A 63-kilodalton cytosolic polypeptide involved in superoxide generation in porcine neutrophils. Purification and characterization.
A cytosolic protein essential for activation of the O2(-)-generating system in neutrophil membrane was highly purified from porcine neutrophils using conventional methods and high performance liquid chromatography. The molecular mass of the protein was estimated as 180 kDa by high performance gel permeation chromatography, and that of subunit as 63 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial amino acid sequence analysis of the 63-kDa polypeptide revealed that it corresponded to a 65-kDa human neutrophil cytosolic factor whose amino acid sequence was recently predicted from the cDNA clone (Leto, T. L., Lomax, K. J., Volpp, B. D., Nunoi, H., Sechler, J. M. G., Nauseef, W. M., Clark, R. A., Gallin, J. I., and Malech, H. L. (1990) Science 248, 727-730). Antibody raised against the porcine 63-kDa polypeptide reacted with a 65-kDa polypeptide in human neutrophils. Neither heme nor flavin was detected in the protein, yet it induced O2- generation when combined with neutrophil membrane in the presence of other cytosolic factors. Furthermore, the antibody diminished the activating effect of cytosol on O2- generation in a cell-free system. Thus, the protein is essential to activate the O2(-)-generating system in neutrophils, acting as a modifier rather than as an electron transport component.
Tanaka
T
T
Department of Biochemistry, School of Medicine, Keio University, Tokyo, Japan.
Imajoh-Ohmi
S
S
Kanegasaki
S
S
Takagi
Y
Y
Makino
R
R
Ishimura
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009504:Neutrophils / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013481:Superoxides / Q000097:blood*; D013552:Swine
D001798:Blood Proteins; D013481:Superoxides; D004247:DNA