The temperature-dependence of human erythrocyte acetylcholinesterase activity is not affected by membrane cholesterol enrichment (original) (raw)
Related papers
Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology
⎯Artificial hypothermic state of homeothermic animals contributes to the stimulation of free radical processes in red blood cells. In order to understand what are the consequences of oxidative damage of erythrocyte membrane, we examined the dependence of the kinetic characteristics of integral membrane enzyme acetylcholinesterase (AChE) and structural and functional state of the membrane on the duration of mild hypothermia. For this purpose we reduced body temperature of adult Wistar rats by external cooling to 30°C (short-term moderate hypothermia) and then prolonged hypothermia up to 1.5 and 3 h. A short-term hypothermia was followed with an increase in V max and a decrease in K m , promoting an increase in the catalysis effectiveness.The optimum point on the graph of the concentration dependence was shifted to the area of lower concentrations, and the character of enzyme-substrate interactions at high concentrations of the enzyme changed. Upon prolongation of hypothermia, changes in the AChE kinetic characteristics favored normalization of the enzyme activity and concentration dependence. To test the hypothesis of a possible influence of the lipid matrix on the kinetic characteristics of AchE, we studied structural properties of the erythrocyte membranes using fluorescent probe pyrene. The observed changes in the structural and dynamic characteristics of erythrocyte membranes after a 1.5-h hypothermia suggested a reduction in microviscosity of both total and annular lipids. Prolongation of hypothermia up to 3 h favored normalization of this parameter. It was found that the indicators of the structural state of erythrocyte membranes at different durations of hypothermia correlate with certain kinetic characteristics of AChE. The data indicate that the prolongation of mild hypothermia up to 3 h triggers adaptive mechanisms directed to normalization of the erythrocytes membrane functioning.
Biophysics
⎯The thermostability of acetylcholinesterase of rat erythrocyte membranes in the norm and moderate hypothermia was studied. It is shown that the kinetics of the thermal denaturation of acetylcholinesterase is nonlinear and corresponds to a model that involves two-step denaturation, fast and slow, of the enzyme's native form. The rate constants of the fast phase, k 1 , are much higher than those of the slow phase, k 2 , while the energy of the fast phase activation is lower by only 19.4% compared to that of the slow one. Short-term moderate hypothermia is shown to increase k 1 and decrease the index of relative activity of the intermediate form of acetylcholinesterase (parameter β), leading to significant lowering of the activation energies of both stages; parameter β becomes more temperature dependent. The prolongation of hypothermia up to 3 h mainly contributes to a decrease in k 1 and k 2 relative to short-term hypothermia and the activation energy of denaturation increases. These data support the hypothesis according to which the structure of acetylcholinesterase is labilized at the initial stages of the development of the hypothermic state and stabilized during prolonged hypothermia.
European biophysics journal : EBJ, 2004
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 suppo...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1972
The allosteric behavior of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from red cell ghosts of rats fed fat-sufficient and fat-deficient diets was investigated. Allosteric type kinetics with n =-1.6 have been obtained for the inhibition by Fin rats fed a fat-sufficient diet. In animals fed a fat-deficient diet the values of n changed from-1.6 to-I.O. When these animals were then fed a fat-sufficient diet the values of n shifted from-I.O to-1.6. This in vivo reversion was obtained after 8 days of refeeding. Two types of changes in the values of n were obtained in vitro in fat-deficient rats: (I) from-I.O to-I.6 by solubilization of the membrane-bound enzyme with Triton X-Ioo, (2) from-1.6 to-I.O by resconstitution of the membrane-like structure from the soluble enzymatic preparation. The possibility that the structure of the membrane could be responsible for the changes in the phenomenon of phenotypic allosteric desensitization in the membrane-bound enzymes is discussed
The Journal of nutrition, 1974
The allosteric character of the acetyl cholinesterase from rat erythrocytes has been described in a previous paper (1). It was also reported there that the enzyme loses its allosteric ‘behavior when bound to EFA-deficient membranes. Similar be havior has been reported for other en zymes ‘boundto erythrocyte (2â€"4) and microsomal (5) membranes. It was later demonstrated that the allosteric behavior of the membrane-bound acetyicholinester ase and (Na@ + K@)-ATPase correlated with the overall fluidity of the membrane fatty acids, where the EFA did not play a particular role (6). However, it was pointed out that a direct correlation exists between the cooperativity of the mem brane-bound acetyleholinesterase and the linoleic fatty acid content of the erythro cyte lipids. This correlation was not found between the enzyme cooperativity and the total EFA content of the membrane (6). We have described (4) that the Arrhenius plot of the (Mg2@+ K@) p-nitrophenyl phosphatase from EFA-deficient and EFA sufficient rats is clearly different. It was of interest to test whether these temperature dependent changes were specific for EFA deficiency or, as the allosterism, correlated with the overall membrane fluidity.
Effects of cholesterol on lipid organization in human erythrocyte membrane
The Journal of cell biology, 1980
The molar ratio of cholesterol to phospholipid (C/P) in human erythrocyte membrane is modified by incubating the cells with liposomes of various C/P ratios. The observed increase in cell surface area may be accounted for by the addition of cholesterol molecules. Fusion between liposomes and cells or attachment of liposomes to cells is not a significant factor in the alteration of C/P ratio. Onset temperatures for lipid phase separation in modified membranes are measured by electron diffraction. The onset temperature increases with decreasing C/P ration from 2 degrees C at C/P = 0.95 to 20 degrees C at C/P = 0.5. Redistribution of intramembrane particles is observed in membranes freeze-quenched from temperatures below the onset temperature. The heterogeneous distribution of intramembrane particles below the onset temperature suggests phase separation of lipid, with concomitant segregation of intramembrane protein into domains, even in the presence of an intact spectrin network.
Cholesterol oxidase susceptibility of the red cell membrane
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1984
We have used the highly variable and conditional susceptibility of cholesterol to cholesterol oxidase to probe molecular rearrangements in the human red cell membrane. Cholesterol in the intact erythrocyte normally is not a substrate for this enzyme. Susceptibility was induced however, by these pretreatments: mild enrichment in membrane cholesterol, exposure to >_ 0.03% (3 mM) glutaraldehyde and warming in dilute salt solutions (# approx. 0.001). Cholesterol reactivity in dilute salt solutions emerged only following a lag of 30 min or more. The lag time was shortened by raising the temperature, by reducing the salt concentration or by treating with glutaraldehyde. The induced sensitivity to the enzyme was inhibited by restoring physiologic ionic strength or by introducing 0.1 mol lysophosphatidyicholine per mol cholesterol into the membrane. (In striking contrast, lysophosphatidylethanolamine and iysophosphatidylserine did not inhibit oxidation.) The various effectors of cholesterol oxidase sensitivity strongly influenced the impact of the others, suggesting that each shifted cholesterol toward or away from an enzyme-sensitive disposition. None of these effects was observed in pure cholesterol or red cell membrane iipids dissolved in detergent, which were uniformly highly reactive with the enzyme. We conclude that the observed variation in cholesterol oxidase sensitivity reflects changes in the organization of the bilayer, perhaps a lateral redistribution of lipids which creates cholesterol-rich phases or domains in which cholesterol is more or less accessible to the enzyme. If so, the time-dependent increase in cholesterol susceptibility during warming at low ionic strength might be a novel indicator of the kinetics of phase changes in the bilayer of the red cell.