic curves are suggestive of strong positive cooperative interactions. The value of the Hill coefficient (5.5) for the free dimeric enzyme drastically changes upon the active center blockage and/or transition to the monomeric enzyme. Acceleration of dephosphorylation induced by Pi in the presence of Mg2+ is observed only in the case of the dimeric protein. The data obtained indicate that phosphorylation of native dimeric pyrophosphatase occurs according to a "flip-flop" mechanism; the Pi binding in the active center exerts a strong influence on individual steps of the reaction.

[Isolation and catalytic properties of the soluble monomeric form of inorganic pyrophosphatase from baker's yeast]. / Poluchenie i kataliticheskie svoistva rastvorimoi monomernoi formy neorganicheskoi pirofosfatazy iz pekarskikh drozhzhei.

Data from sedimentation analysis suggest that modification of about 40% of free amino groups of inorganic pyrophosphatase by maleic anhydride, pH 10.5, results in a loss of the enzyme ability to form dimers at neutral values of pH. The specific activity of monomeric pyrophosphatase is 50-80% of that of the dimeric form. The monomer has a pH optimum of about 7, requires metal ions for activation of both enzyme and substrate and is capable of exergonic synthesis of PPi in the active center. The enzyme binding to PPi is strongly stabilized by fluoride. The experimental data indicate that the individual subunit of inorganic pyrophosphatase possesses all the main catalytic properties of native dimeric molecule.

[Calorimetric assay of yeast inorganic pyrophosphatase interaction with magnesium and phosphate ions]. / Kalorimetrichesko izuchenie vzaimodeistviia neorganicheskoi pirofosfatazy drozhzhei s ionami magniia i fosfatom.

The thermodynamic characteristics for the specific binding of one or two Mg2+ by the yeast inorganic pyrophosphatase and for the enzyme interaction with phosphate were determined. Saturation of the first binding site with Mg2+ causes structural rearrangements in the enzyme molecule without changing the temperature of protein denaturation. On the contrast, saturation of the second binding site results in stabilization of the system, i. e. a considerable fall in the entropy and a rise in the temperature of denaturation. Phosphorylation of the enzyme carboxylic group by inorganic phosphate requires saturation of the first binding site with Mg2+ and is not accompanied by changes in the enthalpy of the system. The pyrophosphate synthesis in the presence of the enzyme saturated with Mg2+ in both binding sites is associated with changes in the enthalpy and, possibly, in the entropy of the system.

[Stable compound of inorganic pyrophosphatase with pyrophosphate obtained by a fluoride-mediated reaction with phosphate]. / Stabil'noe soedinenie neorganicheskoi pirofosfatazy s pirofosfatom, poluchennoe reaktsiei s fosfatom v prisutstvii ftorida.

Incubation of inorganic pyrophosphate from baker's yeast with phosphate and MgCl2 in the presence of fluoride results in a gradual inactivation of the enzyme concomitant with incorporation of PP1 (about 2 moles per mole) into the protein. The rate constant for this process shows an increase with a rise in concentrations of the three reagents, the maximal value of inactivation being 0.11 min-1. The bound PP1 is not separated by gel-filtration. The rate of spontaneous degradation of the enzyme-pyrophosphate complex and the nature of EDTA and Mg2+ effects are similar to those for the analogous compound obtained by inhibition of PP1 hydrolysis by fluoride. The data obtained suggest that during PP1 synthesis and hydrolysis by pyrophosphatase fluoride stabilizes the same intermediate of the enzyme with pyrophosphate.

[Detection and characterization of an additional site for binding of substrate and its analogs by inorganic pyrophosphatase]. / Obnaruzhenie i kharakteristika dopolnitel'nogo tsentra prisoedineniia substrata i ego analogov u neorganicheskoi pirofosfatazy.

Phosphate, pyrophosphate, imidodiphosphate, EDTA and tripolyphosphate increase the rate constant for dissociation of the inorganic pyrophosphatase-substrate intermediate formed after cessation of the reaction by fluoride. The effect is enhanced in the given order 19-fold, the dependence of this effect on ligand concentration being hyperbolic. The values of the dissociation constants of the enzyme-ligand complexes lie within the concentration range of 0.16-1.0 mM. At high concentrations of Na2+ added simultaneously with the ligands this effect is decreased. The value of tau 1/2 for Pi binding to the enzyme-substrate compound is 0.15 min. The data obtained suggest that pyrophosphatase contains an anion ligand binding site, differing from that of the active one. This site does not affect the hydrolytic function of pyrophosphatase, as can be evidenced from the fact that Pi (9.5 mM) does not change the rate of enzymatic cleavage of PPi.

The essential activated carboxyl group of inorganic pyrophosphatase.

1. A carboxyl group of high reactivity has been found in inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from yeast. This group interacts with agents which react neither with carboxyl groups of low molecular weight compounds nor with other carboxyl groups of the protein. 2. The reaction of this activated carboxyl group with inorganic phosphate, hydroxylamine, N-methyl- and O-methylhydroxylamines, and glycine methyl ester has been studied. 3. Homoserine and homoserine lactone were found in the hydrolyzate of phosphorylated and NaBH4-reduced pyrophosphatase, indicating that an aspartyl residue is phosphorylated. 4. Hydroxylamine and other nucleophilic agents cause inactivation of pyrophosphatase as a result of interaction with a carboxyl group. Both diaminobutyric and diaminopropionic acids were seen in the acid hydrolyzate of the protein treated with hydroxylamine and subjected to rearrangement in the presence of carbodiimide. 5. The ways in which the activation of a carboxyl group in the enzyme is achieved and the presumed mechanism of action of inorganic pyrophosphatase are discussed.