Plasma-membrane location of phosphatidylinositol hydrolysis in rabbit neutrophils stimulated with formylmethionyl-leucylphenylalanine

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Abstract
Rabbit peritoneal neutrophils disrupted by sonication, were separated into 3 subcellular fractions by sucrose-step-gradient centrifugation, and these were analyzed 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 labeling intact cells with [125I]di-iodosulfanilic acid diazonium salt and [3H]fMet-Leu-Phe binding}. In cells prelabeled with [3H]glycerol, the hydrolysis of phosphatidylinositol due to cell stimulation with fMet-Leu-Phe (10 nM) occurred in the light particulate fraction. The [32P]Pi-labeling 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.