Rice W G, Ganz T, Kinkade J M, Selsted M E, Lehrer R I, Parmley R T
Blood. 1987 Sep;70(3):757-65.
Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.
防御素是一类新发现的小阳离子多肽，对某些细菌、真菌和病毒具有体外杀菌活性。通过高分辨率Percoll梯度离心法将人中性粒细胞颗粒分离成13个密度级分，并测定这三种防御素多肽在这些级分中的分布。根据总酸可提取蛋白经酸脲和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）后条带的相对染色强度，估算每个级分中防御素和几种颗粒标记蛋白的水平。通过防御素的酶免疫测定以及对典型嗜天青颗粒成分髓过氧化物酶、β-葡萄糖醛酸酶、溶菌酶和弹性蛋白酶的定量测定，证实了这些结果。与八个低密度级分（L1至L8）相比，五个高密度级分（H1至H5）中防御素的相对含量高四倍，约占总蛋白的50%。特别是，级分H5中防御素特别丰富，但髓过氧化物酶、β-葡萄糖醛酸酶、溶菌酶和弹性蛋白酶相对缺乏。超微结构形态显示，级分H5含有最大的颗粒。用甲醇醋酸铀酰-柠檬酸铅染色时，这些颗粒中有70%呈现电子致密边缘和电子透明中心区域，在用二氨基联苯胺对过氧化物酶进行有限反应（30分钟）后，70%呈现相同的边缘染色特征模式。在完整、成熟的外周血中性粒细胞以及人骨髓早幼粒细胞中均发现了这些独特的大的、边缘染色的颗粒，表明它们的合成发生在髓系发育早期。这种不寻常的颗粒类型可能在中性粒细胞的杀菌功能中发挥特殊作用，不同于典型的嗜天青颗粒。
