Membrane Hybridization by Centrifugation Analysed by Lipid Phase Transitions and Reconstitution of NADH-Oxidase-Activity

Abstract

A procedure is described to hybridize cytoplasmic membranes of Escherichia coli. The method involves high-speed contrifugation of two different vesicle preparations at 37 degrees C in the presence of cations such as spermine or Mg2+. The occurrence of hybridization is shown by the following experiments. Firstly, formation of a mixed lipis phase starting from two membranes having a different hydrocarbon chain composition in their phospholipids. Secondly, formation of a hybrid membrane having an intermediate lipid to protein ratio from two membrane fractions having different lipid to protein ratios. Thirdly, reconstitution of NADH oxidase activity by hybridization of two membrane fractions, one lacking active cytochromes and the other being deficient in quinones. It is proposed that hybridization occurs by fusion of vesicles during the tight association of collapsed vesicles under high centrifugal forces. This interpretation is supported by electron microscopy of the membrane pellets after centrifugation. However, lipid transfer as the mechanism of hybridization cannot be excluded and attempts to reconstitute active galactoside transport by complementation of beta-galactoside transport-deficient membranes and cytochrome-deficient membranes have been unsuccessful.