TY - JOUR
T1 - Penicillin-binding proteins and peptidoglycan of Treponema pallidum subsp. pallidum
AU - Radolf, J. D.
AU - Moomaw, C.
AU - Slaughter, C. A.
AU - Norgard, M. V.
PY - 1989
Y1 - 1989
N2 - Penicillin-binding proteins (PBPs) of Treponema pallidum subsp. pallidum (T. pallidum) were characterized by using [3H]penicillin G and a conjugate consisting of ampicillin and 125I-labeled Bolton-Hunter reagent. Both antibiotics specifically rdiolabeled proteins with molecular masses of 94, 80, 63, and 58 kilodaltons (kDa); 125-labeled Bolton-Hunter reagent-ampicillin also radiolabeled several polypeptides, with lower molecular masses. The 94- and 58-kDa proteins demonstrated the highest binding affinities for [3H]penicillin G and were radiolabeled at concentrations of 8 and 40 nM, respectively. Radiolabeling of PBPs was detectable after 1 min of incubation in 1 μM [3H]penicillin G and was nearly maximal within 10 min. The rapidity of penicillin binding contrasted with the observation that only 40% of virulent treponemes became immobilized during prolonged incubation in vitro with a much higher concentration (1 mM) of unlabeled penicillin. Two lines of evidence indicated that most, if not all, of the PBPs are integral cytoplasmic membrane proteins: (i) preincubation of organisms in 0.1% Triton X-100 solubilized nearly all of the outer membranes but did not affect radiolabeling of PBPs, and (ii) except for the 80-kDa protein, the PBPs partitioned into the detergent phase following extraction with the nonionic detergent Triton X-114. The presence of peptidoglycan in T. pallidum was confirmed by the detection of muramic acid in the sodium dodecyl sulfate-insoluble, proteinase K-resistant residue obtained from Triton X-114-extracted organisms.
AB - Penicillin-binding proteins (PBPs) of Treponema pallidum subsp. pallidum (T. pallidum) were characterized by using [3H]penicillin G and a conjugate consisting of ampicillin and 125I-labeled Bolton-Hunter reagent. Both antibiotics specifically rdiolabeled proteins with molecular masses of 94, 80, 63, and 58 kilodaltons (kDa); 125-labeled Bolton-Hunter reagent-ampicillin also radiolabeled several polypeptides, with lower molecular masses. The 94- and 58-kDa proteins demonstrated the highest binding affinities for [3H]penicillin G and were radiolabeled at concentrations of 8 and 40 nM, respectively. Radiolabeling of PBPs was detectable after 1 min of incubation in 1 μM [3H]penicillin G and was nearly maximal within 10 min. The rapidity of penicillin binding contrasted with the observation that only 40% of virulent treponemes became immobilized during prolonged incubation in vitro with a much higher concentration (1 mM) of unlabeled penicillin. Two lines of evidence indicated that most, if not all, of the PBPs are integral cytoplasmic membrane proteins: (i) preincubation of organisms in 0.1% Triton X-100 solubilized nearly all of the outer membranes but did not affect radiolabeling of PBPs, and (ii) except for the 80-kDa protein, the PBPs partitioned into the detergent phase following extraction with the nonionic detergent Triton X-114. The presence of peptidoglycan in T. pallidum was confirmed by the detection of muramic acid in the sodium dodecyl sulfate-insoluble, proteinase K-resistant residue obtained from Triton X-114-extracted organisms.
UR - http://www.scopus.com/inward/record.url?scp=0024536136&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0024536136&partnerID=8YFLogxK
M3 - Article
C2 - 2647634
AN - SCOPUS:0024536136
SN - 0019-9567
VL - 57
SP - 1248
EP - 1254
JO - Infection and immunity
JF - Infection and immunity
IS - 4
ER -