Abstract
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.
Highlights
Feedback inhibition of heme synthesis has been demonstrated in erythroid cells obtained from experimental animals and human subjects, but the mechanism and site of action of cantly inhibited formation of protoporphyrin IX and heme on its own formation has been controversial
Cursor of protoporphyrin IX, 6-aminolevulinate, and The first enzymatic reaction of heme biosynthesis is catahence regulates hemebiosynthesis by limitingthe lysed by 6-aminolevulinicacid synthase (succinyl-CoAglycine availability of the porphyrin, rather than the metal C-succinyltransferase (EC 2.3.1.37)).It has substrate for the ferrochelatase reactionH. emin inte- lately been shown in vertebrates such as chicken and man racts with constituents of the intact reticulocyte sig- that two distinct isozymes of 6-aminolevulinate synthase ocnificantly to reduce6-aminolevulinic acid synthase ac- cur and areencoded separately in thegenome [7,8]
Protoporphyrin formation was quantified by reference to calibrating standards of protoporphyrin IX that were added to unincubated cell lysates to control directly for recovery, which was found to be greater than 60%
Summary
Addition of 80 pmol of [4-"C]6-aminolevulinate to reticulocyte lysates was carried out in the presence or absence of 200 PM 4,6-dioxoheptanoate, and the subsequent recovery of radioactivity was determined after chromatography on Dowex 50W resin, after incubation at 37 "C for 1 h. Total &ALA formed by intact human reticulocytes was measured in the Dowex 50W eluates by means of an enzymatic procedure that utilizes 6-aminolevulinate dehydrase coupled with porphobilinogen deaminase (hydroxymethylbilane synthase) to generate uroporphyrin I that can be determined fluorometrically with a high. The conversion of substrate to uroporphyrin I, as determined by addition of exogenous 6-aminolevulinate to thecell lysates, was linear and found to be 20% in the range of 10-1250 pmol of uroporphyrin I equivalents (80-10,000 pmol of 6-aminolevulinate) These values are closely similar to those obtained under optimal conditions for the coupled enzymatic procedure by Bishop et al [19]
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