Topology of cytochrome b558 in neutrophil membrane analyzed by anti-peptide antibodies and proteolysis.
Cytochrome b558 is an important constituent of the superoxide-generating system in neutrophils and B lymphocytes. In this paper, the topology of the cytochrome in human neutrophil membrane was studied using antibodies raised in rabbits against synthetic peptides corresponding to various regions in the large and small subunits of the cytochrome. The antibodies recognized the cytochrome subunits in immunoblots and the cytochrome in situ. An antibody raised against residues 150-172 in the large subunit (anti-L123) bound to intact neutrophils, indicating that this region is exposed to the outside of the cells. In contrast, antibodies raised against any of the carboxyl-terminal regions of the large and small subunits (anti-LC and anti-SC, respectively) or the amino-terminal region of the small subunit (anti-SN), bound to neutrophils only after the cells were made permeable by freezing and thawing. The region close to the carboxyl terminus of the large subunit was digested by extracellularly added papain and, as a result, an 18-kDa carboxyl-terminal fragment was detected. Thus the carboxyl-terminal region of the large subunit is cytoplasmic and/or buried in the membrane, and the region around residues 369-398 is exposed on the cell surface. In contrast to the large subunits, the small subunit in neutrophils was resistant to any of the proteinases tested, although the subunit in membrane or Triton-solubilized preparation was digestible with papain. These results indicate that the large subunit of cytochrome b558 is a transmembrane protein with at least two regions exposed on the cell surface and that the carboxyl terminus of this subunit and both termini of the small subunit are exposed to the cytoplasmic side.
Imajoh-Ohmi
S
S
Institute of Medical Science, University of Tokyo, Japan.
Tokita
K
K
Ochiai
H
H
Nakamura
M
M
Kanegasaki
S
S
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group / Q000737:chemistry*; D003902:Detergents; D005434:Flow Cytometry; D006801:Humans; D006868:Hydrolysis; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D017830:Octoxynol; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011092:Polyethylene Glycols; D011487:Protein Conformation
D000906:Antibodies; D003573:Cytochrome b Group; D003902:Detergents; D010446:Peptide Fragments; D011092:Polyethylene Glycols; D017830:Octoxynol; C036888:cytochrome b558; D019255:NADPH Oxidases