REGULATION OF HEME-BIOSYNTHESIS IN NORMOBLASTIC ERYTHROPOIESIS - ROLEOF 5-AMINOLEVULINIC ACID SYNTHASE AND FERROCHELATASE

The development of haem biosynthetic enzyme activity during normoblast ic human erythropoiesis was examined in seven patients. The first and last enzymes of the haem biosynthetic pathway, ALA synthase and ferroc helatase, were assayed by radiochemical/high performance liquid chroma tographic (HPLC) methods. An assay for ferrochelatase activity in huma n bone marrow was developed. Enzyme substrates were protoporphyrin IX and Fe-59(2+) ions. Fe-59-labelled haem was isolated by organic solven t extraction/sorbent extraction followed by reversed-phase HPLC. Optim al activity occurred at pH 7.3 in the presence of ascorbic acid, in da rkness and under anaerobic conditions. Haem production was proportiona l to cell number and was linear with time to 30 min. The assay was sen sitive to the picomolar range of haem production. ALA synthase and fer rochelatase activity was assayed in four highly purified age-matched e rythroid cell populations. ALA synthase activity was maximal in the mo st immature erythroid cells and diminished as the cells matured with a n overall five fold loss of activity from proerythroblast to late eryt hroblast development. Ferrochelatase activity was, however, more stabl e with less than a two fold change in activity observed during the sam e period of erythroid differentiation. Maximal activity occurred in er ythroid fractions enriched with intermediate erythroblasts. These resu lts support sequential rather than simultaneous appearance of these en zymes during normoblastic erythropoiesis. Quantitative analysis of rel ative enzyme activity however indicates that at all times during eryth roid differentiation ferrochelatase activity is present in excess to t hat theoretically required relative to ALA synthase activity since ALA and haem are not produced in stoichiometric amounts. The lability of ALA synthase versus the stability and gross relative excess of ferroch elatase activity indicates a far greater role for ALA synthase in the regulation of erythroid haem biosynthesis than for ferrochelatase.