LOW NADPH OXIDASE ACTIVITY IN EPSTEIN-BARR-VIRUS-IMMORTALIZED B-LYMPHOCYTES IS DUE TO A POSTTRANSCRIPTIONAL BLOCK IN EXPRESSION OF CYTOCHROME B(558)

The NADPH oxidase of phagocytes is known to be expressed in Epstein-Ba rr-virus-transformed B-lymphocytes, albeit at levels only approx. 5% o f those found in neutrophils. We have investigated the basis of this l ow level of expression and find that all four specific components of t he NADPH oxidase are expressed in B-lymphocytes, but only p47-phox pro tein attains levels equivalent with those found in neutrophils. This c omponent was shown to phosphorylate and translocate to the membrane no rmally on activation. The other cytosolic component, p67-phox, did sho w a deficit, and by supplementing a B-cell cytosol extract with recomb inant p67-phox, this was shown to account for the somewhat reduced act ivity of B-cell cytosol in a cell-free oxidase system. The cell-free a nalysis also clearly located the major deficiency in superoxide-genera ting capacity of B-lymphocytes to the membrane. Western blotting of me mbrane proteins revealed major reductions in the amount of cytochrome b(558). Analysis of the levels of mRNA for both subunits of cytochrome b(558), however, showed levels greater than expected. Significantly m ore mRNA for gp91-phox was present in B-cells than in undifferentiated HL60 cells, although it was not quite as abundant as in differentiate d HL60 cells, which are capable of producing large amounts of superoxi de. We conclude that the failure of B-lymphocytes to generate amounts of superoxide equivalent to those generated by neutrophils is primaril y due to a post-transcriptionally determined block to the accumulation of cytochrome b(558).