Vesicle penicillinase of Bacillus licheniformis: existence of periplasmic-releasing factor(s)

L J Traficante, J O Lampen
1977 Journal of Bacteriology  
In earlier studies of the membrane-bound penicillinase ofBacillus licheniformis 749/C, the enzyme present in the vesicles that were released during protoplast fornation and the enzyme retained in the plasma membrane ofprotoplasts appeared to differ (i) in their behavior on gel permeation chromatography in the presence or absence of deoxycholate and (ii) in their tendency to convert to the hydrophilic exoenzyme (Sargent and Lampen, 1970) . We have now shown that these vesicle preparations
more » ... a soluble, heat-sensitive enzyme(s) that is released along with the vesicles during protoplast fornation. The enzyme will convert the vesicle penicillinase to a form that resembles exopenicillinase, and this conversion can be inhibited by deoxycholate under certain circumstances. Sedimentation of such vesicle preparations at 100,000 x g produces vesicles which contain penicillinase that behaves as the plasma membrane enzyme obtained from protoplasts. Exopenicillinases released by growing cells at pH 6.5 and by washed cells or protoplasts at pH 9.0 have the same NH2-terminal residues (lysine and some glutamic acid); in addition, the various release systems show a parallel sensitivity to inhibition by deoxycholate, quinacrine, chloroquine, and o-phenanthroline. The formation of exopenicillinase (by cleavage of the membrane-bound enzyme) may well be dependent on the action ofthe releasing enzyme. Bacillus licheniformis 749/C produces penicillinase (EC, penicillin amido-f3-lactamhydrolase) as an extracellular hydrophilic enzyme (9) and as a hydrophobic cell-bound form(s) that is located on the outer surface of the plasma membrane of the protoplast (8) and is also associated with vesicular membrane components ("periplasmic vesicles") believed to arise from mesosomes (12). Although penicillinase appears to occur in three distinct locations, there is only a single structural gene for the enzyme (4). Sargent and Lampen (15) converted exponential-phase cells to protoplasts with lysozyme, and solubilized penicillinase from the plasma membrane of the protoplast and from the vesicle fraction using 0.1% sodium deoxycholate (DOC) in conjunction with 0.05 M pyrophosphate buffer at pH 9.0 (DOC-PP). The extracts from the two fractions were compared by gel permeation chromatography. They reported that the two enzymes eluted with an apparent molecular weight of 45,000 on Bio-Gel A-5M in
doi:10.1128/jb.129.1.184-190.1977 fatcat:4e7dnjltird67edf27ubjvyufa