Fractionation of the Providencia stuartii Cell Envelope

SUMMARY: Cell envelopes (i.e. unfractionated inner and outer membranes) were obtained from Providencia stuartii by following procedures previously applied to the isolation of envelopes from Escherichia coli. The P. stuartii envelopes contained known inner membrane enzymes that included a variety of dehydrogenases and ATPase. The catalytic activity of the ATPase depended upon the concentration of magnesium ions, the substrate (ATP) level and the ratio of magnesium ions to ATP. Cell envelopes from P. stuartii were further fractionated to recover inner and outer membrane polypeptides by treatment with the detergent Sarkosyl. Proteins from the periplasmic region were recovered by a simple osmotic shock procedure also previously applied to E. coli. The purity of the various P. stuartii cell envelope fractions was assessed by a combination of techniques that included one- and two-dimensional gel electrophoresis of proteins, enzyme assays and detection of penicillin-binding proteins.