IN-VITRO PHOSPHORYLATED BOVINE-MILK FAT GLOBULE-MEMBRANE PROTEINS

Incubation of the the milk fat globule membrane (MFGM) with [gamma-P-3 2]ATP, in the presence of protein phosphatase inhibitors and MgCl2 and /or MnCl2, led to protein phosphorylation within this membrane. Analys is of the P-32-labeled proteins of bovine MFGM, by SDS/PAGE and autora diography, showed at least 15 radioactive bands with relative molecula r masses in the range of 10 to 200 kDa. Bovine MFGM had four bands (66 -67, 51, 33, and 24 kDa) labeled more intensively than others. Calcium could not substitute for Mg2+ and Mn2+ in the phosphorylation reactio n. Genistein (100 mu mol/L) reduced protein phosphorylation by 50 to 7 0%, whereas daidzein and heparin did not. The 66-67 and 15 kDa in vitr o phosphorylated bovine MFGM proteins were butyrophilin and fatty-acid -binding protein (FABP), respectively. Phosphoamino acid analysis of P -32-labeled 66, 51, and 15 kDa polypeptide acid hydrolyzates revealed radiolabeled phosphotyrosine, -threonine, and -serine in the 66 kDa pr otein, radiolabeled phosphotyrosine and -threonine in the 51 kDa prote in, and radiolabeled phosphotyrosine in the 15 kDa protein. FABP appea red to be in a complex with its putative kinase. The analysis of in vi tro phosphorylated human MFGM revealed 13-15 radioactive protein bands in the range of 20 to 200 kDa. The 65 and 50 kDa radiolabeled bands w ere prominent in all five individual samples of human MFGM analyzed. F AK, insulin beta-subunit receptor, c-src(60) and MAPK, were detected i n the bovine and human MFGM using Western immunoblotting. MAPKAPK was also found in the bovine MFGM. (C) Elsevier Science Inc. 1997.