Biosynthesis of delta-aminolevulinic acid and the regulation of heme formation by immature erythroid cells in man.
Heme formation in the erythron is subject to end product regulation by negative feedback, but the exact point of metabolic control in human erythroid cells is unknown. To investigate the mode of action of heme on its own formation, the effects of micromolar concentrations of hemin on de novo synthesis of protoporphyrin IX and delta-aminolevulinate (delta-ALA) by intact human reticulocytes were examined in the presence of 1 mM alpha,alpha'-bipyridyl and 200 microM 4,6-dioxoheptanoate to block their further conversion by ferrochelatase or delta-ALA dehydrase, respectively. At final concentrations (25-40 microM), hemin, which is known to reduce incorporation of [2-14C]glycine into cellular heme, significantly inhibited formation of protoporphyrin IX and total delta-aminolevulinate in situ by these cells. Since synthesis of the first committed precursor, delta-aminolevulinate, as well as protoporphyrin (which is derived from it) were diminished, the effects of hemin on delta-aminolevulinate synthase (EC 2.3.1.37) were studied. Hemin, at concentrations up to 40 microM, had no direct effect on enzymatic activity, as measured with [5-14C] alpha-ketoglutarate (in hypotonically lysed cells) or [1,4-14C]succinyl coenzyme A (in deoxycholate lysates), even after preincubation. However, when intact human reticulocytes were incubated with hemin before assay for delta-ALA synthase, there was a rapid, concentration-dependent reduction in enzymatic activity (mean 42 and 23% inhibition after 60 min for these two substrates, respectively). Hemin had no effect on steady-state levels of delta-ALA synthase mRNA, as determined by Northern blot hybridization using an erythroid-specific human cDNA probe. Thus, a mechanism for inducing feedback inhibition of the tetrapyrrole pathway exists in human erythroid cells. It controls formation of the first committed precursor of protoporphyrin IX, delta-aminolevulinate, and hence regulates heme biosynthesis by limiting the availability of the porphyrin, rather than the metal substrate for the ferrochelatase reaction. Hemin interacts with constituents of the intact reticulocyte significantly to reduce delta-aminolevulinic acid synthase activity by an indirect cellular process that does not influence the abundance of erythroid-specific synthase mRNA but may either inhibit its ribosomal translation in an unknown manner or promote degradation of the enzyme itself by specific proteolysis.
Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, United Kingdom.
L C
LC
Gardner
S J
SJ
Smith
T M
TM
Cox
D000624:5-Aminolevulinate Synthetase / Q000037:antagonists & inhibitors / Q000097:blood* / Q000235:genetics; D000622:Aminolevulinic Acid / Q000097:blood*; D015152:Blotting, Northern; D005246:Feedback; D006402:Hematologic Diseases / Q000097:blood; D006418:Heme / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011524:Protoporphyrins / Q000096:biosynthesis / Q000097:blood; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D012156:Reticulocytes / Q000378:metabolism*
D011500:Protein Synthesis Inhibitors; D011524:Protoporphyrins; D012333:RNA, Messenger; D006418:Heme; D000622:Aminolevulinic Acid; C028025:protoporphyrin IX; D000624:5-Aminolevulinate Synthetase