Synthesis and antithrombin activity of phosphoalanine-containing peptides

Boc/Tos-L-Phe-L-Arg-Xaa tripeptides (where Xaa = L-Ala-OBut, L-Ala, or DL-AlaP (OC2H5)2) were synthesized by conventional methods of peptide synthesis in solution. Special features of their interaction with thrombin and trypsin were studied. Unlike trypsin, thrombin did not catalyze the hydrolysis of the L-Arg-L-AlaP-(OC2H5)2 bond. The Tos-L-Phe-L-Arg-DL-AlaP(OC2H5)2 peptide was the most active inhibitor of thrombin among all the compounds studied. The relationship between the structure and inhibitory action of the synthesized peptides is discussed. A part of this study was reported in the Augustusburg Conference of Advanced Science: Nucleic Acids--Targets and Tools, September 17-19, 2000, Germany.     

Inhibiting properties of stable nitroxyl radicals in reactions of linoleic acid and linoleyl alcohol oxidation catalyzed by 5-lipoxygenase

The inhibiting effects of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and its 4-substituted derivatives in reactions of linoleyl acid or linoleyl alcohol oxidation catalyzed by potato tuber 5-lipoxygenase were investigated. Inhibiting properties of stable nitroxyl radicals in presence of lubrol and SDS were reduced at the transition from TEMPO to 4-hydroxy-TEMPO or 4-amino-TEMPO and increased at use of adamantane-1-carboxylic or 3-methyladamantane-1-carboxylic acid 1-oxyl-2,2,6,6-tetramethylpiperidine-4-yl esters. Enzyme activity at saturating concentrations of inhibitor was not suppressed completely, and decreased up to the certain level determined by the substrate nature. The dependence of partial inhibition efficiency on rotational correlation time of stable nitroxides in model micellar systems were analysed. It was supposed that 5-lipoxygenase inhibition includes the interaction of hydrophobic nitroxide with radical intermediate formed in enzymatic process.     

Activity of lipoxygenase incorporated into reversed micelles of aerosol OT in octane

Soybean lipoxygenase (EC 1.13.11.12) incorporated into the reversed micelles of aerosol OT in octane has been studied for its catalytic properties. The enzyme is shown to preserve up to 10% activity as compared with the activity in the aqueous solution. In this case Km of lipoxygenase for linoleic acid increases from 10(-5) M to 5 X 10(-4) M. The activity of lipoxygenase is maximal, the aerosol OT concentration being 0.03 M and a degree of reversed micelle hydratation 40. Cationic detergents of the cetyltrimethyl ammonium bromide type are not good to form reversed micelles of lipoxygenase, since they inhibit the latter with IC50 = (4 divided by 6) x 10(-4) M. The lipoxygenase preparations in reversed micelles of aerosol OT in octane may be used to synthesize natural metabolites of polyunsaturated fatty acids, for instance of eicosanoids.     

Reverse phase high performance liquid chromatography in the synthesis of leukotrienes A4 and C4

The chromatographic (RP HPLC) behaviour of leukotriene C4, its methyl ester, leukotriene A4 methyl ester and some chemicals involved in their synthesis have been investigated. Optimal conditions of separation were determined for the gradient and isocratic HPLC. Parameters of the interaction of the substances with hydrophobic surface are discussed in terms of solvophobic theory.     

Synthesis and Antithrombin Activity of Phosphoalanine-Containing Peptides

Boc/Tos-L-Phe-L-Arg-Xaa tripeptides (where Xaa = L-Ala-OBu ^t , L-Ala, or DL-Ala ^P (OC₂H₅)₂) were synthesized by conventional methods of peptide synthesis in solution. Special features of their interaction with thrombin and trypsin were studied. Unlike trypsin, thrombin did not catalyze the hydrolysis of the L-Arg–L-Ala ^P (OC₂H₅)₂ bond. The Tos-L-Phe-L-Arg-DL-Ala ^P (OC₂H₅)₂ peptide was the most active inhibitor of thrombin among all the compounds studied. The relationship between the structure and inhibitory action of the synthesized peptides is discussed.²     

Inhibition of alkaline phosphatase by thioureido derivatives of methylenebisphosphonic acid

A series of thioureido derivatives of methylenebisphosphonic acid were synthesized by the reaction of aminomethylenebisphosphonic acid with the corresponding isothiocyanates, and their effect on the activity of alkaline phosphatases from bovine small intestine mucosa (BSIM) and human placenta was studied. It was found that (3-phenylthioureido)methylenebisphosphonate is approximately one order of magnitude more effective in inhibiting the activity of alkaline phosphatase from BSIM than the alkyl derivatives of thioureidomethylenebisphosphonic acid with methyl, ethyl, tert-butyl, or cyclohexyl substituents. The introduction of substituents into the benzene ring of (3-phenylthioureido)methylenebisphosphonate decreased the effect of the inhibitor on the activity of the enzyme. The affinity of (3-phenylureido)methylenebisphosphonate to the alkaline phosphatase of BSIM was also weaker as compared with the corresponding thioureidomethylenebisphosphonate. The insertion of thioureidobisphosphonates into the active site of alkaline phosphatase of human placenta by the method of molecular docking indicated that the methylenebisphosphonate residue and the substituted amino groups of the inhibitor are involved in the mechanisms of complex formation with the enzyme. It is supposed that the improvement of the inhibitory activity of (3-phenylthioureido)methylenebisphosphonate toward alkaline phosphatase of BSIM is due to the additional fixation of the phenyl substituent in the active site of the enzyme.     

Hydrophobic nitroxyl radicals inhibit linoleyl alcohol oxidation by 5-lipoxygenase

The linoleyl alcohol oxidation catalyzed by potato tuber 5-lipoxygenase was found to be efficiently inhibited by stable nitroxyl radicals: 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 1-bicyclo[2,2,2]octane-1-carboxylate, 1-adamantylacetate, dodecanoate, and octadecanoate. The dependence of apparent IC50 values on the rotational correlation times of times of 4-hydroxy-1-oxyl-2,2,6,6-tetramethylpiperidine and its derivatives in model micellar systems was analyzed. The inhibition mechanism was proposed; it involves the interaction of hydrophobic nitroxyl radical with the intermediate radical enzyme-substrate complex.     

Inhibition of yeast pyruvate decarboxylase by alkyl phosphates]

It is found that yeast pyruvate decarboxylase is inhibited by alkyl phosphates. Inhibition is competitive with respect to a substrate. The inhibition constants with n-butyl and n-heptyl esters of phosphoric acid are the values of the same order of magnitude. With an increase in the length of the alkyl phosphates hydrocarbon chain from 7 to 10 carbon atoms inhibition constants change drastically. For n-heptyl phosphate and n-decyl phosphate values KI are equal to 1.6 x 10(-4) M and 1.7 x 10(-6) M, respectively. A further increase in the number of carbon atoms in the alkyl substituent of phosphoric acid ester induces no reduction of the inhibition constant. Multiple-inhibitor experiments of pyruvate decarboxylase show that inorganic phosphate and n-decyl ester of phosphoric acid are mutually exclusive. It is suggested that the inhibition mechanism with alkyl phosphates includes the competition of the phosphoric acid residue with alpha-ketocarboxyl group of pyruvate as well as the interaction between a hydrocarbon radical and hydrophobic parts on the enzyme surface, one of them being outside the substrate binding site.     

Potato 5-lipoxygenase. Kinetics of linoleic acid oxidation

The role of main factors influencing the rate of potato 5-lipoxygenase oxidation of linoleic acid was investigated. It was found that nonionic detergent lubrol PX inhibited the potato lipoxygenase. Optimal pH for the linoleic acid oxidation was 6.3 temperature--45 degrees C and substrate concentration--3 x 10(-4) M (if lubrol PX was 0.02%). It was shown that potato 5-lipoxygenase was allosteric enzyme which possessed positive cooperativity for linoleic acid. The Hill coefficient was calculated (n = 1.40 +/- 0.15) with S0.5 = 75 +/- 10 microM.