In vitro phosphorylated bovine milk fat globule membrane proteins

Abstract

Incubation of the milk fat globule membrane (MFGM) with [γ- 32P]ATP, in the presence of protein phosphatase inhibitors and MgCl 2 and/or MnCl 2, led to protein phosphorylation within this membrane. Analysis of the 32P-labeled proteins of bovine MFGM, by SDS PAGE and autoradiography, showed at least 15 radioactive bands with relative molecular 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 Mg 2+ and Mn 2+ in the phosphorylation reaction. Genistein (100 μmol/L) reduced protein phosphorylation by 50 to 70%, whereas daidzein and heparin did not. The 66–67 and 15 kDa in vitro phosphorylated bovine MFGM proteins were butyrophilin and fatty-acid-binding protein (FABP), respectively. Phosphoamino acid analysis of 32P-labeled 66, 51, and 15 kDa polypeptide acid hydrolyzates revealed radiolabeled phosphotyrosine, -threonine, and -serine in the 66 kDa protein, radiolabeled phosphotyrosine and -threonine in the 51 kDa protein, and radiolabeled phosphotyrosine in the 15 kDa protein. FABP appeared to be in a complex with its putative kinase. The analysis of in vitro phosphorylated human MFGM revealed 13–15 radioactive protein bands in the range of 20 to 200 kDa. The 65 and 50 kDa radiolabeled bands were prominent in all five individual samples of human MFGM analyzed. FAK, insulin β-subunit receptor, c-src 60 and MAPK, were detected in the bovine and human MFGM using Western immunoblotting. MAPKAPK was also found in the bovine MFGM.