Isolation and partial polypeptide characterization of bovine neutrophil plasma membranes.
The neutrophil plasma membrane has a major role in migration, phagocytosis, and destruction of microorganisms. Neutrophils isolated from blood and mammary secretions were homogenized, and the plasma membrane fraction was isolated on discontinuous sucrose gradient (20, 32, and 50%). Purity of plasma membrane preparation was determined by use of marker enzyme analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the membrane proteins was performed under reducing conditions for polypeptide characterization. The membrane proteins were also labeled with 125I externally, using 1,3,4,6-tetrachloro-3 alpha-6 alpha-diphenylglycouril, and proteins were separated by SDS-PAGE and autoradiographed. Compared with whole cell homogenate, the plasma membrane fraction obtained at the 20/32% interface was enriched for the marker enzymes, 5'-nucleotidase (16-fold), alkaline phosphatase (5.5-fold), and total phosphatase (26-fold). The membrane fraction had minimal specific activity for beta-glucuronidase (0.4-fold), compared with whole cell homogenate. Plasma membrane protein yield was about 500 micrograms/10(9) bovine blood neutrophils. The SDS-PAGE of plasma membrane proteins revealed 25 protein bands, of which 13 were major bands. There were 3 distinct bands (18, 36, and 65 kd) in the plasma membrane-enriched fraction (20/32 interface) that were not seen in other fractions (30/50% and pellet). Further, 125I-labeling identified 5 distinct protein bands (205, 140, 65, 35, and 30 kd). Blood and mammary neutrophils had similar polypeptide patterns, except that 36- and 65-kd bands were more prominent for blood neutrophils than for mammary neutrophils.
Salgar
S K
SK
Department of Animal Science, University of Maryland, College Park 20742.
Paape
M J
MJ
Alston-Mills
B
B
D000818:Animals; D002417:Cattle / Q000097:blood / Q000276:immunology / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007774:Lactation / Q000097:blood / Q000276:immunology / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D008414:Mastitis, Bovine / Q000209:etiology; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism*
D008565:Membrane Proteins