Separation and Analysis of Subcellular Organelles in a Human Promyelocytic

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 organdIes in postnuclear supernatant separated on a discontinuous gradient of Percoll modified to resolve organdIes 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. I .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 myeloperoxi-