THE ROLE OF HEME BIOSYNTHETIC AND DEGRADATIVE ENZYMES IN ERYTHROID COLONY DEVELOPMENT - THE EFFECT OF HEMIN
THE ROLE OF HEME BIOSYNTHETIC AND DEGRADATIVE ENZYMES IN ERYTHROID COLONY DEVELOPMENT - THE EFFECT OF HEMIN
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DOI：
10.1111/j.1365-2141.1982.tb01886.x
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发表时间：
1982-01-01
影响因子：
6.5
通讯作者：
LEVERE, RD
中科院分区：
文献类型：
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作者：
IBRAHIM, NG;LUTTON, JD;LEVERE, RD
小鼠骨髓的体外培养已被证明是确定红细胞集落 (CFU-E) 生长和发育过程中血红素生物合成和降解酶途径的有用系统。先前阐明红细胞生成过程中这些途径的尝试受到可用生物材料的量以及特定酶测定的敏感性的限制。这些条件在这里都被克服了。为了富集 CFU-E，使用含有促红细胞生成素的改良甲基纤维素培养基在特殊培养板中培养非贴壁小鼠骨髓细胞。 CFU-E 产量增加约。 3倍。利用灵敏的放射化学测定，在非常早期发育的CFU-E培养物以及成熟的培养物中直接测量ALAS[δ-氨基乙酰丙酸合酶]活性。 ALAS 活性在培养物生长 60 小时后达到峰值，然后活性开始下降。测定了血红素的细胞合成(用14C-ALA)，并且还对标准测定法进行了微小修改，以在开发CFU-E培养物中通过分光光度法测定δ-氨基乙酰丙酸脱水酶(ALAD)和血红素加氧酶活性。这些测定只需 4 或 5 个培养板即可重现。培养物生长 36 小时后，ALAD 活性逐渐上升，并在 .apprx 处达到稳定水平。 60小时的生长。在培养物生长的后期，观察到 14C-ALA 掺入血红素的持续增加，表明 ALAS 之外的其他血红素酶的诱导仍然限制血红素合成的速率。相反，血红素加氧酶活性在生长 60 小时内下降，并在培养后期升高，但随后 ALAS 和 ALAD 没有增加。通过向培养物中添加放线菌酮完全抑制了观察到的由氯高铁血红素引起的血红素酶的增加。血红素合成酶的 mRNA 的正确翻译对于红细胞分化是必需的。此外，血红素加氧酶活性和血红素可用性的下降可触发 CFU-E 的发育和分化。
In vitro culture of murine bone marrow has proved to be a useful system for defining the heme biosynthetic and degradative enzymatic pathways during erythroid colony (CFU-E) growth and development. Previous attempts to elucidate these pathways during erythropoiesis were limited by the amount of biological material available as well as the sensitivity of specific enzyme assays. These conditions were overcome here. To obtain an enrichment of CFU-E, nonadherent mouse bone marrow cells were cultured in special culture plates using a modified methyl-cellulose medium with erythropoietin. CFU-E yields were increased .apprx. 3-fold. Utilizing the sensitive radiochemical assay, direct measurement of ALAS [.delta.-aminolevulinic acid synthase] activity was determined in very early developing CFU-E cultures as well as mature cultures. ALAS activity reached a peak after 60 h of culture growth and then started to decline in activity. Cellular synthesis of heme was determined (with 14C-ALA) and minor modifications of standard assays were also made to determine spectrophotometrically .delta.-aminolevulinic acid dehydratase (ALAD) and heme oxygenase activity in developing CFU-E cultures. These assays were reproducible with as few as 4 or 5 culture plates. ALAD activity rose progressively after 36 h of culture growth and reached a plateau at .apprx. 60 h of growth. A continuous increase in 14C-ALA incorporation into heme was seen at later hours of culture growth, suggesting that the induction of other heme enzymes beyond ALAS is still rate limiting in heme synthesis. Conversely, heme oxygenase activity declined up to 60 h of growth and was elevated at later culture periods without a subsequent increase in ALAS and ALAD. The observed increase in heme enzymes brought about by hemin was completely suppressed by addition of cycloheximide to the cultures. The proper translation of mRNA for the heme synthetic enzymes are necessary for erythroid cell differentiation. Furthermore, a drop in heme oxygenase activity and availability of heme act as a trigger for CFU-E development and differentiation.