Organization of the membrane-bound penicillinases of Bacillus licheniformis

Abstract

Previously we have shown that cell-bound penicillinase of Bacillus licheniformis strain 749/C is found in the plasma membrane and in a membranous vesicle fraction, and also that vesicle fraction penicillinase was a precursor of at least a part of the secreted enzyme whereas the plasma-membrane penicillinase was not available for secretion. We have therefore undertaken an investigation of the penicillinase from these two cell fractions and compared them with the exo-form of the enzyme in order to reveal any changes in structure or conformation that may have occurred during secretion. Both forms of penicillinase have been solubilized using sodium deoxycholate (DOC) or sodium taurocholate (STC) in the presence of pyrophosphate at pH 9.0. Pyrophosphate apparently acts as a chelating agent as it can be replaced by other chelating agents, and reassociation with membrane components occurs on addition of magnesium ions to DOC-solubilizates from which DOC has been removed. After solubilization with either DOC or STC, plasma-membrane penicillinase elutes on Biogel A5M in 0.05 m NaCl with an apparent molecular weight of 600,000, but when chromatographed in the presence of DOC, penicillinase is depolymerized reversibly into a 45,000 molecular weight form. STC is unable to depolymerize the molecule. Vesicle fraction penicillinase behaves in a similar manner in the presence of DOC, eluting with a molecular weight of 45,000, but on removal of DOC from this fraction, the penicillinase elutes with a molecular weight of 24,000. However, if STC or DOC-solubilized vesicle fraction is chromatographed on Biogel A5M in 0.05 m NaCl directly, two peaks are obtained at molecular weights of 24,000 and 210,000. We consider the latter to be an artifact resulting from interaction of the 24,000 form with contaminating proteins. The behaviour of these two forms of penicillinase contrasts strongly with that of exopenicillinase which elutes at a molecular weight of 24,000 in the presence and absence of DOC. The gel filtration behaviour of four other proteins is unaffected by the presence or absence of DOC. Our interpretation of these data is that vesicle fraction penicillinase is a conformational variant of the exo-form that has a hydrophobic surface capable of reversibly binding DOC to give a molecule of 45,000 molecular weight. The plasma-membrane penicillinase we regard as an aggregate composed of monomers that are similar to the vesicle fraction penicillinase in that they bind DOC, but with a conformation of greater hydrophobicity so that they polymerize in the absence of DOC. Highly cross-linked, gel-filtration media and sucrose cause aggregation of the vesicle fraction monomer to a form which is excluded by Biogel A5M. It can subsequently be depolymerized by bile salts.