Biological characterization of a new radioactive labeling reagent for bacterial penicillin-binding proteins

Preston, David A.; Wu, C. Y. E.; Blaszczak, Larry C.; Seitz, David E.; Halligan, Noreen G.

doi:10.1128/aac.34.5.718

Cited by 56 publications

(43 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The binding of penicillin to purified PBP2x enzymes was measured as described previously (38) with 125 I-labeled penicillin V (5). For the determination of binding constants of S-PBP2x and R-PBP2x enzymes for 125 I-labeled penicillin V and of 50% inhibitory concentrations of other antibiotics, 0.2 g of each purified S-PBP2x and R-PBP2x enzyme was used.…”

Section: Methodsmentioning

confidence: 99%

Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics

et al. 1997

View full text Add to dashboard Cite

To understand the biochemical basis of resistance of bacteria to ␤-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k cat /K m values for the two purified enzymes (3,400 M ؊1 s ؊1 for S-PBP2x and 11.2 M ؊1 s ؊1 for R-PBP2x) suggests that they are significantly different kinetically. Implications of this finding are discussed. We also found that the two purified enzymes did not possess a detectable level of ␤-lactam hydrolytic activity. Finally, we show that the expression levels of both PBP2x enzymes were similar during different growth phases.

show abstract

Section: Methodsmentioning

confidence: 99%

Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics

et al. 1997

View full text Add to dashboard Cite

show abstract

“…PBPs were radiolabelled by a modification of the method of Spratt (1977), as described by Preston et al (1990) using 1251-labelled penicillin V as the labelled p-lactam (Blaszczak & Halligan, 1988). Thep-(trimethylstanny1)penicillin V was kindly provided by Larry C. Blaszczak (Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, IN, USA), and was iodinated by using Nal 251 from Amersham.…”

Section: Labelling Of Penicillin-binding Proteins (Pbps)mentioning

confidence: 99%

Effects of antibiotics on the growth and morphology of Pasteurella multocida

Jacques

Lebrun

Foiry

et al. 1991

Journal of General Microbiology

View full text Add to dashboard Cite

The effects of subminimal inhibitory concentrations (subMICs) of certain antibiotics, namely penicillin G, tetracycline and trimethoprim/sulphamethoxazole, on the growth and morphology of Pasteurella multocida were evaluated. SubMICs of penicillin markedly reduced the growth of P. multocida. Tetracycline and trimethoprim/ sulphamethoxazole had no effect on its growth. SubMICs of penicillin greatly affected the morphology of P. multocida. At the highest concentrations tested (1/2 and 1/4 MIC) cells were acapsulate, and long filamentous cells (4-6 pm) were observed with some isolates. There was no correlation between the observed differences in the penicillin-binding proteins of the P. multocida isolates, and the extent of cell filamentation induced by penicillin G. SubMICs of tetracycline and trimethoprim/sulphamethoxazole did not seem to affect capsule production although filamentation was observed. Our results indicate that subMICs of penicillin can reduce growth of P. multocida. Furthermore, results also indicate that subMICs of antibiotics can affect the production of capsular material and the morphology of P. multocida.

show abstract

“…PBPs of T. pallidum were identified by using '2iodine-labeled penicillin V as follows. Sodium p-(trimethylstannyl)phenoxyacetamidopenicillin, kindly provided by Larry C. Blaszczak, Lilly Laboratories, Eli Lilly and Company, Indianapolis, Ind., was labeled with Na125I by using chloramine-T as previously described (41). Equal volumes of 125I-penicillin V were combined with 1 X 108 Ficollpurified treponemes, 1 X 108 protoplasmic cylinders, and outer membrane derived from 5 x 10 treponemes.…”

mentioning

confidence: 99%

Isolation of the outer membranes from Treponema pallidum and Treponema vincentii

et al. 1994

View full text Add to dashboard Cite

The outer membranes from Treponema pallidum subsp. pa~idum and Treponema vincentii were isolated by a novel method. Purified outer membranes from T. palidum and T. vincentii following sucrose gradient centrifugation banded at 7 and 31% (wt/wt) sucrose, respectively. Freeze fracture electron microscopy of purified membrane vesicles from T. pallidum and T. vincentii revealed an extremel low density of protein particles; the particle density of T. pallidum was approximately six times less than that of T. vincentii. The great majority of T. vincentii lipopolysaccharide was found in the outer membrane preparation. The T. vincentii outer membrane also contained proteins of 55 and 65 kDa. 1251-penicillin V labeling demonstrated that T. pallidum penicillin-binding proteins were found exclusively with the protoplasmic cylinders and were not detectable with purified outer membrane material, indicating the absence of inner membrane contamination. Isolated T.pallidum outer membrane was devoid of the 19-kDa 4D protein and the normally abundant 47-kDa lipoprotein known to be associated with the cytoplasmic membrane; only trace amounts of the periplasmic endoflagella were detected. Proteins associated with the T. pallidum outer membrane were identified by one-and two-dimensional electrophoretic analysis using gold staining and immunoblotting. Small amounts of strongly antigenic 17-and 45-kDa proteins were detected and shown to correspond to previously identified lipoproteins which are found principally with the cytoplasmic membrane. Less antigenic proteins of 65, 31 (acidic pl), 31 (basic pl), and 28 kDa were identified. Compared with whole-organism preparations, the 65-and the more basic 31-kDa proteins were found to be highly enriched in the outer membrane preparation, indicating that they may represent the T. pallidum rare outer membrane proteins. Reconstitution of solubilized T. paUidum outer membrane into lipid bilayer membranes revealed porin activity with two estimated channel diameters of 0.35 and 0.68 nm based on the measured single-channel conductances in 1 M KCI of 0.40 and 0.76 nS, respectively.The outer membranes of spirochetes, including that of Treponema pallidum subsp. pallidum, the agent of syphilis, are fragile compared with those of typical gram-negative bacteria (17,22,24,39,42,49). Spirochetal outer membranes bleb from the underlying protoplasmic cylinder under relatively mild conditions, including dilute detergents and hypotonic environments (24). Freeze fracture electron microscopy has revealed that the outer membranes of pathogenic spirochetes contain amounts of integral membrane protein that are 1 to 2 orders of magnitude less than those of gram-negative bacteria (45,54,55). This striking feature has been suggested to be a key factor in the pathogenicity of these organisms by contributing to their ability to evade the host immune response and to establish chronic infection (11,15,38,45,54,55).The outer membrane of T. pallidum has an extremely low content of membrane-spanning protein (45, 55), a finding...

show abstract

Biological characterization of a new radioactive labeling reagent for bacterial penicillin-binding proteins

Abstract: Radiolabeled penicillin G is widely used as the imaging agent in penicillin-binding protein (PBP)

Cited by 56 publications

References 6 publications

Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics

Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics

Effects of antibiotics on the growth and morphology of Pasteurella multocida

Isolation of the outer membranes from Treponema pallidum and Treponema vincentii

Contact Info

Product

Resources

About