The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes

Abstract

A method has been described for isolation of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. Homogeneous suspensions of leucocytes were disrupted by lysis in 0.34 M sucrose. This procedure liberated the cytoplasmic contents of the cell and dissolved a considerable proportion of the nuclei. Following disruption, the sucrose lysate was separated into three fractions by differential centrifugation, i.e. 400 g or nuclear pellet, 8,200 g or granule pellet and the postgranule supernate. Microscopic examination revealed that the 8,200 g pellet was composed of intact granules as well as occasional mitochondria. The other two fractions were morphologically heterogeneous. Studies with isolated granules demonstrated their lysis by a variety of weak acids and surface-active agents. When buffered solutions were employed between the ranges of pH 2.0 and 9.0, granule lysis began at pH 5.5 and was complete at pH 4.0. Chemical analysis disclosed that the granule pellet contained protein and phospholipid with only traces of nucleic acids. Approximately 70 to 80 per cent of the total cellular antimicrobial agent phagocytin was present in the granule fraction. This material was liberated from the granules by acid (pH 5.0 or lower). Studies on selected enzymes showed that acid phosphatase, alkaline phosphatase, nucleotidase, ribonuclease, deoxyribonuclease, and beta glucuronidase were predominantly localized in the granule fraction. Approximately 50 per cent of total cellular lysozyme and cathepsin were also present in the 8,200 g pellet. Disruption of the granules was associated with the release of the majority of granule protein and enzymes in a non-sedimentable form. The properties and composition of rabbit polymorphonuclear leucocyte granules seem to be analogous to those of liver lysosomes.