Functional expression of NADPH oxidase components (alpha- and beta-subunits of cytochrome b558 and 45-kDa flavoprotein) by intrinsic human glomerular mesangial cells.
Recently, we have shown that human glomerular mesangial cells (HMCs) release oxygen radicals from the plasma membrane in response to cytokines. Now we have used diphenylene iodonium, a covalent binding inhibitor of activated 45-kDa flavoprotein, in neutrophils radiolabeled with 125I and could identify a 45-kDa protein band in a separated HMC plasma membrane fraction. Low temperature difference spectroscopy showed a peak absorbance at 428 and 558 nm. Direct potentiometry of HMC membranes (-340 to -160 mV) showed the presence of a low potential cytochrome (76 pmol/mg to HMC membrane protein) identified as cytochrome b558. In slot blots, mouse monoclonal antibody (mAb) 7D5, specific for the extracellular domain of the alpha-subunit, showed a positive reaction with HMCs. In Western blots, mAb 449, directed against the cytoplasmic epitope of the alpha-subunit, identified a 23-kDa protein; and mAb 48, raised against the large (beta) subunit of cytochrome b558 of human neutrophils (Verhoeven, A. J., Bolscher, B. G. J. M., Meerhof, L. J., van Zwieten, R., Keijer, J., Weening, R. S., and Roos, D. (1989) Blood 73, 1686-1694), detected a smear between 75 and 100 kDa in denatured HMC membrane protein. These data determined with HMCs, suggest for the first time the expression of three essential components of NADPH:O2- oxidoreductase in mesenchymal cells.
Radeke
H H
HH
Institute of Molecular Pharmacology, Medical School, Hannover, Federal Republic of Germany.
Cross
A R
AR
Hancock
J T
JT
Jones
O T
OT
Nakamura
M
M
Kaever
V
V
Resch
K
K
D000911:Antibodies, Monoclonal / Q000276:immunology; D003573:Cytochrome b Group / Q000737:chemistry* / Q000276:immunology; D005420:Flavoproteins / Q000737:chemistry* / Q000276:immunology; D005609:Free Radicals; D005920:Glomerular Mesangium / Q000201:enzymology*; D006801:Humans; D015151:Immunoblotting; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D013057:Spectrum Analysis; D013481:Superoxides / Q000378:metabolism
D000911:Antibodies, Monoclonal; D003573:Cytochrome b Group; D005420:Flavoproteins; D005609:Free Radicals; D013481:Superoxides; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases