Irreversible inhibition of lysyl oxidase by homocysteine thiolactone and its selenium and oxygen analogues. Implications for homocystinuria.

Homocysteine thiolactone, selenohomocysteine lactone, and homoserine lactone were found to be competitive, irreversible inhibitors of lysyl oxidase, with KI values of 21 +/- 3 microM, 8.3 +/- 2.2 microM, and 420 +/- 56 microM, respectively. The first order rate constants for inactivation (k2) of the enzyme varied over a much smaller range, ranging from 0.12 to 0.18 to 0.28 min-1 for the Se-, thio-, and O-lactones, respectively. Mutually exclusive labeling of the enzyme by [1-14C]beta-aminopropionitrile, [U-14C]phenylhydrazine, or [35S]homocysteine thiolactone was observed. These labeling results, together with the closely similar perturbations of the near UV-visible spectra of lysyl oxidase and of a model of its lysine tyrosylquinone cofactor by the thiolactone, indicate that the lactones likely derivatize and reduce the active site carbonyl cofactor. Substitution with deuterium at the alpha-carbon of the thiolactone caused a deuterium kinetic isotope effect on k2 of 3.2 +/- 0.2, consistent with the involvement of rate-limiting alpha-proton abstraction during lactone-induced inactivation of the enzyme. The activities of plasma amine oxidase and diamine oxidase were only minimally reduced at concentrations of the sulfur or selenium lactones that fully inhibited lysyl oxidase. Thus, these lactones constitute a new category of mechanism-based inactivators selective for lysyl oxidase. Further, these results may relate to the development of connective tissue defects seen in homocystinuria.

Mitochondrial malate dehydrogenase and malic enzyme of a filarial worm Setaria digitata: some properties and effects of drugs and herbal extracts.

Mitochondrial malate dehydrogenase (mMDH) and malic enzyme (mME) of a filarial worm Setaria digitata were studied. mMDH exhibited the highest activities in the oxidation and reduction reactions at pH 9.5 and pH 6.2, respectively, while mME did so in the malate decarboxylation reaction at pH 6.8. mME showed no detectable activity on the pyruvate carboxylation direction. The Km values for malate (1.7 mM) and oxaloacetate (0.17 mM) and the ratio of Vmax oxidation: Vmax reduction (2.73) tend to favor the oxaloacetate reduction by mMDH. mME showed a relatively high Km value of 8.3 mM, for malate decarboxylation. A drug, diethylcarbamazine citrate (DEC-C), did not change appreciably the activity of either mMDH or mME, while filarin (a drug of herbal origin) effectively inhibited mMDH. The leaf extracts of Ocimum sanctum, Lawsonia inermis and Calotropis gigantea and leaf and flower extracts of Azadirachta indica were, however, found to inhibit both mMDH and mME.

Lysolecithin--a potent activator of prophenoloxidase from the hemolymph of the lobster, Homarus americanas.

The phenoloxidase system, which is involved in encapsulation and melanization of foreign objects in crustacean, is found to be present in an inactive proenzyme form in the hemocytes of the lobster, Homarus americanas. Activation of the enzyme could be achieved either by treatment with an anionic detergent such as sodium dodecyl sulfate, or by a cationic detergent such as cetylpyridinium chloride, but not by either nonionic detergent or zwitterionic detergent. In addition, a number of fatty acids also activated the proenzyme. However, phospholipids, especially lysolecithin proved to be the most potent activator of prophenoloxidase. Therefore, it is proposed that apart from the well established proteolytic mode of activation, prophenoloxidase can also be activated by this alternative mode involving lipids.

Some properties of beta-D-galactosidase from the adult filarial nematode Setaria digitata.

beta-D-galactosidase (beta-D-galactoside galactohydrolase, E.C. 3.2.1.23) activity was localised in the digestive tract of Setaria digitata. The enzyme extract shows maximum activity in the pH range between 3.5 and 5.0 and at 45 degrees C. The enzyme shows the Km value of 3.636 mM for the substrate 6-bromo-2-naphthyl beta-D-galactoside and Vmax of 28.57 nmol 6-bromo-2-naphthol liberated mg-1 protein min-1. Activation/inhibition of the enzyme by various ions, medicinal plants and drugs has been studied. Polyacrylamide gel electrophoresis revealed that the enzyme exists as single form. The medicinal plants and the drug filarin effectively inhibit the enzyme. The significance of these results are discussed in relation to chemotherapy.

Lactate dehydrogenase from adult Setaria digitata (Nematoda: Onchocercidae).

Lactate dehydrogenase of Setaria digitata exhibited an optimum pH of 7.0-8.0 and showed resistance to high temperature. The inhibition/activation of various anions differed in both the forward and backward directions. Filarin (a drug used in Siddha medicine) and diethylcarbamazine (DEC) inhibited pyruvate reduction rather than lactate oxidation. High pyruvate reduction:lactate oxidation at Vmax and Vmax/Km favoured pyruvate reduction in vivo. The enzyme exists as isozymes (four in the female and three in the male) and their separation depended on the percentage of gel and on pH. The mobility of the 700 X g supernatant fraction in the gel was less than that of the 10,000 X g supernatant.