Identification of the milk fat globule membrane proteins. II. Isolation of major proteins from electrophoretic gels and comparison of their amino acid compositions.
The fat globule membranes of milk are derived from the apical plasma membrane of the mammary secretory cells. The nature of the membrane proteins, as isolated from cows' milk, has been studied by the use of discontinuous and continuous SDS-gel electrophoresis. Six methods of preparation of milk fat globule membrane suggested by various authors were tested; gel electrophoresis showed that five major bands were present, independent of the method of preparation. The apparent molecular masses of these proteins as determined on SDS-gels (15% T) were 167, 142, 64, 49 and 46 kDa, respectively. The 167 kDa band stained only with periodic acid-Schiff reagent, while the 142 kDa band stained only with Coomassie blue; the last three bands stained with both. Delipidated membranes were extracted stepwise with water, 0.02 M NaCl and 0.6 M NaCl. The 64 kDa band appears to be nearly insoluble, while the bands of 142, 49 and 46 kDa are fractionated by this procedure. The resolution of all of these proteins by electrophoresis was superior to that achieved by molecular sieve chromatography, and so electrophoretic extraction was used to isolate the major proteins. Dansyl chloride derived proteins were used as markers. Amino acid compositions of the recovered proteins were obtained and are compared.