Gardner L C, Cox T M
Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom.
J Biol Chem. 1988 May 15;263(14):6676-82.
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 59Fe transferrin, 2-[14C]glycine, or 4-[14C]delta-aminolevulinate, isotopic incorporation into extracted heme was determined. Addition of cycloheximide to increase endogenous free heme, reduced incorporation of labeled glycine and iron but not delta-aminolevulinate into cell heme. Incorporation of glycine and iron was also sensitive to inhibition by exogenous hematin (Ki, 30 and 45 microM, respectively) i.e. at concentrations in the range which affect cell-free protein synthesis in reticulocyte lysates. Hematin treatment rapidly diminished incorporation of intracellular 59Fe into heme by human erythroid cells but assimilation of 4-[14C]delta-aminolevulinate into heme was insensitive to inhibition by hematin (Ki greater than 100 microM). 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 delta-aminolevulinate or protoporphyrin IX greatly stimulated utilization of cell 59Fe 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 delta-aminolevulinate formation. Hence in man the regulatory process affects generation of the first committed precursor of porphyrin biosynthesis by delta-aminolevulinate synthetase, whereas in the rabbit separate regulatory mechanisms exist which control the incorporation of iron into protoporphyrin IX.
细胞内“游离”血红素已被证明可控制转铁蛋白铁用于血红素合成的获取。为了确定人类红细胞生成中血红素生物合成的控制位点，从未成熟的红细胞系细胞从外周血和抽取的骨髓中获取。在用人类59Fe转铁蛋白、2-[14C]甘氨酸或4-[14C]δ-氨基乙酰丙酸孵育后，测定同位素掺入提取的血红素中的情况。添加环己酰亚胺以增加内源性游离血红素，可减少标记甘氨酸和铁掺入细胞血红素，但不影响δ-氨基乙酰丙酸掺入细胞血红素。甘氨酸和铁的掺入也对外源性血晶素的抑制敏感（Ki分别为30和45微摩尔），即在影响网织红细胞裂解物中无细胞蛋白质合成的浓度范围内。血晶素处理迅速减少人类红细胞系细胞将细胞内59Fe掺入血红素，但4-[14C]δ-氨基乙酰丙酸掺入血红素对血晶素的抑制不敏感（Ki大于100微摩尔）。在人类网织红细胞中（与家兔的不同），添加水杨醛异烟酰腙铁以独立于转铁蛋白循环增加血红素前体铁池，未能促进血红素合成或改变血晶素诱导的反馈抑制。在人类红细胞系细胞中（但在家兔网织红细胞中不是），用未标记的δ-氨基乙酰丙酸或原卟啉IX预孵育极大地刺激了细胞59Fe用于血红素合成的利用，也减弱了终产物抑制。因此，在人类红细胞系细胞中，血红素生物合成主要在导致δ-氨基乙酰丙酸形成的一个或多个步骤受反馈抑制调节。因此，在人类中，调节过程影响由δ-氨基乙酰丙酸合成酶产生的卟啉生物合成的第一个定向前体，而在家兔中存在单独的调节机制来控制铁掺入原卟啉IX。
