Subcellular fractionation of porcine neutrophils by nitrogen cavitation and sucrose‐density‐gradient centrifugation

Abstract

Neutrophils, isolated in large quantities from porcine blood were disrupted by N cavitation and separated by differential centrifugation into a nuclear fraction and a post-nuclear supernatant. The latter was subfractionated by sucrose density gradient centrifugation into cytosol, a fraction consisting of membrane vesicles and 2 granule-rich fractions. The membrane fraction accounted for 1.9% of the protein in the post-nuclear supernatant, the light granule fraction for 2.2% and the dense granule fraction for 4.2%. Catalase, lactate dehydrogenase and malate dehydrogenase were largely confined to the cytosol. The dense granule fraction contained the highest quantities of the hydrolytic enzymes, although the membrane fraction was also rich in alkaline and acid phosphatase and .gamma.-glutamyl transpeptidase activities. EM of the membrane fraction showed intact membrane vesicles, whereas the granular fractions consisted of electron-dense, membrane-bound granules. Two granular fractions were isolated which contained granules of differing size and density. 3H-labeled wheat germ agglutinin bound to the surface of intact neutrophils and when these were disrupted and fractionated the membrane fraction showed a specific binding activity 16-times greater than that of the cavitated sample. The membrane fraction interacted with the detergent digitonin and as a result underwent density perturbation increasing from 1.13 g .cntdot. cm-3 to 1.18 g .cntdot. cm-3. Dodecylsulfate-polyacrylamide gel electrophoresis showed the membrane fraction to consist of at least 40 protein bands, with relative molecular masses ranging from 200,000-16,000. The granule fractions contained less protein bands, with a protein composition quite distinct from that of the membrane fraction.