Effect of membrane fluidity upon binding of Electrophorus acetylcholinesterase to lipid vesicles.

A previous report (Watkins, M.S., Hitt, A.S. and Bulger, J.E. (1977) Biochem. Biophys. Res. Commun. 79, 640-647) has indicated that the asymmetric forms of Electrophorus acetylcholinesterase bind exclusively to sphingomyelin vesicles through interaction with the collagen-like 'tail' portion of the enzyme. We report here that acetylcholinesterase also binds to phosphatidylcholine vesicles containing saturated fatty acyl chains and to egg phosphatidylcholine vesicles containing cholesterol. This suggests preferential binding of acetylcholinesterase to membranes of lower fluidity. Surface charge of vesicles and density of zwitterionic lipid headgroups do not significantly affect binding of native acetylcholinesterase. The presence of chondroitin sulfate or hyaluronic acid slightly increases the binding of native acetylcholinesterase to sphingomyelin vesicles, while the presence of 1 M NaCl, bovine serum albumin, or tissue fractions enriched in basement membrane diminish binding. The dissociation constant for native acetylcholinesterase and sphingomyelin vesicles is (1.0-1.5) X 10(-7) M, as measured by a flotation binding assay. The globular, 11S form of acetylcholinesterase also binds to lipid vesicles, although not to the same degree as native acetylcholinesterase. This suggests that the collagen tail of the enzyme enhances binding, but is not essential for binding to occur. These results are consistent with the location of acetylcholinesterase on the surface of the postsynaptic plasma membrane in vivo.

Hydrogenation of triton X-100 eliminates its fluorescence and ultraviolet light absorption while preserving its detergent properties.

The ultraviolet-light absorption and fluorescence of Triton X-100 were virtually eliminated by hydrogenation to its reduced cyclohexyl analog, RTX-100. The critical micelle concentration of RTX-100 was 12% higher than that of Triton X-100. RTX-100 and Triton X-100 were quite similar in their abilities to extract proteins from human erythrocyte membranes.

9-(5-Carboxypentylamino)-acridine. An affinity adsorbent ligand for Electrophorus acetylcholinesterase.

We have developed a simple method for the synthesis of the ligand 9-(5-carboxypentylamino)-acridine and the resulting affinity adsorbent using Sepharose CL-4B. This affinity adsorbent is efficient and specific for purification acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from Electrophorus electricus electric organ.

Inhibition of lactose transport in E. coli by N-ethylmaleimide-beta-galactoside, N-ethylsuccinimide-beta-galactoside and N-ethylmaleimide.

N-ethylmaleimide (NEM) inhibits lactose uptake in E. coli by reacting with the M protein component of the lac permease system. In an attempt to estimate the distance between the NEM reactive site and the substrate binding site, we have synthesized a beta-galactoside with NEM as the aglycon moiety (NEM-gal). NEM-gal was a more effective inhibitor of lactose transport than was NEM. Part of the inhibition by NEM-gal was caused by competition with lactose for the substrate binding site. To estimate this part of the inhibition, we synthesized the saturated and thus the unreactive N-ethylsuccinimide (NES) analog of NEM-gal. Nes-gal was a competitive inhibitor of lactose uptake. The remainder of the inhibition by NEM-gal followed first-order kinetics with the same rate constant as NEM. In addition, the protective effect of thiodigalactoside against the inhibition of transport by NEM was also observed against irreversible inhibition by NEM-gal. We suggest that the reactivity of NEM was unaltered by bringing it near the beta-galactoside binding site by way of covalent attachment to galactose. We conclude that the distance between the NEM reactive site and the position of the glycosidic oxygen of beta-galactosides bound to the lactose site is greater than 8 A.

The characterization of energized and partially de-energized (respiration-independent) beta-galactoside transport into Escherichia coli.

Unidirectional fluxes of [14C]lactose by whole cells of Escherichia coli under highly energized and partially de-energized (in the presence of CN-) conditions are analyzed kinetically. When the cells are energized, the value for V influx is 0.45 +/- 0.01 mM internal concentration increment/s and Kt is 0.26 +/- 0.03 mM. At an external concentration of 0.61 mM the steady-state internal concentration is 0.25 M, reached after about 1h. The maximum steady-state concentration ratio is 2-10(3). The efflux process under these conditions is non-saturable, being linearly dependent upon internal concentration over the range 25-250 mM with a first-order rate constant of 8.8 +/- 0.2-10(-4) S-1. The transport in the presence of CN- is active, with a maximum concentration ratio (internal concentration/external concentration) of 104, and the uptake is mimicked by anoxia (less than 70 ppm O2). The effects of CN- are to lower the V for influx and to change the efflux from a non-saturable to a saturable process with a value for Kt (60 mM) intermediate between that for energized efflux (greater than 250 mM) and influx (0.3-0.6 mM), the latter value not changing appreciably. Partial de-energization thus affects both the influx and efflux processes.