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. 1972 Dec;69(12):3751–3755. doi: 10.1073/pnas.69.12.3751

Isolation by Covalent Affinity Chromatography of the Penicillin-Binding Components from Membranes of Bacillus subtilis

Peter M Blumberg 1, Jack L Strominger 1
PMCID: PMC389864  PMID: 4630162

Abstract

An affinity chromatography technique was developed to isolate the five penicillin-binding components present in Bacillus subtilis membranes. The proteins were solubilized by the detergent Nonidet P-40, bound covalently to penicillin-substituted Sepharose, and subsequently eluted from the matrix with neutral hydroxylamine, which cleaves the penicilloyl-enzyme bond. Penicillin binding-component V, the D-alanine carboxypeptidase, makes up 1% of the total membrane protein. A modification of the above procedure enabled this enzyme to be obtained from the membrane in pure form in a single step with 50% overall recovery of enzymatic activity.

Keywords: D-alanine carboxypeptidase, transpeptidase, detergent solubilization

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Araki Y., Shirai R., Shimada A., Ishimoto N., Ito E. Enzymatic synthesis of cell wall mucopeptide in a particulate preparation of Escherichia coli. Biochem Biophys Res Commun. 1966 May 25;23(4):466–472. doi: 10.1016/0006-291x(66)90751-0. [DOI] [PubMed] [Google Scholar]
  2. Ashani Y., Wilson I. B. A covalent affinity column for the purification of acetylcholinesterase. Biochim Biophys Acta. 1972 Jul 13;276(1):317–322. doi: 10.1016/0005-2744(72)90034-4. [DOI] [PubMed] [Google Scholar]
  3. Blumberg P. M., Strominger J. L. Inactivation of D-alanine carboxypeptidase by penicillins and cephalosporins is not lethal in Bacillus subtilis. Proc Natl Acad Sci U S A. 1971 Nov;68(11):2814–2817. doi: 10.1073/pnas.68.11.2814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cuatrecasas P. Affinity chromatography. Annu Rev Biochem. 1971;40:259–278. doi: 10.1146/annurev.bi.40.070171.001355. [DOI] [PubMed] [Google Scholar]
  5. Cuatrecasas P., Parikh I. Adsorbents for affinity chromatography. Use of N-hydroxysuccinimide esters of agarose. Biochemistry. 1972 Jun 6;11(12):2291–2299. doi: 10.1021/bi00762a013. [DOI] [PubMed] [Google Scholar]
  6. EAGLE H., LEVY M., FLEISCHMAN R. The binding of penicillin in relation to its cytotoxic action. IV. The amounts bound by bacteria at ineffective, growth-inhibitory, bactericidal, and maximally effective concentrations. J Bacteriol. 1955 Feb;69(2):167–172. doi: 10.1128/jb.69.2.167-172.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. EAGLE H. The binding of penicillin in relation to its cytotoxic action. I. Correlation between the penicillin sensitivity and combining activity of intact bacteria and cell-free extracts. J Exp Med. 1954 Mar;99(3):207–226. doi: 10.1084/jem.99.3.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. EAGLE H. The multiple mechanisms of penicillin resistance. J Bacteriol. 1954 Nov;68(5):610–616. doi: 10.1128/jb.68.5.610-616.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Edwards J. R., Park J. T. Correlation between growth inhibition and the binding of various penicillins and cephalosporins to Staphylococcus aureus. J Bacteriol. 1969 Aug;99(2):459–462. doi: 10.1128/jb.99.2.459-462.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Izaki K., Matsuhashi M., Strominger J. L. Biosynthesis of the peptidoglycan of bacterial cell walls. 8. Peptidoglycan transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reaction in strains of Escherichia coli. J Biol Chem. 1968 Jun 10;243(11):3180–3192. [PubMed] [Google Scholar]
  11. Izaki K., Matsuhashi M., Strominger J. L. Glycopeptide transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reactions. Proc Natl Acad Sci U S A. 1966 Mar;55(3):656–663. doi: 10.1073/pnas.55.3.656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Izaki K., Strominger J. L. Biosynthesis of the peptidoglycan of bacterial cell walls. XIV. Purification and properties of two D-alanine carboxypeptidases from Escherichia coli. J Biol Chem. 1968 Jun 10;243(11):3193–3201. [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Lawrence P. J., Strominger J. L. Biosynthesis of the peptidoglycan of bacterial cell walls. XV. The binding of radioactive penicillin to the particulate enzyme preparation of Bacillus subtilis and its reversal with hydroxylamine or thiols. J Biol Chem. 1970 Jul 25;245(14):3653–3659. [PubMed] [Google Scholar]
  15. Lawrence P. J., Strominger J. L. Biosynthesis of the peptidoglycan of bacterial cell walls. XVI. The reversible fixation of radioactive penicillin G to the D-alanine carboxypeptidase of Bacillus subtilis. J Biol Chem. 1970 Jul 25;245(14):3660–3666. [PubMed] [Google Scholar]
  16. Leyh-Bouille M., Nakel M., Frère J. M., Johnson K., Ghuysen J. M., Nieto M., Perkins H. R. Penicillin-sensitive DD-carboxypeptidases from Streptomyces strains R39 and K11. Biochemistry. 1972 Mar 28;11(7):1290–1298. doi: 10.1021/bi00757a027. [DOI] [PubMed] [Google Scholar]
  17. Pollock J. J., Ghuysen J. M., Linder R., Salton M. R., Perkins H. R., Nieto M., Leyh-Bouille M., Frere J. M., Johnson K. Transpeptidase activity of Streptomyces D-alanyl-D carboxypeptidases. Proc Natl Acad Sci U S A. 1972 Mar;69(3):662–666. doi: 10.1073/pnas.69.3.662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rowley D., Cooper P. D., Roberts P. W. The site of action of penicillin. 1. Uptake of penicillin on bacteria. Biochem J. 1950 Feb;46(2):157–161. doi: 10.1042/bj0460157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Strominger J. L., Blumberg P. M., Suginaka H., Umbreit J., Wickus G. G. How penicillin kills bacteria: progress and problems. Proc R Soc Lond B Biol Sci. 1971 Dec 31;179(1057):369–383. doi: 10.1098/rspb.1971.0103. [DOI] [PubMed] [Google Scholar]
  20. Suginaka H., Blumberg P. M., Strominger J. L. Multiple penicillin-binding components in Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Escherichia coli. J Biol Chem. 1972 Sep 10;247(17):5279–5288. [PubMed] [Google Scholar]
  21. Tipper D. J., Strominger J. L. Biosynthesis of the peptidoglycan of bacterial cell walls. XII. Inhibition of cross-linking by penicillins and cephalosporins: studies in Staphylococcus aureus in vivo. J Biol Chem. 1968 Jun 10;243(11):3169–3179. [PubMed] [Google Scholar]
  22. Tipper D. J., Strominger J. L. Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-D-alanyl-D-alanine. Proc Natl Acad Sci U S A. 1965 Oct;54(4):1133–1141. doi: 10.1073/pnas.54.4.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  24. Wickus G. G., Strominger J. L. Penicillin-sensitive transpeptidation during peptidoglycan biosynthesis in cell-free preparations from Bacillus megaterium. II. Effect of penicillins and cephalosporins on bacterial growth and in vitro transpeptidation. J Biol Chem. 1972 Sep 10;247(17):5307–5311. [PubMed] [Google Scholar]
  25. Wise E. M., Jr, Park J. T. Penicillin: its basic site of action as an inhibitor of a peptide cross-linking reaction in cell wall mucopeptide synthesis. Proc Natl Acad Sci U S A. 1965 Jul;54(1):75–81. doi: 10.1073/pnas.54.1.75. [DOI] [PMC free article] [PubMed] [Google Scholar]

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