FIG 3 .

SLO prevents proteolytic cleavage of NADase. (A) Western blot using antiserum to NADase revealed two distinct bands in culture supernatant of a mutant strain deficient in SLO production (Δslo) but not in supernatants from the wild type or a mutant producing NADase G330D. (B) NADase enzymatic activity associated with the Δslo mutant was not significantly different from that of wild-type 854 as determined by one-way analysis of variance with Tukey’s posttest. (C) NADase cleavage in the Δslo mutant is zinc dependent; it is inhibited by EDTA and enhanced by the addition of ZnCl₂ (1 mM) but not MgCl₂ (1 mM) or MnCl₂ (1 mM). Experiments were performed three times, and where appropriate, the mean ± standard deviation is represented (U, untreated; PI, protease inhibitor). (D) The NADase cleavage site in the Δslo mutant was determined by extraction of full-length and processed NADase from SDS-PAGE and analysis by tryptic digest and LC–MS-MS for fragment identification. The processing site was found to be between amino acids T53 and K54 (vertical arrow). The region of the protein identified in the full-length NADase but not the processed form is highlighted in red. Underlining denotes peptides identified in the analysis for both forms of NADase.