Taylor K L, Guzman G S, Burgess C A, Kinkade J M
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
Biochemistry. 1990 Feb 13;29(6):1533-9. doi: 10.1021/bi00458a026.
Myeloperoxidase is a major protein component of the azurophilic granules (specialized lysosomes) of normal human neutrophils and serves as part of a potent bactericidal system in the host defense function of these cells. In normal, mature cells, myeloperoxidase occurs exclusively as a dimer of Mr 150,000 while in immature leukemia cells, there are both monomeric (Mr 80,000) as well as dimeric species. Like other lysosomal enzymes, myeloperoxidase is synthesized as a larger glycosylated precursor (Mr 91,000) that undergoes processing through single-chain intermediates (Mr 81,000 and 74,000) to yield mature heavy (Mr 60,000) and light (Mr 15,000) subunits. To study the assembly of dimeric myeloperoxidase, azurophilic granules were isolated from either unlabeled or pulse-labeled ([35S]methionine/cysteine) HL-60 cells, and myeloperoxidase was extracted and separated into monomeric and dimeric forms by FPLC gel filtration chromatography. Steady-state levels of dimeric and monomeric myeloperoxidase were found to account for 67% and 33%, respectively, of the total peroxidase activity and were correlated with the levels of associated heme as measured by absorption at 430 nm. Labeled myeloperoxidase polypeptides were immunoprecipitated using a monospecific rabbit antibody and were identified and quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography and liquid scintillation counting. After a 2-h pulse, labeled myeloperoxidase species of Mr 74,000 and 60,000 were found in fractions coeluting with the monomeric form of myeloperoxidase.(ABSTRACT TRUNCATED AT 250 WORDS)
髓过氧化物酶是正常人中性粒细胞嗜天青颗粒（特殊溶酶体）的主要蛋白质成分，在这些细胞的宿主防御功能中作为强效杀菌系统的一部分。在正常成熟细胞中，髓过氧化物酶仅以150,000道尔顿的二聚体形式存在，而在未成熟白血病细胞中，既有单体形式（80,000道尔顿）也有二聚体形式。与其他溶酶体酶一样，髓过氧化物酶以更大的糖基化前体（91,000道尔顿）形式合成，该前体通过单链中间体（81,000和74,000道尔顿）进行加工，产生成熟的重链（60,000道尔顿）和轻链（15,000道尔顿）亚基。为了研究二聚体髓过氧化物酶的组装，从未标记或脉冲标记（[35S]甲硫氨酸/半胱氨酸）的HL-60细胞中分离嗜天青颗粒，提取髓过氧化物酶，并通过快速蛋白质液相色谱凝胶过滤色谱法将其分离为单体和二聚体形式。发现二聚体和单体髓过氧化物酶的稳态水平分别占总过氧化物酶活性的67%和33%，并且与通过430nm处的吸收测量的相关血红素水平相关。使用单特异性兔抗体对标记的髓过氧化物酶多肽进行免疫沉淀，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳/荧光自显影和液体闪烁计数进行鉴定和定量。在2小时脉冲后，在与髓过氧化物酶单体形式共洗脱的级分中发现了74,000和60,000道尔顿的标记髓过氧化物酶种类。（摘要截短至250字）
