Biosynthesis of Heme in Immature Erythroid Cells

Heme formation in reticulocytes from rabbits and rodents is subject to end product negative feedback regulation: intracellular “free” heme has been shown to control acquisition of transferrin iron for heme synthesis. To identify the site of control of heme biosynthesis in the human erythron, immature erythroid cells were obtained from peripheral blood and aspirated bone marrow. After incubation with human “Fe transferrin, 2-[‘4C]glycine, or 4-[‘4C]G-aminolevulinate, isotopic incorporation into extracted heme was determined. Addition of cycloheximide to increase endogenous free heme, reduced incorporation of labeled glytine and iron but not d-aminolevulinate into cell heme. Incorporation of glycine and iron was also sensitive to inhibition by exogenous hematin (Ki, 30 and 45 pM, respectively) i.e. at concentrations in the range which affect cell-free protein synthesis in reticulocyte lysates. Hematin treatment rapidly diminished incorporation of intracellular “Fe into heme by human erythroid cells but assimilation of 4-[‘4C]6-a~inolevulinate into heme was insensitive to inhibition by hematin (Ki > 100 PM). In human reticulocytes (unlike those from rabbits), addition of ferric salicylaldehyde isonicotinoylhydrazone, to increase the pre-heme iron pool independently of the transferrin cycle, failed to promote heme synthesis or modify feedback inhibition induced by hematin. In human erythroid cells (but not rabbit reticulocytes) pre-incubation with unlabeled a-aminolevulinate or protoporphyrin IX greatly stimulated utilization of cell “Fe for heme synthesis and also attenuated end product inhibition. In human erythroid cells heme biosynthesis is thus primarily regulated by feedback inhibition at one or more steps which lead to d-aminolevulinate formation. Hence in man the regulatory process affects generation of the first committed precursor of porphyrin biosynthesis by b-aminolevulinate synthetase, whereas in the rabbit separate regulatory mechanisms exist which control the incorporation of iron into protoporphyrin TV