Factors influencing the yield and purity of goat sperm plasma membranes isolated by means of an aqueous two-phase polymer system.
An improved aqueous two-phase polymer method has been developed for the isolation of sperm plasma membranes by manipulating various parameters that influence markedly the purity as well as yield of the membrane. The method consists of hypotonic shock of intact spermatozoa with 1.25 mM EDTA to dissociate the plasma membrane and dispersion of these cells to a two-phase polymer system consisting of 5.5% 252-Kd dextran and 4.2% 20-Kd polyethylene glycol prior to centrifugation at 9700 X g for 30 min when the two polymer phases are separated; the membrane fraction sediments at the interphase. The resulting membrane fraction was purified further by repeating the two-phase fractionation step. The yield of the membranes was approx. 35-40%, based on the recovery of the membrane-bound marker enzymes alkaline phosphatase and 5'-nucleotidase. The isolated membranes showed a high degree of purity as evidenced by phase contrast and electron microscopic studies and analyses of marker enzymes characteristic of cellular organelles. The yield and purity of the membranes have been found to be markedly dependent on the conditions of the hypotonic shock, obtained as a function of, EDTA concentration and on the molecular sizes of the dextran and polyethylene glycol that constitute the two-phase polymer system, as well as on the centrifugal force used for the sedimentation of the membrane.
D015720:5'-Nucleotidase; D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D002498:Centrifugation; D003911:Dextrans; D004492:Edetic Acid; D006041:Goats / Q000033:anatomy & histology*; D007038:Hypotonic Solutions; D008297:Male; D008854:Microscopy, Electron; D009708:Nucleotidases / Q000032:analysis; D011092:Polyethylene Glycols; D011108:Polymers; D013094:Spermatozoa / Q000648:ultrastructure*
D003911:Dextrans; D007038:Hypotonic Solutions; D011108:Polymers; D011092:Polyethylene Glycols; D004492:Edetic Acid; D009708:Nucleotidases; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase