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Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
14:58

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Published on: November 12, 2012

Purification and properties of penicillin-binding proteins 5 and 6 from Escherichia coli membranes

H Amanuma, J L Strominger

    The Journal of Biological Chemistry
    |December 10, 1980
    PubMed

    Insights

    Penicillin-binding proteins (PBPs) 5 and 6 from Escherichia coli were purified and found to possess distinct D-alanine carboxypeptidase I and transpeptidase activities. These purified PBPs are separate proteins, highlighting their unique roles in bacterial cell wall synthesis.

    Area of Science:

    • Microbiology
    • Biochemistry
    • Molecular Biology

    Background:

    • Penicillin-binding proteins (PBPs) are crucial enzymes involved in bacterial cell wall synthesis.
    • PBPs 5 and 6 from Escherichia coli K12 were previously identified as a co-purified penicillin-sensitive D-alanine carboxypeptidase IA complex.

    Purpose of the Study:

    • To purify Penicillin-binding proteins (PBPs) 5 and 6 to homogeneity.
    • To characterize the enzymatic activities and biochemical properties of purified PBP 5 and PBP 6.
    • To confirm the distinct polypeptide nature of PBP 5 and PBP 6.

    Main Methods:

    • Selective membrane solubilization using Triton X-100.
    • Covalent penicillin affinity chromatography.
    • CM-cellulose column chromatography for protein purification.
    • Enzymatic assays for D-alanine carboxypeptidase I and transpeptidase activities.
    • Peptide mapping using SDS-PAGE, proteolysis, and cyanogen bromide treatment.

    Main Results:

    • Purified PBP 5 and PBP 6 exhibited D-alanine carboxypeptidase I activity on various substrates, with PBP 5 showing higher specific activity.
    • Both PBPs catalyzed a model transpeptidase activity and displayed similar pH profiles and MgCl2 sensitivities.
    • Stoichiometric binding of [14C]penicillin G was observed for both purified PBPs.
    • Peptide mapping confirmed that PBP 5 and PBP 6 are distinct polypeptides.

    Conclusions:

    • Penicillin-binding proteins (PBPs) 5 and 6 are separate enzymes with distinct D-alanine carboxypeptidase I and transpeptidase activities.
    • These findings elucidate the individual biochemical functions of PBP 5 and PBP 6 in Escherichia coli.
    • The distinct nature of PBP 5 and PBP 6 suggests specialized roles in peptidoglycan metabolism and cell wall integrity.

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