Reconstitution of mycoplasma membranes

Abstract

Detergent-solubilized proteins and lipids of mycoplasma membranes reassemble spontaneously into membranous structures on t h e removal or dilution of the detergent in the presence of divalent cations. The cations seem t o function by neutralizing the negatively charged groups on membrane lipids and proteins which interfere by electrostatic repulsion with membrane reassembly. Moreover, salt bridges formed by the divalent cation between acidic groups on membrane proteins and lipids seem t o play an important role in the reconstituted membrane stability. Electron transport activity, as measured by the transport of electrons from NADH t o oxygen, has been demonstrated in reconstituted Acholeplasma laidlawii membranes. However, restoration of active transport of sugars or ions has not been achieved so far. The conditions for obtaining properly sealed vesicles, which are obligatory for demonstrating transport activity, are still rather poorly defined. The reassembled membranous structures cannot be distinguished from the native membranes in chemical composition, density, and thin sections. However, probe techniques, x-ray diffraction, and freeze-fracturing electron microscopy indicate that the proteins are organized differently in the reassembled membranes, though the lipid bilayer is restored. The results obtained so far leave little hope for successfully reconstituting the molecular organization of membranes as complex as those of mycoplasmas by a single-step reassembly of detergent-solubilized membrane components. The prospects appear brighter with membranes having only a few protein species, such as the outer membrane of gram-negative bacteria. In spite of the failure t o reconstitute fully active mycoplama membranes, the reassembly procedure was found valuable in studying the interactions of detergent-solubilized membrane proteins with lipids, the effects of a hydrophobic environment on hydrophilic enzymes, and the production of ''hybrid" membranes having selected membrane components.

The mycoplasmas.

“…The use of membrane reconstitution to study protein disposition has not been successful with mycoplasmas. Reconstituted membranes, produced from mycoplasma membrane material solubilized by sodium dodecyl sulfate or by bile salts, were found to differ radically from the native membranes in disposition of their protein components, apparently due to the denaturation of the proteins by the detergents (215,236,238).…”

Reduced nicotinamide adenine dinucleotide oxidase activity in membranes and cytoplasm of Acholeplasma laidlawii and Mycoplasma mycoides subsp. capri

“…capri could be incorporated into reaggregated membranes, but to a much tower extent than the corresponding membrane-bound enzyme system of A. laidlawii. The fact that the susceptibility to heat inactivation of the reaggregated enzyme systems was still higher than that of the native systems may be explained by residual detergent and/or by different molecular organization of the reaggregated membranes (11).…”