UVC activation of the HeLa cell membrane “TGFαASE,” a metalloenzyme

We have investigated the effect of UVC irradiation on “TGFαase” activity using both intact HeLa cells and isolated membrane fragments with an assay based on the previously described nonapeptide substrate method [Brown et al. (1992): J Cell Biochem 48:411–423]. This method allows recognition of cleavage at the scissile bond cognate with that of the TGFα N‐terminal cleavage site from its membrane‐bound precursor. The level of ectoendopeptidase (including “TGFαase”) activity observed on intact cells was lower than that of ectoaminopeptidases. Addition of foetal bovine serum (FBS) enhanced aminopeptidase and dipeptidyl peptidase activity but inhibited “TGFαase” activity, while phorbol 12‐myristate 13‐acetate (PMA) had no significant effect on the ectopeptidases monitored, except for “TGFαase,” which was also inhibited, in contradistinction to their effects in other cell systems. Sublethal UVC irradiation (10 Jm 2 ) of the cultures resulted in activation of the ectoaminopeptidase and ectoendopeptidases which was maximal 16 and 20–24 h after irradiation, respectively. The addition of FBS to these irradiated cells appeared to reduce the increase in endopeptidase products, due in part to increased aminopeptidase activity but also to the direct inhibitory effect of FBS on the “TGFαase.” The activation of these proteases by UVC, even at high fluences (500 Jm 2 ), was not observed within the first 30 min after the cells were irradiated. Purified plasma membrane fragments were prepared from suspension cultures of HeLa cells and displayed high levels of “TGFαase” activity. The rate of “TGFαase” activity using 140 nM peptide substrate (P9) was 5.6 pmol/min/mg membrane protein, which was elevated to 13.7 pmol/min/mg membrane protein, 20 h after the cells had been irradiated with 10 Jm 2 UVC. Inhibition studies indicate that the plasma membrane “TGFαase” is a metalloenzyme, as it was inhibited by EDTA, EGTA, and 1,10‐phenanthroline but not by elastase or serine protease inhibitors. “TGFαase” activity on intact cells was shown to be inhibited by 1,10‐phenanthroline, which further supports this suggestion. Treatment of the membranes with Triton X‐100 resulted in a loss of “TGFαase” activity, raising the possibility that this enzyme may require a cofactor to be fully functional. We show that in purified membrane preparations of HeLa cells there is evidence for the presence of a “TGFαase” which can be activated by UV irradiation, which differs from the putative “TGFαase” described in various other cell lines, and which does not seem dependent on protein kinase C (PKC) activity. J. Cell. Biochem. 64:353–368. © 1997 Wiley‐Liss, Inc.