ELUCIDATION OF BIPHASIC ALTERATIONS ON ACETYLCHOLINESTERASE (ACHE) ACTIVITY AND MEMBRANE FLUIDITY IN THE STRUCTURE-FUNCTIONAL EFFECTS OF TETRACAINE ON ACHE-ASSOCIATED MEMBRANE-VESICLES

Tetracaine-induced biphasic structure-functional alterations were inve stigated in acetylcholinesterase (AChE)-associated membrane vesicles f rom the electric organ of Torpedo californica. Enzyme assays showed th at tetracaine exhibits a biphasic effect on the activity of membrane-b ound AChE: increasing it at low concentrations (<12 mM) and decreasing it at high concentrations (>12 mM). FIuorescence-polarization experim ents demonstrated that tetracaine affects the fluidity of lipid hydroc arbon chains of these membranes in a biphasic manner: increasing it at <20 mM and decreasing it at >20 mM. This small molecule also alters t he fluidity of the negatively charged lipid head group: increasing it at <13 mM and remaining essentially at the same level at >13 mM. The p ositively charged lipid head group is unaffected, Contrasting effects on AChE activity with changes in membrane fluidity showed that [tetrac aine] for AChE activity is comparable to that for the fluidity of the negatively charged lipid head group (12 mM versus 13 mM), but lower th an that for a biphasic effect on the fluidity of lipid hydrocarbon cha ins (12 mM versus 20 mM). Differential scanning microcalorimetry showe d that, due to membrane protein-lipid interaction, the lipid-phase tra nsition temperature (t(ml)) is higher for AChE-associated membrane ves icles than for isolated lipids from these membranes, An overall disord ering of the membranes by tetracaine, as inferred from the lowering of t(ml), was also demonstrated, These findings suggested that binding o f tetracaine to the lipid polar head group and membrane protein-lipid interaction may contribute to a higher [tetracaine] in inducing a comp arable biphasic effect on membrane fluidity. At high [tetracaine], cha rge interactions between the tetracaine cation and the negatively char ged lipid head group may result in a new lipid phase in the membranes, which could reverse the increase in membrane fluidity, resulting in t he observed biphasic effect, Although both tetracaine and alcohol are amphiphilic species, they exhibit distinctive structure-functional eff ects on the membranes, as shown by comparing the results obtained on t etracaine with those previously reported for alcohol, The present obse rvations may have significant physiological implications and may be of importance in understanding the biochemical effects of tetracaine in correlation with its physiological impact. (C) 1998 Academic Press.