Penicillin-binding proteins of Streptococcus pneumoniae: characterization of tryptic peptides containing the beta-lactam-binding site.
Penicillin-binding proteins of Streptococcus pneumoniae were labeled with [3H] propionyl-ampicillin and treated with trypsin. The fragments were separated on sodium dodecyl sulfate/polyacrylamide gels, and peptides containing the beta-lactam-binding site visualized by fluorography. From native penicillin-binding proteins (PBP), either membrane-bound or solubilized with Triton X-100, relatively stable end products of proteolysis were obtained. The smallest radioactive peptides from PBP 1a (92 kDa), PBP 2b (77 kDa), and PBP 3 (43 kDa ) had sizes of 36.5 kDa, 26 kDa, and 29 kDa, respectively. When the PBP were trypsin treated prior to labeling with the radioactive beta-lactam, these small peptides were still able to bind the antibiotic. Under conditions of limited proteolysis, membrane-bound PBP 2b and PBP 3 were converted into soluble, hydrophilic derivatives after loss of a peptide of only 2 kDa and 1.5 kDa, respectively. These two PBP are therefore anchored in the membrane by a small terminal peptide. In contrast, PBP 1a could be digested to a Mr of 48000 without becoming water-soluble; the only hydrophilic tryptic peptide that could be found was the 36.5 kDa fragment. Therefore, large domains of this PBP seem to be embedded in the membrane.
D000667:Ampicillin / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001426:Bacterial Proteins; D001665:Binding Sites; D002268:Carboxypeptidases / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D005470:Fluorometry; D006602:Hexosyltransferases; D008970:Molecular Weight; D002267:Muramoylpentapeptide Carboxypeptidase / Q000032:analysis* / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D010458:Peptidyl Transferases; D013296:Streptococcus pneumoniae / Q000032:analysis*; D014357:Trypsin / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010446:Peptide Fragments; D000667:Ampicillin; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002268:Carboxypeptidases; D002267:Muramoylpentapeptide Carboxypeptidase; D014357:Trypsin