RESUMO
Mg-protoporphyrin IX monomethyl ester cyclase activity was assayed in isolated developing cucumber (Cucumis sativus L. var. Beit Alpha) chloroplasts [Chereskin, Wong & Castelfranco (1982) Plant Physiol. 70, 987-993]. The presence of both 6- and 7-methyl esterase activities was detected, which permitted the use of diester porphyrins in a substrate-specificity study. It was found that: (1) the 6-methyl acrylate derivative of Mg-protoporphyrin monomethyl ester was inactive as a substrate for cyclization; (2) only one of the two enantiomers of 6-beta-hydroxy-Mg-protoporphyrin dimethyl ester had detectable activity as a substrate for the cyclase; (3) the 2-vinyl-4-ethyl-6-beta-oxopropionate derivatives of Mg-protoporphyrin mono- or di-methyl ester were approx. 4 times more active as substrates for cyclization than the corresponding divinyl forms; (4) at the level of Mg-protoporphyrin there was no difference in cyclase activity between the 4-vinyl and 4-ethyl substrates; (5) reduction of the side chain of Mg-protoporphyrin in the 2-position from a vinyl to an ethyl resulted in a partial loss of cyclase activity. This work suggests that the original scheme for cyclization proposed by Granick [(1950) Harvey Lect. 44, 220-245] should now be modified by the omission of the 6-methyl acrylate derivative of Mg-protoporphyrin monomethyl ester and the introduction of stereo-specificity at the level of the hydroxylated intermediate.
Assuntos
Oxigenases/metabolismo , Clorofila/metabolismo , Cloroplastos , Cromatografia Líquida de Alta Pressão , Deuteroporfirinas/metabolismo , Plantas , Porfirinas/metabolismo , Protoclorifilida/análogos & derivados , Protoclorifilida/metabolismo , S-Adenosilmetionina/metabolismo , Espectrofotometria , Especificidade por SubstratoRESUMO
A method was developed to separate the monovinyl and divinyl forms of protochlorophyllide and chlorophyllide by high pressure liquid chromatography using a silicic acid column coated with dodecyl residues and a moving phase containing the lipophilic cation, tetrabutyl ammonium. The solvent was 70% methyl alcohol containing varying amounts of methyl ethyl ketone. The separation was carried out at 0 degrees C. This method was used to test and confirm a previous report that, in cucumber cotyledons, divinyl protochlorophyllide is phototransformed to give divinyl chlorophyllide, which is biologically unstable and disappears rapidly in the dark.
RESUMO
High pressure liquid chromatography was used to demonstrate that chelation of Mg(2+) into protoporphyrin IX precedes methylation in isolated greening etioplasts from cucumber (Cucumis sativus L. var. Beit Alpha) cotyledons. Mg-protoporphyrin IX synthesized in vitro from protoporphyrin IX, Mg(2+), and ATP or exogenous Mg-protoporphyrin IX could serve as substrates for the methylation step. In either case, S-adenosylmethionine was the methyl donor and could not be replaced by ATP plus methionine.