UVC ACTIVATION OF THE HELA-CELL MEMBRANE TGF-ALPHA-ASE, A METALLOENZYME

We have investigated the effect of UVC irradiation on ''TGF alpha ase' ' activity using both intact HeLa cells and isolated membrane fragment s with an assay based on the previously described nonapeptide substrat e 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 TGF alpha N-terminal cleavage site from its membrane-bound pre cursor. The level of ectoendopeptidase (including ''TGF alpha ase'') a ctivity observed on intact cells was lower than that of ectoaminopepti dases. Addition of foetal bovine serum (FBS) enhanced aminopeptidase a nd dipeptidyl peptidase activity but inhibited ''TGF alpha ase'' activ ity, while phorbol 12-myristate 13-acetate (PMA) had no significant ef fect on the ectopeptidases monitored, except for ''TGF alpha ase,'' wh ich was also inhibited, in contradistinction to their effects in other cell systems. Sublethal UVC irradiation (10 Jm(-2)) Of the cultures r esulted 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 incr ease in endopeptidase products, due in part to increased aminopeptidas e activity but also to the direct inhibitory effect of FBS on the ''TG F alpha ase.'' 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 pre pared from suspension cultures of HeLa cells and displayed high levels of ''TGF alpha ase'' activity. The rate of ''TGF alpha ase'' activity using 140 nM peptide substrate (P9) was 5.6 pmol/min/mg membrane prot ein, which was elevated to 13.7 pmol/min/mg membrane protein, 20 h aft er the cells had been irradiated with 10 Jm(-2) UVC. Inhibition studie s indicate that the plasma membrane ''TGF alpha ase'' is a metalloenzy me as it was inhibited by EDTA, EGTA, and 1,10-phenanthroline but not by elastase or serine protease inhibitors. ''TGF alpha ase'' activity on intact cells was shown to be inhibited by 1,10-phenanthroline, whic h further supports this suggestion. Treatment of the membranes with Tr iton X-100 resulted in a loss of ''TGF alpha ase'' activity, raising t he possibility that this enzyme may require a cofactor to be fully fun ctional. We show that in purified membrane preparations of HeLa cells there is evidence for the presence of a ''TGF alpha ase'' which can be activated by UV irradiation, which differs from the putative ''TGF al pha ase'' described in various other cell lines, and which does not se em dependent on protein kinase C (PKC) activity. (C) 1997 Wiley-Liss, Inc.