Characterization of two monoclonal antibodies against cytochrome b558 of human neutrophils.
Monoclonal antibodies (MoAbs) were raised against cytochrome b558, a membrane-bound component of the NADPH:O2 oxidoreductase in human neutrophils. This cytochrome consists of a low-molecular-weight (low-mol-wt) subunit of 22 to 23 Kd, probably encoded by an autosomal gene, and a high-mol-wt subunit of 75 to 90 Kd, encoded on the X-chromosome. MoAb 449 reacts with the low-mol-wt subunit and MoAb 48 with the high-mol-wt subunit on Western blots of purified cytochrome b558 and on blots of whole neutrophil extracts. In extracts of neutrophils from patients with chronic granulomatous disease (CGD) in which cytochrome b558 is not detectable by spectrophotometric methods, the low-mol-wt subunit is present, albeit in a much smaller amount. The high-mol-wt subunit is not detected by MoAb 48 in neutrophils of patients with X-linked CGD and in neutrophils of patients with the autosomal, cytochrome-b558-negative form of the disease. These results can be explained by a marked instability of these subunits when the synthesis of either of the two is disturbed. In differentiated HL-60 cells, the high-mol-wt subunit appears to be present in a different form. Cloning of the low-mol-wt subunit with the help of MoAb 449 suggests the presence of a heme-binding site on this subunit. By comparison of the binding characteristics of MoAb 449 to intact and permeabilized neutrophils with those of MoAb 7D5, recently isolated by Nakamura et al (Blood 69:1404, 1987), the low-mol-wt subunit was established as a transmembrane protein.
Verhoeven
A J
AJ
Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Bolscher
B G
BG
Meerhof
L J
LJ
van Zwieten
R
R
Keijer
J
J
Weening
R S
RS
Roos
D
D
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000276:immunology*; D003902:Detergents; D000939:Epitopes; D005434:Flow Cytometry; D006105:Granulomatous Disease, Chronic / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019255:NADPH Oxidases; D009504:Neutrophils / Q000276:immunology*
D000911:Antibodies, Monoclonal; D003573:Cytochrome b Group; D003902:Detergents; D000939:Epitopes; C036888:cytochrome b558; D019255:NADPH Oxidases