RESUMO
Crithidia fasciculata poly(ADP-ribose)polymerase (PARP) has been isolated and partially purified. This is the first PARP isolated from trypanosomatids; it requires DNA and histone for activity, using NAD(+) as substrate. Thiol compounds specially dithiothreitol essentially contributed to PARP stability during purification and to PARP activity during assays. Nicotinamide, 3-aminobenzamide, theophylline, histamine, histidine, N-ethylmaleimide, p-chloromercuribenzoic acid, p-chloromercuriphenylsulfonic acid and o-iodosobenzoate inhibited PARP, thus confirming enzyme identity. PARP was also inhibited by the Fe(II)/H(2)O(2) Fenton system. beta-Lapachone inhibited PARP, apparently by direct interaction with the enzyme.
Assuntos
Antiprotozoários/farmacologia , Crithidia fasciculata/enzimologia , Naftoquinonas/farmacologia , Poli(ADP-Ribose) Polimerases/isolamento & purificação , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Peróxido de Hidrogênio/farmacologia , Ferro/farmacologia , Inibidores de Poli(ADP-Ribose) PolimerasesRESUMO
Several structurally related ortho-naphthoquinones isolated from Mansonia altissima Chev (mansonones C, E and F) (a) inhibited NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (b) prevented NADPH-dependent cytochrome P450 destruction; (c) inhibited NADPH-supported aniline 4-hydroxylase activity; (d) inhibited Fe(III)ADP reduction by NADPH-supplemented microsomes; (e) stimulated superoxide anion generation by NADPH-supplemented microsomes; and (f) stimulated ascorbate oxidation. ESR investigation of ascorbate-reduced mansonone F demonstrated semiquinone formation. Mansonone C had a greater effect than mansonones E and F on NADPH-dependent lipid peroxidation, O2- production and ascorbate oxidation, whereas mansonone E was more effective than mansonones C and F on aniline 4-hydroxylase activity. Mansonones E and F did not inhibit hydroperoxide-dependent lipid peroxidation, cytochrome P450 destruction or microsomal aniline 4-hydroxylase activity. Mansonone C inhibited to a limited degree tert-butyl hydroperoxide-dependent lipid peroxidation, this inhibition being increased by NADPH. Mansonone A, a tetrahydro orthonapthoquinone derivative, was in all respects relatively less effective than mansonones C, E and F. It is postulated that mansonones C, E and F inhibited microsomal lipid peroxidation and cytochrome P450 catalyzed reactions by diverting reducing equivalents from NADPH to dioxygen, but mansonone C (including its reduced form) may also exert direct antioxidant activity.