Biochemical and immunochemical properties of B lymphocyte cytochrome b558.
Like neutrophils, Epstein-Barr virus (EBV)-immortalized B lymphocytes express all constituents of the NADPH oxidase complex necessary to generate superoxide anion O2-. The NADPH oxidase activity in EBV-B lymphocytes is only 5% of that measured in neutrophils upon PMA stimulation. Cytochrome b558 is the sole redox membrane component of NADPH oxidase; it is the protein core around which cytosolic factors assemble in order to mediate oxidase activity. In the present study, we have compared the structural and functional properties of cytochrome b558 from EBV-B lymphocytes and neutrophils. Cytochrome b558 from EBV-B lymphocyte plasma membrane, like that from neutrophils, is characterized by a heterodimeric structure with a highly glycosylated beta subunit, known as gp91-phox. While the amount of cytochrome b558 recovered after purification from EBV-B lymphocytes (approximately 0.24 nmol from 1010 cells) was low compared to that recovered from neutrophils (approximately 10 nmol), the biochemical properties of purified cytochrome b558 from both EBV-B lymphocytes and neutrophils were quite similar with respect to their differential spectra, redox potential, and FAD binding site. Once cytochrome b558 was extracted from the EBV-B lymphocyte membrane, it was able to mediate, in a reconstituted system of O2- production the same oxidase turnover as that found for cytochrome b558 extracted from neutrophils. A comparison between membrane bound and soluble cytochrome b558 suggested that the weak oxidase activity measured in intact EBV-B cells might be the result not only of the small amount of expressed cytochrome b558, but also of a defect of the activation process in lymphocyte membrane.
Batot
G
G
GREPI, JE-UJF, Laboratoire d'Enzymologie, CHU, Grenoble, France.
Paclet
M H
MH
Doussière
J
J
Vergnaud
S
S
Martel
C
C
Vignais
P V
PV
Morel
F
F
D001402:B-Lymphocytes / Q000201:enzymology* / Q000821:virology; D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation; D005420:Flavoproteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D004854:Herpesvirus 4, Human; D006801:Humans; D008055:Lipids; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000302:isolation & purification; D011199:Potentiometry; D012995:Solubility; D013481:Superoxides / Q000378:metabolism
D003573:Cytochrome b Group; D005420:Flavoproteins; D008055:Lipids; D008565:Membrane Proteins; D010750:Phosphoproteins; C085072:neutrophil cytosol factor 40K; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; C036888:cytochrome b558; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1