Isolation of plasma membrane from human blood monocytes. Subcellular fractionation and marker distribution.
The isolation of plasma membrane from human peripheral blood monocytes is described. Monocytes were isolated by centrifugal elutriation, to eliminate an adherence step, thus minimizing functional and surface antigenic alterations to the cells. Monocytes were surface-labelled with a radiolabelled monoclonal antibody, 125I-WVH-1, and then disrupted by nitrogen cavitation. Membranes were separated according to equilibrium buoyant density by isopycnic centrifugation on a sucrose gradient. The subcellular membranes were localized using marker enzymes for the plasma membrane, 5'-nucleotidase and leucine 2-naphthylamidase (leucine aminopeptidase), and for intracellular membranes: galactosyltransferase (Golgi), arylsulfatase C (endoplasmic reticulum), monoamine oxidase (mitochondria), catalase (peroxisomes), beta-hexosaminidase and beta-glucuronidase (lysosomal vesicles) and lactate dehydrogenase (cytosol). The monoclonal antibody 125I-WVH-1 was shown to label the plasma membrane, as judged by known markers, and represents a highly specific trace label, applicable to the use of plasma membrane as an immunogen for monoclonal antibody production. The NAD-splitting enzyme, NAD+ nucleosidase, was detected and its presence on the plasma membrane was demonstrated. The subcellular localization of non-specific esterase in human mononuclear phagocytes is controversial. No evidence was found for alpha-naphthyl acetate esterase activity on the plasma membrane or in lysosomal vesicles. However, a membrane-bound esterase in fractions with properties similar to the smooth endoplasmic reticulum was detected.
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000276:immunology / Q000648:ultrastructure; D002500:Centrifugation, Isopycnic; D004072:Digitonin; D006801:Humans; D009000:Monocytes / Q000276:immunology / Q000648:ultrastructure*; D013347:Subcellular Fractions / Q000276:immunology / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D004072:Digitonin