Isolation and characterization of alkaline phosphatase-containing granules (phosphasomes) from human polymorphonuclear leucocytes

Abstract

Human polymorphonuclear leucocytes were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by centrifugation on a discontinuous sucrose density gradient to produce a phosphasome-enriched fraction, contaminated primarily by plasma membrane. Experiments to separate these membrane fractions by centrifugation on gradients of Nycodenz or Percoll, or by toroidal coil counter current chromatography, were unsuccessful. Fractionation carried out on neutrophils previously suspended in isotonic sucrose containing a low concentration of digitonin resulted in the preparation of a highly purified phosphasome fraction, free of plasma membrane components. Electron-microscope cytochemistry of the purified fraction identified the phosphasomes as regular and irregular-shaped spheres and rods, the alkaline phosphatase being associated with the inner aspect of the vesicle membrane. These granule structures were very similar to phosphasomes observed in intact neutrophils. A proportion of the endoplasmic reticulum and Golgi membrane marker enzymes remained associated with the phosphasomes throughout the separation procedures.