Myeloperoxidase: its structure and expression during myeloid differentiation.
Myeloperoxidase: its structure and expression during myeloid differentiation.
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DOI：
10.1182/blood.v65.2.484.bloodjournal652484
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发表时间：
1985-02
期刊：
影响因子：
20.3
通讯作者：
H. Koeffler;J. Ranyard;M. Pertcheck
中科院分区：
文献类型：
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作者：
H. Koeffler;J. Ranyard;M. Pertcheck
髓过氧化物酶（MPO）是存在于髓样细胞中的主要蛋白质，并被这些细胞用来帮助杀死微生物。人早幼粒细胞HL-60系可被诱导分化为粒细胞或巨噬细胞样细胞。从HL-60粒细胞、HL-60巨噬细胞、HL-60胚细胞和正常人粒细胞中分离含有Poly（A）的RNA。在35 S-甲硫氨酸存在下，在网织红细胞裂解物系统中翻译mRNA。用兔抗人MPO IgG抗血清从裂解物中沉淀MPO。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上，HL-60原始细胞产生的沉淀物产生一条约77，000道尔顿的放射性主带和另一条约46，000道尔顿的带。标记条带的MPO身份通过冷竞争来确认。相对mRNA活性表示为掺入MPO（77，000-道尔顿条带）的放射性与总三氯乙酸（TCA）可沉淀放射性相比的百分比，为0.2%。在HL-60粒细胞、HL-60巨噬细胞和正常人粒细胞中MPO的mRNA活性可忽略不计。脉冲追踪实验表明，用~（35）S-蛋氨酸标记HL-60细胞1/2小时后，细胞裂解液经免疫沉淀和SDS-PAGE后，MPO呈约75，000-道尔顿的放射性主带和77，000-道尔顿的放射性次带。追踪实验（1至24小时）显示，77，000和75，000道尔顿放射性条带被两个主要条带（55，000和15，000道尔顿）和一个次要条带（约39，000道尔顿）放射性替代。6小时35 S-蛋氨酸标记实验表明，MPO合成与总TCA可沉淀放射性相比的相对速率在HL-60母细胞中为0.5%，在HL-60巨噬细胞和粒细胞、正常人粒细胞和B淋巴细胞中几乎可以忽略不计。KG-1成髓细胞和KG-1a早期成髓细胞合成少量的75，000-道尔顿MPO蛋白。虽然HL-60细胞分化后不再合成MPO，但HL-60粒细胞和HL-60巨噬细胞继续含有MPO，如通过酶活性测量的。（400字处截断摘要）
Myeloperoxidase (MPO) is a major protein present in myeloid cells and is used by these cells to help kill microbes. The human promyelocytic HL-60 line can be induced to differentiate to granulocytes or macrophagelike cells. Poly (A) containing RNA was isolated from HL-60 granulocytes, HL-60 macrophages, HL-60 blasts, and normal human granulocytes. The mRNA was translated in a reticulocyte lysate system in the presence of 35S-methionine. The MPO was precipitated from the lysate with rabbit IgG antiserum to human MPO. The resulting precipitate from HL-60 blasts gave a major band of radioactivity of approximately 77,000 daltons and another band at approximately 46,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The MPO identity of the labeled bands was confirmed by cold competition. The relative mRNA activity expressed as a percentage of radioactivity incorporated into MPO (77,000-dalton band) as compared with total trichloracetic acid (TCA) precipitable radioactivity was 0.2%. Negligible mRNA activity for MPO was present in HL-60 granulocytes, HL-60 macrophages, and normal human granulocytes. Pulse-chase experiments showed that MPO was an approximate 75,000-dalton major band and 77,000-dalton minor band of radioactivity after HL-60 blasts were labeled for 1/2 hour with 35S-methionine and the cell lysate immunoprecipitated and subjected to SDS-PAGE. The chase experiments (one to 24 hours) showed that the 77,000- and 75,000-dalton bands of radioactivity were replaced with two major bands (55,000 and 15,000 daltons) and one minor band (approximately 39,000 daltons) of radioactivity. Six-hour 35S-methionine labeling experiments showed that the relative rate of MPO synthesis compared with total TCA precipitable radioactivity was 0.5% in HL-60 blasts and almost negligible in HL-60 macrophages and granulocytes, normal human granulocytes, and B-lymphocytes. The KG-1 myeloblasts and KG-1a early myeloblasts synthesized a small amount of the 75,000-dalton MPO protein. Although HL-60 cells no longer synthesized MPO after differentiation, HL-60 granulocytes and HL-60 macrophages continued to contain MPO as measured by enzyme activity.(ABSTRACT TRUNCATED AT 400 WORDS)