The incorporation of radioactive phosphorus in the leucocyte during the extrusion of protein induced by staphylococcal leucocidin

Abstract

It has been shown that treatment of the polymorphonuclear leucocyte with leucocidin stimulates the incorporation of radioactive phosphorus into the phospholipids of the cell (Woodin, 1962). The radioactivity of the precursors was not known and so it was uncertain if this represented an increased turnover. The present paper shows that the stimulated incorporation of radioactive phosphorus is found in the nucleotides and other acidsoluble compounds of the leucocidin-treated leucocyte. There is no evidence for a causal connexion between this stimulated incorporation and the extrusion of protein from the granules of the leucocyte. METHODSThe two components of leucocidin were crystallized as described by Woodin (1960); unless otherwise indicated they were added to cell suspensions to give 2 jig./108 cells.Polymorphonuclear leucocytes were obtained from peritoneal exudates of rabbits (Woodin, 1962) and will be referred to below as leucocytes. ATP and ADP were obtained from C. F. Boehringer und Soehne, Mannheim, Germany; GTP and GDP were obtained from the Sigma Chemical Co., St Louis, Mo., U.S.A. The solutions used for suspending the cells were Hanks (1948) Incorporation of 32p into the nucleotide& of the leucocyte.Tubes containing 5 x 109 cells in 10 ml. of Hanks solution were incubated for 10 min. at 370 and then leucocidin was added to one tube and 82p (0.05 ml. containing 107 counts/ min.) was added to both tubes. Incubation was continued for 10 min. and the tubes were cooled in ice. They were centrifuged at 0°for 5 min. at 2000 rev./min. and the supernatant was rejected. The cell pellets were extracted for 2 hr. at room temperature with 20 vol. of chloroformmethanol (2:1, v/v). The suspensions were centrifuged and the supernatants used for the preparation of the phospholipids (see below). The cell residues were washed with ether and dried in a stream of air. The powder was then extracted with 10 ml. of ice-cold HC104 (3 %, w/v), residual ether and occluded air being removed by evacuating the tubes until the cell residues no longer floated on the surface. Extraction was continued for 30 min. and then the suspensions were centrifuged at 20 000 rev./min. for 20 min. The slightly opalescent supernatant was passed down a column containing 100 mg. of Norit and 100 mg. of Celite (Threlfall, 1957), which was then washed three times with 3 ml. of water. The effluent from the column was collected; it contained the nucleotide-free, acid-soluble fraction. The nucleotides were eluted from the washed Norit-Celite column in 7 ml. of pyridine and freeze-dried. The solid was dissolved in a small volume of water and put on paper for chromatography. After separation as described below the specific activities of the nucleotides were determined.In a similar experiment, 2 x 109 leucocytes in 12 ml. of Hanks medium were incubated alone or with added leucocidin for 10 min. 32P (01 ml.; 2 x 108 counts/min.) was added and incubation at 37°continued for 20 sec. The suspensions of cells were then diluted with 75 ml. of icecold Hanks mediu...

Morphological changes associated with the extrusion of protein induced in the polymorphonuclear leucocyte by staphylococcal leucocidin

“…Table 1 shows that this is not the case. The retention of much of the acid phosphatase within the granule fraction of the leucocidin-treated cell was reported by Woodin (1962). The increased concentration of this enzyme within the vesicles is taken as evidence that they are not produced by pinocytotic uptake of the medium proceeding independently of the extrusion of the contents of the granules.…”

“…of phosphorus/hr. The assay and unit of activity of peroxidase is given by Woodin (1962). The assays and units of activity of fl-glucuronidase and aldolase are described by Woodin & Wieneke (1963).…”

“…The preparation of leucocidin, polymorphonuclear leucocytes (referred to in this paper as leucocytes) and media for suspending the cells are described by Woodin (1961).…”

“…It has been suggested that the release of protein may have features in common with the extrusion phase of secretion in mammalian cells (Woodin, 1963). In the leucocyte the process is completed within 10 min.…”

“…A survey of biochemical changes that occur when polymorphonuclear leucocytes are treated with leucocidin showed that although the normal impermeability of the cell membrane to many small molecules is retained there is a release into the medium of protein derived from the cytoplasmic granules (Woodin, 1961(Woodin, , 1962. When calcium is omitted from the medium the release of the granule proteins is prevented but the cell membrane simultaneously becomes more permeable to the soluble components of the cytoplasm (Woodin & Wieneke, 1963).…”