Penicillin-binding proteins of Streptococcus pneumoniae: characterization of tryptic peptides containing the beta-lactam-binding site

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Abstract
Penicillin-binding proteins of S. 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 [kilodalton]), PBP 2b (77 kDa) and PBP 3 (43 kDa) had sizes of 36.5, 26 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 and 1.5 kDa, respectively. These 2 PBP are therefore anchored in the membrane by a small terminal peptide. PBP 1a could be digested to a MW of 48,000 without becoming water-soluble; the only hydrophilic tryptic peptide that could be found was the 36.5 kDa fragment. Thus, large domains of this PBP seem to be embedded in the membrane.
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