Abstract
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.
Highlights
From the Slnstitute of Molecular Pharmacology, Medical School0, -3000 Hannouer 61, Federal Republic of Germany, the §Department of Biochemistv, University of Bristol Medical School,Bristol BS8 ITD, United Kingdom, and the Tlnstitute of Medical Science, Universityof Tokyo, Tokyo 108, Japan
Low temperature difference spectros- showed an oxygen radical release induced by soluble stimuli copy showed a peak absorbance at 428 and 558 nm. independent of phagocytosis
In Western blots, monoclonal antibody (mAb) 449, di- al., 1987).Whether these specific effects are due to an oxidarected against thceytoplasmic epitopeof the a-subunit, tive modification of phospholipids (Sevanian et al, 1988), identified a 23-kDaprotein;and mAb 48,raised protein kinase C(Gopalakrishnaand Anderson, 1989), or againstthelarge (@) subunit of cytochrome bsss of other components of cellular signaling systems remains unhuman neutrophils
Summary
Grant SFB244/B1 and by grants from the Medical Research Council and theRheumatism Council (United Kingdom). The calculated low potential cytochrome b55S content of crude HMC membranes amounted to 76 pmol/mg of protein, which represented 23% (Cross et al, 1984) or 76% (Segal, 1989) of the concentrations observed in neutrophils. The specific cytochrome b558containing compartment may be more diluted.* In addition, this may explain the lower intensity of the PMNbands despite equal amounts of transferred protein It is, still possible that the HMC cytochrome b558moiety is only antigenically related, but not identical to the phagocytic cytochrome b558despite the complete similarity of the biophysical properties of the PMN and HMC cytochromes b558 as demonstrated by our spectral and potentiometricanalysis.
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