Localization of diphtheria toxin nuclease activity to fragment A.
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.
Lessnick
S L
SL
Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Los Angeles 90024.
Lyczak
J B
JB
Bruce
C
C
Lewis
D G
DG
Kim
P S
PS
Stolowitz
M L
ML
Hood
L
L
Wisnieski
B J
BJ
D002852:Chromatography, Ion Exchange; D003851:Deoxyribonucleases / Q000737:chemistry*; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D004167:Diphtheria Toxin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003851:Deoxyribonucleases