| Abstract: | The C-5 of 5-aminolaevulinate, a tetrapyrrole precursor which accumulates when inhibitory laevulinate is present, is derived from either the C-2 of glycine by the 5-aminolaevulinate-synthase-mediated Shemin pathway or the C-1 of 2-oxoglutarate by the C5 pathway. Thin-layer-radiochromatographic procedures are described for determining whether 2-14C]glycine or 2-1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a, in addition to or rather than the methyl ester or phytyl ester moieties of the side-chains. The method was also used for detecting whether the same substrates label the formaldehyde (C-5) or the succinate (C-1 to C-4) fragments, obtained by periodate cleavage of 5-aminolaevulinate. These methods therefore can readily distinguish between the Shemin and C5 pathways as was demonstrated by using Rhodopseudomonas spheroides and Zea mays (maize), respectively, as examples of each pathway. Both 2-14C]glycine and, to a lesser extent 2-1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a formed during adaptation of respiring R. spheroides cells to photosynthetic (anaerobic, illuminated) conditions. This and earlier evidence suggested augmentation of the Shemin pathway by a minor C5 pathway contribution. The present studies revealed only Shemin pathway activity: with laevulinate present, 2-14C]glycine formed 5-5-14C]aminolaevulinate as proved by H14CHO production during periodate cleavage. These methods were sufficiently sensitive also to detect the incorporation of 14CO2, from degradation of either substrate, into 5-aminolaevulinate via the Shemin pathway thus labelling the succinate fragment produced with periodate: this explains bacteriochlorophyll a labelling by 2-1-14C]oxoglutarate and proves double labelling of 5-aminolaevulinate by 2-14C]glycine. The same techniques were applied to etiolated maize leaves exposed to aerobic illuminated conditions with laevulinate and either 2-1-14C]oxoglutarate or 2-14C]glycine as substrates. Only the C5 pathway was detected: 2-1-14C]oxoglutarate was converted to 5-5-14C]aminolaevulinate, which yielded H14CHO on periodate cleavage. This is not inconsistent with our earlier 13C-NMR studies Porra, R.J., Klein, O. and Wright, P. E. (1983) Eur. J. Biochem. 130, 509-516] showing that the C5 pathway formed all the 5-aminolaevulinate for chlorophyll biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS) |
