A rapid immunological procedure for the isolation of hormonally sensitive rat fat-cell plasma membrane.
1. A rapid method for the isolation of hormonally sensitive rat fat-cell plasma membranes was developed by using immunological techniques. 2. Rabbit anti-(rat erythrocyte) sera were raised and shown to cross-react with isolated rat fat-cells. 3. Isolated rat fat-cells were coated with rabbit anti-(rat erythrocyte) antibodies, homogenized and the homogenate made to react with an immunoadsorbent prepared by covalently coupling donkey anti-(rabbit globulin) antibodies to aminocellulose. Uptake of plasma membrane on to the immunoadsorbent was monitored by assaying the enzymes adenylate cyclase and 5'-nucleotidase and an immunological marker consisting of a 125I-labelled anti-(immunoglobulin G)-anti-cell antibody complex bound to the cells before fractionation. Contamination of the plasma-membrane preparation by other subcellular fractions was also investigated. 4. By using this technique, a method was developed allowing 25-40% recovery of plasma membrane from fat-cell homogenates within 30 min of homogenization. 5. Adenylate cyclase in the isolated plasma-membrane preparation was stimulated by 5 mum-adrenaline.
D000262:Adenylyl Cyclases / Q000032:analysis; D000273:Adipose Tissue / Q000032:analysis; D000327:Adsorption; D000818:Animals; D000906:Antibodies; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology; D004822:Epididymis / Q000032:analysis; D007074:Immunoglobulin G; D007158:Immunologic Techniques; D008297:Male; D009708:Nucleotidases / Q000032:analysis; D051381:Rats
D000906:Antibodies; D007074:Immunoglobulin G; D009708:Nucleotidases; D000262:Adenylyl Cyclases