Localization of diphtheria toxin nuclease activity to fragment A

Abstract

We describe a series of experiments that aimed to establish whether nuclease activity is actually associated with diphtheria toxin (DTx) and its A subunit (DTA), as we originally reported (M. P. Chang, R. L. Baldwin, C. Bruce, and B. J. Wisnieski, Science 246:1165-1168, 1989). Here we show that (i) trypsinization of DTx does indeed produce nucleolytically active DTA, (ii) reduction of electroeluted, unreduced, cleaved DTx (58 kDa) yields nuclease-active DTA (24 kDa), and (iii) fractionation of DTx and DTA by anion-exchange chromatography leads to coelution of nuclease activity with both forms of the toxin, even though each form elutes at a distinct salt concentration. In addition, we show that Escherichia coli-derived DTA also expresses nuclease activity. These studies confirm our initial assertion that the nuclease activity observed in DTx preparations is intrinsic to the DTA portion of DTx.

Files

Additional details