Nauseef W M, Clark R A
Blood. 1986 Aug;68(2):442-9.
We describe a system for analysis of the intracellular pathways in the biosynthesis and packaging of functionally important proteins in human myeloid cells. The human promyelocytic cell line HL-60 was used since peripheral blood neutrophils are terminally differentiated and do not actively synthesize protein. Cells were disrupted by nitrogen cavitation and subcellular organelles in postnuclear supernatant separated on a discontinuous gradient of Percoll modified to resolve organelles important in protein synthesis. This Percoll gradient separated azurophilic granules from less dense organelles and partially separated the less dense organelles from one another. Approximate densities of organelles identified by electron microscopy and by biochemical markers are azurophilic granules, 1.102 g/mL; endoplasmic reticulum, 1.039 g/mL; Golgi apparatus, 1.032 g/mL; and plasma membrane, 1.027 g/mL. We validated the utility of this method of subcellular fractionation by examining intracellular transport of myeloperoxidase, a myeloid lysosomal enzyme present in azurophilic granules. The subunits of mature myeloperoxidase (molecular weight [mol wt] = 59,000 and 13,500) cosediment with biochemical markers for lysosomes, whereas the large-mol wt (89,000) precursor forms cosediments with biochemical markers of less dense organelles. Within the limits of assay sensitivity, the 89,000-mol wt precursor is enzymatically inactive and has no spectral evidence for a heme group, suggesting that precursors of myeloperoxidase may undergo proteolytic maturation in a prelysosomal compartment with concomitant incorporation of a heme group and acquisition of enzymatic activity. This system of analysis should be suitable for the identification, subcellular localization, and maturational analysis of other myeloid lysosomal enzymes as well as functionally important membrane proteins.
我们描述了一种用于分析人类髓细胞中功能重要蛋白质生物合成和包装过程中细胞内途径的系统。使用人类早幼粒细胞系HL - 60，因为外周血中性粒细胞已终末分化且不活跃合成蛋白质。细胞通过氮空化破碎，核后上清液中的亚细胞器在经改良以分离对蛋白质合成重要的细胞器的不连续Percoll梯度上进行分离。这种Percoll梯度将嗜天青颗粒与密度较低的细胞器分开，并部分地将密度较低的细胞器彼此分开。通过电子显微镜和生化标记鉴定的细胞器的近似密度为：嗜天青颗粒，1.102 g/mL；内质网，1.039 g/mL；高尔基体，1.032 g/mL；质膜，1.027 g/mL。我们通过检测髓过氧化物酶（一种存在于嗜天青颗粒中的髓系溶酶体酶）的细胞内转运，验证了这种亚细胞分级分离方法的实用性。成熟髓过氧化物酶的亚基（分子量[mol wt]=59,000和13,500）与溶酶体的生化标记共沉降，而大分子重量（89,000）的前体形式与密度较低的细胞器的生化标记共沉降。在检测灵敏度范围内，89,000 - mol wt的前体无酶活性且无血红素基团的光谱证据，这表明髓过氧化物酶的前体可能在溶酶体前区室中经历蛋白水解成熟，同时结合血红素基团并获得酶活性。这种分析系统应适用于其他髓系溶酶体酶以及功能重要的膜蛋白的鉴定、亚细胞定位和成熟分析。
