Proteolytic and chemical modification of colicin E3 activity.
Proteolyses of colicin E3 by both trypsin and subtilisin yield fragments of various molecular weights. On the basis of sodium dodecyl sulfate gel electrophoresis, tryptic cleavage yields peptides of molecular weight about 42 000 and 18 000, while the comparable pieces in a subtilisin digest have apparent weights of about 36 000 and 24 000. The digests lose almost all of their in vivo cell killing activity but the in vitro activity leading to ribosomal inactivation is augmented. Trypsin-treated colicin E3 shows a 20-30-fold increase in its ability to release the 52 nucleotide fragment from the 16S ribosomal ribonucleic acid (rRNA) and this activity is associated with the smaller fragment. Subtilisin-treated colicin E3 is only about two to three fold more active than the native protein in vitro, and the peptides obtained upon cleavage cannot be separated by gel filtration or polyacrylamide gel electrophoresis without sodium dodecyl sulfate. However, in the presence of 0.1% sodium dodecyl sulfate, subtilisin-treated E3 shows a 20-30-fold augmentation in in vitro activity which is again associated with the smaller fragment extracted from the sodium dodecyl sulfate gel. Amino terminal end-group studies showed that the two larger fragments and intact E3 have the same N-terminal residue, valine. These fragments presumably originate from the amino end of the native protein. The smaller tryptic fragment has an N-terminal alanine, while the smaller subtilisin piece has an N-terminal leucine. In addition, modification of a single carbosyl group in intact colicin E3 abolishes more than 90% of the in vivo activity with a simultaneous increase in in vitro activity. This carboxyl group is located in the larger fragments obtained in both trypsin and subtilisin cleavage. Binding of E3 to sensitive cells is drastically reduced or eliniated by this chemical modification and by both of the limited proteolytic cleavages.
D000596:Amino Acids / Q000032:analysis; D002264:Carboxylic Acids; D003087:Colicins / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000494:pharmacology; D011485:Protein Binding; D012335:RNA, Ribosomal / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013381:Subtilisins; D014357:Trypsin
D000596:Amino Acids; D002264:Carboxylic Acids; D003087:Colicins; D010446:Peptide Fragments; D012335:RNA, Ribosomal; D012967:Sodium Dodecyl Sulfate; D013381:Subtilisins; D014357:Trypsin