A novel membrane lateral domain approach was used to test whether the activity of the membrane-bound enzyme acetylcholinesterase (AChE) depends on the local properties (e.g. local lipid ordering) of ...bovine erythrocyte-ghost membrane. This issue has an additional aspect of interest due to an alternative mode of insertion of AChE molecules into the membrane by the glycosylphosphatidylinositol (GPI) anchor. In our experiments the lateral domain membrane structure was influenced by temperature and by the addition of n-butanol, and was quantitatively characterized using the method of EPR spectrum decomposition. The activity of AChE was determined by a colorimetric assay in the same samples. The results show that the membrane stabilizes the conformation of the membrane-bound AChE compared to the isolated AChE. In addition, a correlation was observed between the temperature dependence of order parameter of the most-ordered domain type and the activity of AChE. Therefore, our findings support the idea that the function of GPI proteins can be modulated by the lipid bilayer. Based on the assumption that the overall activity of AChE depends on the order parameters of particular domain types as well as their proportions, two models for AChE activity were introduced. In the first, a random distribution of enzyme molecules was proposed, and in the second, localization of enzyme molecules in a single (cholesterol-rich) domain type was assumed. Better agreement between measured and calculated activity values speaks in favor of the second model.
Using EPR spectroscopy a typical lateral domain structure was detected in the membranes of spin-labeled bovine erythrocyte ghosts. The spectral parameters were determined by decomposing the EPR ...spectrum into three spectral components and tuned by a hybrid-evolutionary-optimization method. In our experiments the lateral domain structure and its properties were influenced by the variation in the temperature and by the addition of
n-butanol. The specific responses of the particular domain types were detected. For the most-ordered domain type a break was seen in the temperature dependence of its order parameter, while the order parameters of the two less-ordered domain types exhibited a continuous decrease. Below the break-point temperature the alcohol-induced membrane fluidity variation is mainly a consequence of the change in the proportions of the least- and the most-ordered domain type and not the change of the domain-type ordering or dynamics (with
n-butanol concentration). On the other hand, the fluidity variation above the break-point temperature arises from both types of changes. Interestingly, the proportion of the domain type that has its order parameter between that of the least- and the most-ordered domain type remains almost constant with concentration as well as with temperature, which implies its stability. Such characterization of the lateral membrane domain structure could be beneficial when considering the lipid–protein interactions, because it can be assumed that the activity of the membrane-bound enzyme depends on the properties of the particular domain type.
We monitored electrooxidation of noradrenaline and α-melanocyte stimulating hormone (α-MSH) at a carbon-fibre microelectrode (CFME). The solution of noradrenaline (1 mM) or α-MSH (1 mM) was applied ...by a pressure pulse (2 s) from a micropipette to a voltage-clamped (850 mV) CFME immersed into bathing solution of an inverted microscope chamber. The distance between the CFME and micropipette was 2 to 12 μm. The maximal currents recorded for these two agents were 8.0 ± 0.5 pA (N = 9) and 3.0 ± 1.1 pA (N = 9), respectively. Pressure application of control solution did not affect the measured current. The noradrenaline-evoked anodic current was characterized by a monotonic increase that attained the maximum at the end of the pressure pulse. In contrast, the time-course of the α-MSH-evoked current was biphasic. The maximum amplitude of this current was attained in 0.59 ± 0.15 s (N = 9) and then it declined with a time constant of 7.5 ± 4.0 s (N = 9) until the pressure pulse was terminated. We explain this phenomenon to be due to an interaction between the peptide oxidation products and the CFME which results in its desensitization.
