Separation of the proteins of bovine milk-fat-globule membrane by electrofocusing with retention of enzymatic and immunological activity.
1. Proteins of fat-globule membrane from bovine milk were solubilized with the non-ionic detergent Triton X-100 in the presence of protease inhibitors. Approximately 25% of the total membrane protein was solubilized and the extracts were shown to contain a sample of most of the major membrane proteins and glycoproteins. 2. The solubilized proteins were separated in flat-beds of Ultrodex by electrofocusing and the pI values for the major proteins, glycoproteins and certain enzymes determined. Several of the proteins displayed marked heterogeneity indicating the existence of protein variants and isoenzymes. Principal pI values for the enzymes assayed were as follows: xanthine oxidase, 7.35--7.55; NADH2: iodonitrotetrazolium reductase, less than 4.5; 5'-nucleotidase, 7.15--7.4; alkaline phosphatase, 5.4--5.7; phosphodiesterase, 4.6--4.8; gamma-glutamyl transpeptidase, 4.4--4.55. 3. Fractions after electrofocusing were analyzed by 'fused rocket' immunoelectrophoresis and crossed immunoelectrophoresis after separation in polyacrylamide gels containing sodium dodecyl sulphate. Major antigens of the membrane include xanthine oxidase and glycoproteins of apparent molecular weights 67 000, 49 500 and 46 000. The latter two components share common antigenic determinants and could not be separated by gel filtration, ion-exchange chromatography, lectin-affinity chromatography or preparative electrofocusing.
D015720:5'-Nucleotidase; D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D002417:Cattle; D005260:Female; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007525:Isoelectric Focusing; D008055:Lipids; D008565:Membrane Proteins / Q000302:isolation & purification*; D008892:Milk / Q000032:analysis* / Q000201:enzymology; D008894:Milk Proteins / Q000302:isolation & purification*; D009246:NADH Tetrazolium Reductase / Q000032:analysis; D009708:Nucleotidases / Q000032:analysis; D017830:Octoxynol; D010727:Phosphoric Diester Hydrolases / Q000032:analysis; D011092:Polyethylene Glycols; D014969:Xanthine Oxidase / Q000032:analysis; D005723:gamma-Glutamyltransferase / Q000032:analysis
D008055:Lipids; D008565:Membrane Proteins; D008894:Milk Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; D014969:Xanthine Oxidase; D009246:NADH Tetrazolium Reductase; D005723:gamma-Glutamyltransferase; D009708:Nucleotidases; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase; D010727:Phosphoric Diester Hydrolases