[Role of calcium ions in the development of the catalytic activity of phospholipase D]. / Rol' ionov kal'tsiia v proiavlenii kataliticheskoi aktivnosti fosfolipazy D.

The peculiarities of the reaction of mycellar lecithine cleavage by phospholipase D and the role of Ca2+ in the catalytic activity of the enzyme were studied. It was shown that Ca2+ participates in the formation of a catalytically active intermediary enzyme-substrate complex with a strict stoichiometric ratio of substrate, phospholipase activator (Na-DS) and Ca2+. The kinetics of inhibition for this reaction by lantane and fluoride ions and EDTA were studied. The inhibition of the reaction in a lecithine-Na-Ca2+-phospholipase D system by La3+ is due to substitution of bivalent Ca2+ by trivalent La3+ within the intermediary complex. The inhibiting effect of F- is due to penetration of the electron-negative ion into the coordination sphere of the intermediary complex and to disturbances in the hydrophobic binding between the ligands. The inhibitory action of EDTA is revealed during protein binding to the substrate and is not due to the chelating capacity of EDTA with respect to Ca2+. It was demonstrated that the role of Ca2+ is not restricted by its binding to phospholipase C. Ca2+ are also necessary for neutralization of the extra charge on the surface of substrate mycelles, production of an appropriate ionic strength and modification of the substrate phase surface at the interface. The catalytic function of Ca2+ consists in a formation of a "recognition" site in the active center of phospholipase D.

[Separation and properties of molecular forms of phospholipase D from plants]. / Razdelenie i svoistva molekuliarnykh form fosfolipazy D iz rastenii.

The optimal conditions for isolation and purification of phospholipase D (EC 3.1.4.4) from Middle-Asian radish tubers, cotton plant seeds and cabbage leaves using biospecific chromatography on polyamide absorbent with covalently linked phosphatidylethanolamine, were elaborated. The synthetic absorbent can be used for separation of molecular enzyme forms differing in their affinity for immobilized phosphatidylethanolamine (forms I, II, and III). Experimental evidence for the interconvertibility and interrelationship of different forms of plant phospholipase D is given. It is demonstrated that forms I and III separated by biospecific chromatography are represented by enzyme conformers distinguishable by their thermal stabilities (phospholipases D1 and Ds, respectively), while form II is a high molecular weight phospholipase D.

[The dependence of the effects of calcium ions on enzymatic hydrolysis of phospholipids on the physical state of the substrate]. / Vliianie ionov kal'tsiia na fermentativnyi gidroliz fosfolipidov v zavisimosti otfizicheskogo sostoianiia substrata.

The role of calcium ions in the phospholipid hydrolysis by phospholipase D was studied. It was shown that the enzyme does not split egg lecithine in the absence of Ca2+. In the presence of Ca2+ the reaction occurs via different routes, depending on the type of initiation of the reaction. The optimal concentrations of Ca2+ necessary for activation of phospholipase D are different in the systems activated by various treatments (organic solvents, detergents and solid adsorbents). Optimal concentrations of Ca2+ for the hydrolysis and methanolysis catalyzed by phospholipase D are also different. It was found that the need for Ca2+ and their optimal concentrations are determined by the state of phospholipids at the substrate phase. The data suggest that the enzymatic hydrolysis may occur in the absence of Ca2+. Thus, Ca2+-induced activation is merely an alternative pathway of catalytically active conformation of lypolytic enzymes.

[Identification and properties of 2 forms of plant phospholipase D]. / Obnaruzhenie i svoistva dvukh form fosfolipazy D rastenii.

Effect of different factors (pH, temperature etc.) on the activity of phospholipase D is studied. It is found that plant phospholipase D has two forms: thermolabile (D1) and thermostable (Ds). Both forms are shown to differ in some characteristics (pH optimum, temperature optimum, calcium ions activation, activator effect, kinetic parameters, thermostavility). The contradictive literature data on phospholipase D properties are suggested to be due to the existence of two enzyme forms.

[The state of phospholipase D in solution and its catalytic activity]. / Sostoianie fosfolipazy D v rastvore i EE kataliticheskaia aktivnost'

Functioning of water-soluble phospholipase D from cotton seeds is studied on two phases contact area (liquid-liquid, liquid-solid substance) and on the surface of mixed lecitine and sodium dodecylsulphate micelles. It is found that water-soluble phospholipase D, which normally has no catalytic activity, is capable to hydrolyse its substrates in the presence of organic solvents, solid adsorbents and sodium dodecylsulphate. The data obtained show that in all the cases studied the activation observed is due to adsorption immobilization of the enzyme. K lambda and K alpha constants are introduced, which are characteristics of immobilyzing ability of agents-matrices for immobilization. Phase transitions, which take place in heterogenous system (enzyme-activator-substrate-water solution), are found to be a necessary condition for the enzyme activation. A hypothesis, that catalytical activity of water-soluble phospholipase D is inherent of the adsorbed enzyme, is discussed on the basis of the data on comparative study of adsorbed and water-soluble enzymes.

[Immobilized phospholipase D]. / Immobilizovannaia fosfolipaza D

Conditions of phospholipase D adsorption on silica gels have been studied. The immobilized phospholipase D is shown to differ from the soluble form in thermostability, pH optima and activation conditions. A question is discussed as to the connection of the use of activators and the adsorption immobilization. It is assumed that phospholipase D belongs to enzymes, functioning only in the immobilized state.

[Several properties of cotton seed phospholipase D]. / Nekotorye svoistva fosfolipazy D iz semian khlopchatnika

Properties of phospholipase D were studied using purified enzyme preparation from cotton seeds. The results obtained differ from those described in literature. It has been shown that the promoting action is exerted not only by diethyl ether and sodium dodecyl sulfate commonly used as initiators, but by some organic solvents in the presence of calcium ions as well. The activation of phospholipase D is also possible in the presence of other bivalent cations, e.g. Sr2+, Ba2+, Mn2+ and Mg2+. It is assumed that the enzyme activation occurs only in the presence of the stable heterogenous system: water-soluble enzyme--phospholipid--non-aqueous phase. Another important factor is the type of modification of the surface of the phospholipid phase, responsible for the enzyme adsorption and its subsequent activation. Comparison is made of the properties of phospholipases D isolated from cotton seeds and some other sources.

[Isolation of phospholipase D from cotton seeds]. / Vydelenie fosfolipazy D iz semian khlopchatnika

A method of isolating homogenous phospholipase D from cotton seeds is described. The homogeneity of the enzyme preparation is demonstrated by means of polyacrylamide disc electrophoresis, gel filtration through Sephadex G-200 and determination of N-terminal amino acid residue, which turned to be glutamic acid. Molecular weight of the enzyme is found to be 71000 +/- 3000. Several equilibrium forms of the enzyme are observed.

[Activators of phospholipase D]. / Ob aktivatorakh fosfolipazy D

The activation of phospholipase D from cotton seeds by diethyl ester, sodium dodecyl sulphate and silica gel during egg lecithin hydrolysis was investigated. In the presence of silica gel the enzymic reaction developed without calcium ions. The data were accumulated indicating that the main characteristics of phospholipase D obtained from the in vitro study of the enzymic hydrolysis of phospholipids depended at large on the activator applied.