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. We have used the fatty acid spin label, 5-nitroxy steanc acid to measure fluidity changes in the plasma membrane of carrot suspension culture celi 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, 2Am.) in the presence of Mg from 0 to 10 millimolar or Ca from 0 to 5 millinolar. Protoplasts in 10 millimolar Ca, however, showed a dramatic increase of 5 gauss in 2Am.a and evidence ofexchange-broadening. The original (control) spectrum was regained by removing bound Ca with a Ca chelator. Polyeth-ylene 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 millimolar Ca. Pretreatment with polyethylene glycol did, however, inhibit the Ca-induced phase separation. These data on a lving system describe membrane structural changes under conditions similar to those used for protoplast fusion. ESR2 has played an important role in the understanding of molecular motion of lipids in model and biological membrane systems (2, 4, 17). We chose the fatty acid spin label 5-NS to measure fluidity changes of the plasma membrane of carrot suspension culture cell protoplasts. The 5-NS probe has the nitroxide moiety on the fifth carbon from the carboxy terminus of stearic acid and the probe intercalates into the membrane bilayer so that the nitroxide is located in the region of the glycerol backbone of phospholipids (4, 15). The ESR signal from the probe is thus sensitive to perturbations near the surface of the bilayer. Ca stabilizes membranes and enhances membrane fusion in both artificial and biological systems (3, 7, 14). Ca binds to the negatively charged phospholipids near the membrane surface resulting in altered membrane fluidity and phase separation of the bilayer (11-13). Reported here are the effects of Ca on membrane fluidity of living carrot protoplasts as determined through ESR using the 5-NS probe. MATERIALS AND METHODS Protoplast Isolation. A cell suspension culture of carrot, Daucus carota L., was maintained as previously described (1). Protoplaststetraacetic acid; G, gauss; 2Am81, maximum hyperfme splitting constant. were released enzymically from cells in the exponential phase of growth (3 to 4 days after transfer) (1). The protoplasts were filtered through glass wool and washed once by centrifugation (40g, 5 min) through 1% (w/v) dextran in osmoticum A, which consisted of 0.45 molal sorbitol and 0.5 millimolal Mes (pH 5.5). Debris was further removed by three additional washes in osmoticum A without dextran. Spin-Labeling of Protoplasts. A freshly prepared stock solution of 5-NS spin-label probe (SYVA Co. Palo Alto, CA) in methanol (1.0 mg/0.1 ml) was kept in a darkened tube on ice during an experiment. Aliquots (4 ,ul) were pipetted into 10-ml glass tubes; 0.1 ml settled volume of protoplasts (approximately 105 proto-plasts) was added, and the tube was tapped gently to mix the protoplasts and the spin-label. Labeled protoplasts were immediately drawn up into a quartz capillary (1 mm i.d., Wilmad Glass Co., Buena, N. J.). and the ESR spectrum was recorded. The protoplasts then were expelled from the capillary and washed once by centrifugation in an appropriate osmoticum with varying treatment (Table I) and rescanned. At least three replicate scans were made after each treatment. The sorbitol concentration of the treatment osmoticum was adjusted so that the osmolality remained the same in each treatment, except in the case of PEG. Protoplasts were gently manipulated at each stem to minimize breakage, and the 40g centrifugation during the wash removed suspended debris. The spectra were recorded on a JEOL MES-3X spectrometer at 2 mw power, 50-to 100-G scan width, 0.3-to 1.0-s response, 5-to 10-min scan time, modulation frequency of 2 G, and a variable gain. The 2Amaz values were based on Tempo as a standard. RESULTS Effect of Ca and Mg on Membrane Fluidity. A typical first-derivative spectrum of carrot suspension culture cell protoplasts labeled with 5-NS is shown in Figure I (control). Protoplasts released and/or washed in the presence of 0.5 mm Ca(H2PO4)2 or CaCl2 gave an ESR spectrum identical to those in osmoticum A without Ca added. Even at 5.0 mm Ca, there was no change in membrane fluidity as determined by 2Am.2 of the 5-NS spin label (Table I). However, when spin-labeled protoplasts were washed in 10 mm Ca [either as CaCl2 or Ca(H2PO4)2J, there was a marked increase (5 G) in 2A,,1 indicative of decreased fluidity of the membrane lipid in the region of the fatty acid probe (Figure 1 and Table I). Along with the increase in 2Amax, the 10 mm Ca also caused a change in the spectrum indicated by the arrow (Fig. 1), This peak loss is indicative of exchange-broadening which is caused by interaction between the nitroxide free radicals. Such exchange-broadening is commonly seen as the concentration of spin label in protoplasts is increased in the absence of Ca (i.e. overlabeling). The effect of Ca was not simply to increase the concentration of spin label in the membrane since 2Am. did not increase when protoplasts were deliberately over labeled. These data suggest that Ca decreased membrane fluidity and, at the same time or as a 835