Characterization of rat lymphocyte cell membranes by analytical isopycnic centrifugation

Lymph node cell homogenates were fractionated by differential or isopycnic centrifugation and the fractions analyzed for biochemical markers with particular focus on plasma membrane constituents. Markers for the nucleus (DNA), mitochondria (cytochrome oxidase), and lysosomes (acid hydrolases) showed the expected distributions which were different from those of membrane-bound enzymes. 5'-Nucleotidase, alkaline phosphodiesterase, gamma-glutamyltranspeptidase, and cholesterol were membrane-bound and distributed identically after isopycnic centrifugation with peaks at 1.15. The distributions of the enzymes were all shifted to higher densities by digitonin treatment, confirming their association with plasma membrane-derived elements. The distribution of galactosyltransferase (ovalbumin acceptor), largely overlapped those of plasma membrane markers but it was only slightly shifted by digitonin, suggesting its localization in Golgi apparatus. The distribution of mannosyltransferase (dolichyl phosphate acceptor) also overlapped those of plasma membrane and Golgi markers but it was centered at higher density (1.18) and was unaffected by digitonin. It is a useful marker for endoplasmic reticulum. 50% of the activity was in low speed "nuclear" sediments where it was associated with the nuclear membrane. A number of other putative and previously used markers for the endoplasmic reticulum of lymphocytes were shown not to be localized in these membranes. In particular, NADH-cytochrome c reductase was only partly associated with the endoplasmic reticulum (56%) and the remainder of the activity was in mitochondria (44%). The results show the heterogeneity in equilibrium density of plasma membrane vesicles and the considerable overlap of their distribution with those of other cellular membranes; they should provide a basis for the more rational design of preparative procedures for the lymphocyte plasma membrane.