Bennett J P, Cockcroft S, Caswell A H, Gomperts B D
Biochem J. 1982 Dec 15;208(3):801-8. doi: 10.1042/bj2080801.
Rabbit peritoneal neutrophils, disrupted by sonication, were separated into three subcellular fractions by sucrose-step-gradient centrifugation and these were analysed with respect to biochemical markers. They comprised a high-speed supernatant containing the cytosol, a light particulate fraction enriched in Golgi and plasma membranes and a heavy particulate fraction enriched in granules and nuclei. The light particulate fraction was further separated into its components, which were identified as Golgi membranes (galactosyltransferase activity) and plasma membranes ((radioactivity derived from labelling intact cells with [125I]di-iodosulphanilic acid diazonium salt and [3H]formylmethionyl-leucylphenylalanine ([3H]fMet-Leu-Phe) binding)). In cells prelabelled with [3H]glycerol, the hydrolysis of phosphatidylinositol due to cell stimulation with fMet-Leu-Phe (10 nM) was shown to occur in the light particulate fraction. The [32P]Pi-labelling of phosphatidate, which is an early consequence of phosphatidylinositol hydrolysis, also occurred in this fraction. Analytical sucrose-gradient centrifugation of the light particulate fraction showed that the stimulated increment in [32P]phosphatidate (and thus by implication the initial phosphatidylinositol breakdown) was localized in the plasma membrane.
通过超声破碎处理的兔腹膜中性粒细胞，经蔗糖阶梯梯度离心法分离为三个亚细胞组分，并针对生化标记物进行了分析。它们包括含有胞质溶胶的高速上清液、富含高尔基体和质膜的轻颗粒组分以及富含颗粒和细胞核的重颗粒组分。轻颗粒组分进一步分离为其各个成分，分别鉴定为高尔基体膜（半乳糖基转移酶活性）和质膜（源自用[125I]二碘磺胺酸重氮盐标记完整细胞的放射性以及[3H]甲酰甲硫氨酰 - 亮氨酰 - 苯丙氨酸（[3H]fMet - Leu - Phe）结合）。在用[3H]甘油预标记的细胞中，显示由于用fMet - Leu - Phe（10 nM）刺激细胞导致的磷脂酰肌醇水解发生在轻颗粒组分中。磷脂酸的[32P]Pi标记，这是磷脂酰肌醇水解的早期结果，也发生在该组分中。对轻颗粒组分进行分析性蔗糖梯度离心表明，[32P]磷脂酸的刺激增量（因此意味着最初的磷脂酰肌醇分解）定位于质膜中。
