Analytical subcellular fractionation of guinea pig peritoneal macrophages: preparation of purified plasma membranes.

Abstract

A method using sucrose gradient centrifugation is described for the purification of plasma membranes of guinea pig peritoneal macrophages. The subcellular fractions obtained have been submitted to a biochemical and ultrastructural analysis. Two plasma membrane markers, 5'-nucleotidase and alkaline phosphodiesterase I, have been assayed at the same time as markers for other subcellular organelles, DNA (nuclei), succinic dehydrogenase (mitochondria), inosine diphosphatase (endoplasmic reticulum), and acid phosphatase (lysosomes). The exposure of the plasma membranes to a low concentration of digitonin allowed us to obtain their high purification. They are only contaminated by 2-3% of other cell components present in the macrophages homogenate. The representative ultrastructural technique used has confirmed the purity of the plasma membranes isolated.