An Alternatively Spliced Form of NQOj (DT-Diaphorase) Messenger RNA Lacking the Putative Quinone Substrate Binding Site Is Present in Human Normal and Tumor Tissues1

DT-diaphorase is a ubiquitously expressed flavoenzyme responsible for the two-electron reduction of a number of quinone and other anticancer drugs. The majority of DT-diaphorase enzyme activity in human tissues is the product of the NQOt gene. We have now identified a novel alterna tively spliced form of human NQO, niRN'A lacking exon 4 at levels equal to or exceeding those of wild-type NQO, mRNA. Exon 4 codes for the putative quinone substrate binding site of DT-diaphorase derived from NQO, and the recombinant protein from alternatively spliced NQO, mRNA lacking exon 4 has minimal enzyme activity with quinoid and other known substrates of DT-diaphorase. The physiological substrate of DTdiaphorase is unknown, and it is possible that the protein derived from the alternatively spliced NQO, mRNA could have enzyme activity with an appropriate substrate. We found full-length DT-diaphorase protein but could not detect expression of an appropriately smaller form of DTdiaphorase in human tissues using polyclonal antibody to DT-diaphorase, suggesting that alternatively spliced NQO, mRNA lacking exon 4 may not be translated or that the protein product is rapidly degraded. Alternative splicing of NQO, RNA could provide an important mechanism for regu lating NQOt gene expression.