Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase.

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by alpha-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.

The efficiency of various detergents for extraction and stabilization of acetylcholinesterase from bovine erythrocytes.

The efficiency of several nonionic detergents and a homologous series of zwitterionic detergents for the extraction of acetylcholinesterase (EC 3.1.1.7) from bovine erythrocyte membranes was examined. Of the nonionic detergents examined, the polyoxyethylene-based Tweens were the least effective solubilizing agents. Within this series, increasing the length of the saturated fatty acid chain progressively decreased the efficiency of enzyme recovery, while unsaturation in the side chain reversed this trend. In the Lubrol detergents, where the chain length of the alcohol group is variable, an increase in the length of the polyoxyethylene glycol group decreased the recovery of acetylcholinesterase in the solubilized state, without affecting the efficiency of extraction of total erythrocyte protein. As with the other nonionic detergents examined, Triton X-100 and octyl beta-D-glucoside were maximally effective in solubilizing acetylcholinesterase activity at concentrations greater than their respective critical micelle concentrations. In the sulfobetaine (N-alkyldimethylaminopropane sulphonate) zwitterionic detergent series, the longer alkyl chain zwittergents Z 316 and Z 314 were more efficient than the shorter chain length members of the series (Z 310 and Z 312). In contrast to the higher chain length compounds, short chain analogs were maximally effective at or below their critical micelle concentrations. After purification by ion-exchange chromatography and affinity chromatography, the enzyme extracted with the various detergents gave sedimentation coefficients between 6.8S and 7.6S, consistent with a dimeric structure. Acetylcholinesterase could also be efficiently released by 0.2 mM EDTA or 0.5 M NaCl from bovine erythrocyte membranes previously depleted of 70-80% of the membrane lipids by butanol. Nonlinear Arrhenius plots of enzyme activity were found whether acetylcholinesterase was solubilized with Tween 20, Lubrol PX, or Triton X-100. The present work confirms that bovine erythrocyte acetylcholinesterase requires detergents to solubilize it from membranes and that its activity depends on the structure of the amphiphiles used to solubilize the enzyme.

Quantitative study of the fluidity of Escherichia coli membranes using deuterium magnetic resonance.

Specifically deuterated palmitic acid was incorporated into the membrane phospholipids of the L51 strain of Escherichia coli. The cytoplasmic and outer membranes were separated by using standard techniques and studied by deuterium nuclear magnetic resonance between 0 and 40 degrees C. Distinctive liquid-crystalline and gel spectra were observed to coexist over a wide temperature range. The relative intensities of these spectra provided a direct measure of the fraction of the deuterium-labeled phospholipids in the fluid state as a function of temperature. Above 37 degrees C, the amount of immobilized or gel-phase phospholipid is estimated to be less than 3% of the total phospholipid. The gel to liquid-crystalline transition region for the outer membrane was shifted upwards by approximately 7 degrees C relative to that of the cytoplasmic membrane, in agreement with previous studies [Davis, J. H., Nichol, C. P., Weeks, G., & Bloom, M. (1979) Biochemistry 18, 2103]. The orientational order in the fluid phase of both membranes decreased gradually with increasing temperature and was greater in the outer membrane than in the cytoplasmic membrane. The orientational order of the gel-phase component was the same for both membranes, within an experimental uncertainty of 10%, and was independent of temperature from 0 to 30 degrees C for the outer membrane and from 10 to 30 degrees C for the cytoplasmic membrane.

Study of the cytoplasmic and outer membranes of Escherichia coli by deuterium magnetic resonance.

The cytoplasmic and outer membranes of Escherichia coli were studied between 0 and 40 degrees C by deuterium magnetic resonance quadrupolar echo spectroscopy. The L51 strain of E. coli was used to incorporate perdeuterated palmitic acid into the membrane phospholipids. The cytoplasmic and outer membranes were separated using standard techniques. The spectrum of each membrane preparation was dominated at high temperatures (greater than or equal to 37 degrees C) by the characteristic liquid-crystalline plateau previously observed for perdeuterated palmitate chains in model phospholipid membranes. At low temperatures, the shape and width of the spectrum were characteristic of the gel phase. The relative intensities of the liquid-crystalline and gel features varied systematically with temperature. A quantitative analysis of the acyl chain orientational order was carried out by using the method of moments. The orientational order at each temperature was greater in the outer membrane sample than in that of the cytoplasmic membrane, indicating that the liquid-crystalline-gel transition region in the outer membrane is shifted to higher temperatures than that of the cytoplasmic membrane by about 7 degrees C. It is clear from the results that most of the phospholipid molecules participate in the phase transition.

Fatty acyl chain order in lecithin model membranes determined from proton magnetic resonance.

Proton magnetic resonance (1H NMR) has been used to compare the local orientational order of acyl chains in phospholipid bilayers of multilamellar and small sonicated vesicular membranes of dipalmitoyllecithin (DPL) at 50 degrees C and egg yolk lecithin (EYL) at 31 degrees C. The orientational order of the multilamellar systems was characterized using deuterium magnetic resonance order parameters and 1H NMR second moments. 1H NMR line shapes in the vesicle samples were calculated using vesicle size distributions, determined directly using electron microscopy, and a theory of motional narrowing, which takes into account the symmetry properties of the bilayer systems. The predicted non-Lorentzian line shapes and widths were found to be in good agreement with experimental results, indicating that the local orientational order (called "packing" by many workers) in the bilayers of small vesicles and in multilamellar membranes is substantially the same. This results was found to be true not only for the largest 1H NMR line associated with the nonterminal methylene protons but also for the resolved 1H NMR lines due to the alpha-CH2 and the terminal CH3 positions on the acyl chain. Analysis of the vesicle 1H NMR spectra of EYL taken with different medium viscosities yielded a value of approximately 4 X 10(-8) cm2 s-1 for the lateral diffusion constant of the phospholipid molecules at 31 degrees C.