Effects of Divalent Cations and Polyethylene Glycol on the Membrane Fluidity of Protoplast

Abstract

Calcium is often used to stabilize membranes and enhance membrane fusion. The fatty acid spin label, 5-nitroxy stearic acid was used to measure fluidity changes in the plasma membrane of carrot [Daucus carota] suspension culture cell protoplasts in response to divalent cations. Electron spin resonance spectra from spin-labeled protoplasts showed no membrane fluidity changes (as determined by the hyperfine splitting constant, 2Amax) in the presence of Mg from 0-10 mM or Ca from 0-5 mM. Protoplasts in 10 mM Ca showed a dramatic increase of 5 gauss in 2Amax and evidence of exchange-broadening. The original (control) spectrum was regained by removing bound Ca with a Ca chelator. Polyethylene glycol, which enhances protoplast fusion, did not alter the membrane fluidity in the region of the 5-nitroxy stearic acid probe if added simultaneously with or following 10 mM Ca. Pretreatment with polyethylene glycol did inhibit the Ca-induced phase separation. These data on a living system describe membrane structural changes under conditions similar to those used for protoplast fusion.