Pinnix I B, Guzman G S, Bonkovsky H L, Zaki S R, Kinkade J M
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
Arch Biochem Biophys. 1994 Aug 1;312(2):447-58. doi: 10.1006/abbi.1994.1331.
Myeloperoxidase (MPO) is a hemoprotein that is synthesized in the lumen of the endoplasmic reticulum (ER) as a single-chain precursor and undergoes a complex series of post-translational modifications prior to packaging into azurophilic granules. We and others have previously observed that treatment of human myeloid leukemic cells with succinylacetone (SA), a potent inhibitor of 5-aminolevulinic acid dehydratase (ALA-D), and hence of heme biosynthesis, resulted in loss of MPO enzyme activity, inhibition of the appearance of mature MPO, and accumulation of enzymatically unreactive, but immunoreactive, MPO in the ER. The present study using HL-60 cells was undertaken to establish the nature and specificity of the inhibition by SA and to identify and quantify the biochemical changes in the post-translational pathway of MPO processing. Dose-response studies showed that SA (250 microM) did not affect cell viability or growth up to 72 h, but resulted in inhibition of ALA-D activity (> 93%) and decreased cellular levels of both heme and MPO (approximately 25% of control). There were no effects on the level of total cellular protein or on the activities of lactate dehydrogenase or several other nonheme enzymes colocalized with MPO in azurophilic granules. Northern blot analyses confirmed the nontoxic nature of the conditions and indicated there was no effect on transcription of MPO mRNA. The kinetics of processing in the presence and absence of 250 microM SA were determined using pulse-chase and Percoll density gradient centrifugation methods, followed by identification and quantification of MPO species by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The initial rate of disappearance of precursor MPO was identical for control and SA-treated cells and, after a lag of 2-3 h, there was a fourfold decrease in the rate of appearance of mature MPO in SA-treated cells. In the presence of SA, precursor apoMPO remained in the ER, did not undergo proteolytic processing and, compared to control cells, about 50% was degraded. The disruption in MPO processing was reversible by the addition of exogenous heme. We conclude that the availability of heme is important in the complex maturation of MPO that occurs in the ER, events which precede exit from this compartment and subsequent proteolytic processing and transport to the azurophilic granule.
髓过氧化物酶（MPO）是一种血红素蛋白，在内质网（ER）腔中作为单链前体合成，并在包装到嗜天青颗粒之前经历一系列复杂的翻译后修饰。我们和其他人之前观察到，用琥珀酰丙酮（SA）处理人髓系白血病细胞，SA是5-氨基酮戊酸脱水酶（ALA-D）的有效抑制剂，因此也是血红素生物合成的抑制剂，会导致MPO酶活性丧失，成熟MPO的出现受到抑制，并且在ER中积累无酶活性但有免疫反应性的MPO。本研究使用HL-60细胞来确定SA抑制的性质和特异性，并识别和量化MPO加工翻译后途径中的生化变化。剂量反应研究表明，SA（250 microM）在长达72小时内不影响细胞活力或生长，但会导致ALA-D活性受到抑制（>93%），血红素和MPO的细胞水平降低（约为对照的25%）。对总细胞蛋白水平、乳酸脱氢酶活性或与MPO共定位于嗜天青颗粒中的其他几种非血红素酶的活性没有影响。Northern印迹分析证实了这些条件的无毒性质，并表明对MPO mRNA的转录没有影响。使用脉冲追踪和Percoll密度梯度离心方法，然后通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和荧光显影来识别和量化MPO种类，确定了在存在和不存在250 microM SA的情况下加工的动力学。对照细胞和SA处理细胞中前体MPO消失的初始速率相同，并且在延迟2 - 3小时后，SA处理细胞中成熟MPO出现的速率下降了四倍。在存在SA的情况下，前体脱辅基MPO保留在ER中，未经历蛋白水解加工，并且与对照细胞相比，约50%被降解。通过添加外源性血红素，MPO加工中的破坏是可逆的。我们得出结论，血红素的可用性在ER中发生的MPO复杂成熟过程中很重要，这些事件发生在从该隔室退出以及随后的蛋白水解加工和运输到嗜天青颗粒之前。
