Biosynthesis of porphyrins and heme from y , & dioxovalerate by intact hepatocytes


The purpose of this study was to assess the ability of hepatocytes to synthesize porphyrins and heme from 6-aminolevulinic acid derived from ,8-dioxovalerate and alanine. An alternate pathway for 8-aminolevulinic acid synthesis, in contrast to the condensation of succinate and glycine by 8-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC] has been suggested. This has been supported by the isolation of y,8-dioxovalerate transaminase from liver mitochondria (Varticovski, L., Kushner, J. P. & Burnham, B. F. (1980) J. BioL Chem. 255, 3742-3747). y,8-Dioxovalerate transaminase catalyzes the formation of f-aminolevulinic acid by a transamination reaction involving y;8-dioxovalerate and alanine. To assess the significance of this alternate route, we prepared suspensions of respiring rat' hepatocytes, which were incubated with optimal concentrations of various additives and then analyzed spectrophotometrically for synthesized porphyrins. No porphyrin synthesis was detected in cell suspensions incubated with succinate (1 mM) and glycine' (20 mM). Cell suspensions incubated with y,6-dioxovalerate (0 5-1.0 mM) andalanine (20 mM) synthesized 0.19 nmol of porphyrin per 1 cells per 2 hr (SD, 0.057). Cell suspensions incubated with 6-aminolevulinic acid (0.1 mM) synthesized 1.26 nmol of porphyrin per 107 cells per 2 hr (range, 1.18-1.32). Incubations with chemically synthesized y,8dioxo[5-'4C]valerate were followed by extraction and purification of porphyrin esters and heme. Liquid scintillation counting revealed radiolabel incorporation into both porphyrins and heme. These studies demonstrate significant tetrapyrrole synthesis by the 'y,8-dioxovalerate transaminase reaction. That y,8-dioxovalerate is an important precursor of heme in vivo must be considered. 8-Aminolevulinic acid is the first committed precursor of porphyrins and heme' (1). The biosynthesis of 8-aminolevulinic acid in mammalian cells has been thought to occur exclusively through a condensation reaction involving glycine and succinate (2). The enzyme catalyzing this condensation is 8-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), 'EC]. Recently, several lines ofevidence have led to the conclusion that not all 8-aminolevulinic acid is synthesized by the condensation of glycine and succinate. First, although 8-aminolevulinic acid is an obligatory precursor inthe biosynthesis ofchlorophyll, 8-aminolevulinate synthase has not yet been demonstrated in green plants. It now seems established that'plants form 8-aminolevulinic acid directly from the intact skeleton of a 5-carbon precursor (3-6). Secondly, we 'recently reported the isolation and characterization of the enzyme L-alanine:y,&dioxovaleric acid aminotransferase from mammalian liver mitochondria (7, 8). This enzyme catalyzes a transamination reaction between LCOOH

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@inproceedings{KushnerBiosynthesisOP, title={Biosynthesis of porphyrins and heme from y , & dioxovalerate by intact hepatocytes}, author={James P. Kushner and Bruce F. Burnham and W . JAMES HORTONt} }