Preparation of Everted Membrane Vesicles from Escherichia Coli for the Measurement of Calcium Transport Article

Rosen, BP, Tsuchiya, T. (1979). Preparation of Everted Membrane Vesicles from Escherichia Coli for the Measurement of Calcium Transport . METHODS IN ENZYMOLOGY, 56(C), 233-241. 10.1016/0076-6879(79)56026-1

cited authors

  • Rosen, BP; Tsuchiya, T

authors

abstract

  • This chapter discusses number of methods that are available for the isolation of the cytoplasmic or inner membrane of Escherichia coli. With each procedure, it is necessary to consider the orientation of the membrane of the vesicles relative to that of the inner membrane in vivo. In this chapter, the term “orientation” refers to the localization of membrane proteins, as determined by a number of means. For example, if a substrate of an enzymatically catalyzed reaction is converted to product only when it is in contact with the cytoplasmic side of the membrane, then the membrane-associated enzyme responsible for that catalysis is considered to be localized on the cytoplasmic side of the membrane. In fact, that is a functional definition for the location of the active site of the enzyme; it is possible and often probable that a protein or complex of proteins spans the membrane. In such a case, the protein might be “localized” on either side of the membrane depending on what reagent was used for the localization. It is frequently difficult to make absolute determinations, but there are instances in which active sites, antigenic sites or morphologically identifiable structures have been localized on either the cytoplasmic or the periplasmic side of the inner membrane. By such criteria, there have been attempts to categorize in vitro vesicle preparations into four specific types: fight-side-out, everted, hybrid, and mosaic. The two most commonly used methods for the preparation of membrane vesicles from E. coli utilize (1) osmotic lysis of spheroplasts or (2) rupture of intact cells by extrusion at high pressure through a narrow aperture using a French press. © 1979, Elsevier Inc. All rights reserved.

publication date

  • January 1, 1979

published in

Digital Object Identifier (DOI)

start page

  • 233

end page

  • 241

volume

  • 56

issue

  • C