Electrophoretic analysis of protein selectively extracted with chaotropic agents from membranes of rat sperm.
Sperm from rat cauda epididymis was washed, sonicated and centrifuged to obtain fractions sedimenting at 600 x g for 5 min, 27.000 x g for 5 min, and 100.000 x g for 40 min. All fractions were observed with the electron microscopy and assayed for cytochrome c oxidase activity. The 100.000 x g fraction contained only small membranous vesicles and less than 0.5% of the total enzymatic activity. This fraction was considered to represent sperm plasmalemma and it was extracted with Tris-HCl buffer before treating it with one of the following chemicals: acetate buffer, pH: 4.5; 0.6 M KCl; bicarbonate buffer, pH 11.0; Triton X-100, and Sodium Dodecyl Sulfate (SDS). After centrifuging, the residual sediments were solubilized in hot 2% SDS. The extracts and the solubilized sediments (hot SDS) were analyzed in SDS-PAGE. The extracts obtained with the first three chemicals contained 11,9, and 25% of total proteins respectively. The bicarbonate buffer solubilized 45%, and the detergents 55% and 65% respectively. A total of 30 bands were seen in the extracts and sediments. Acid pH extracted a low number of bands of high mobility and low molecular weight. Instead, the KCl and bicarbonate buffer, extracted a great number of bands over a wide range of molecular weights (23, 38.5, 55, 100, and 140 KD). The detergents had similar effects: both solubilized four new bands. In residual sediments there were no new proteins and the bands corresponded to those extracted with the detergents, but they varied in staining intensity. According to the results obtained with the mild chaotropic agents of 0.6 M KCl and bicarbonate buffer, 50% of the mass of membraneous proteins may be peripheric. Proteins partially extracted with the detergents were also found in the residual sediment, and they may constitute the skeleton of sperm membrane.
Instituto de Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo-CONICET, Mendoza, Argentina.
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Fornés
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Barbieri
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Bertini
D000818:Animals; D002021:Buffers; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification*; D008854:Microscopy, Electron; D051381:Rats; D013094:Spermatozoa / Q000737:chemistry* / Q000648:ultrastructure
D002021:Buffers; D003902:Detergents; D008565:Membrane Proteins