Characterization of biomimetic surfaces formed from cell membranes

A method for fabricating biomimetic surfaces from intact cell membranes is described. A monolayer of alkanethiol on gold is covered by a second layer derived from the components of erythrocyte membranes either by self-assembly or by Langmuir-Blodgett methods. The resulting asymmetric hybrid layer was characterized by ellipsometry, surface plasmon resonance (SPR), contact angle, capacitance, voltammetry, and electron and atomic force microscopy. The erythrocyte membrane layer was measured to be approximately 30-40 A in thickness. Using SPR, the presence of erythrocyte components on the surface was demonstrated by their selective removal by enzymatic action. The uniform deposition of membranous material on the substrate was shown by electron and atomic force microscopy. Demonstration of acetylcholinesterase (AChase) activity, a membrane-anchored enzyme, on the surface for at least 8 days, suggests that the outer leaflet of the erythrocyte membrane is present in its native form. Cyclic voltammetry demonstrates that enhanced electron transport from a solution redox species accompanies formation of the erythrocyte layer at the surface. This enhanced electron transport is blocked by 4,4'-diisothiocyanate stilbene-2,2'-disulfonic acid, a well known blocker of anion transport, suggesting that an erythrocyte anion transporter protein is incorporated into the surface layer in an active conformation.     

生物电化学及其在环境监测中的应用

简要概述了生物电化学的研究领域,包括生物体系和生物界面模拟、生物分子的电化学、生物电催化、光合作用模拟和活组织电化学;总结了生物电化学传感器、生物芯片和生物电化学反应器在环境监测中的应用现状,并提出了其发展趋势,即不断向商品化方向发展,实现环境污染物的在线检测;利用基因技术,创造出检测能力更强的生物传感器和生物芯片;与其他精密分析仪器相结合,向多功能、集成化、智能化、微型化方向发展。     

Ion flux across lipid bilayer membranes with charged surfaces

Summary In this paper we derive expressions for the ion flux across lipid bilayer membranes with charged surfaces treating the membrane as a continuous phase interposed between two electrolyte solutions and calculating the ion flux with the Nernst-Planck equations. The theoretical results are compared with experiments of Seufert and Hashimoto on lipid bilayer membranes with charged surface active agents added to the membranes. If the charge of both membrane surfaces has the same sign the flux of the gegenions is greatly increased whereas the flux of the coions decreases to a small amount. For oppositely charged membrane surfaces the membrane behaves like a np semiconductor and typical rectification voltage-current characteristics are obtained.     

Evidence for tubulin-binding sites on cellular membranes: plasma membranes, mitochondrial membranes, and secretory granule membranes

We describe the interaction of pure brain tubulin with purified membranes specialized in different cell functions, i.e., plasma membranes and mitochondrial membranes from liver and secretory granule membranes from adrenal medulla. We studied the tubulin-binding activity of cellular membranes using a radiolabeled ligand-receptor assay and an antibody retention assay. The tubulin-membrane interaction was time- and temperature-dependent, reversible, specific, and saturable. The binding of tubulin to membranes appears to be specific since acidic proteins such as serum albumin or actin did not interfere in the binding process. The apparent overall affinity constant of the tubulin- membrane interaction ranged between 1.5 and 3.0 X 10(7) M-1; similar values were obtained for the three types of membranes. Tubulin bound to membranes was not entrapped into vesicles since it reacted quantitatively with antitubulin antibodies. At saturation of the tubulin-binding sites, the amount of reversibly bound tubulin represents 5-10% by weight of membrane protein (0.4-0.9 nmol tubulin/mg membrane protein). The high tubulin-binding capacity of membranes seems to be inconsistent with a 1:1 stoichiometry between tubulin and a membrane component but could be relevant to a kind of tubulin assembly. Indeed, tubulin-membrane interaction had some properties in common with microtubule formation: (a) the association of tubulin to membranes increased with the temperature, whereas the dissociation of tubulin- membrane complexes increased by decreasing temperature; (b) the binding of tubulin to membranes was prevented by phosphate buffer. However, the tubulin-membrane interaction differed from tubulin polymerization in several aspects: (a) it occurred at concentrations far below the critical concentration for polymerization; (b) it was not inhibited at low ionic strength and (c) it was colchicine-insensitive. Plasma membranes, mitochondrial membranes, and secretory granule membranes contained tubulin as an integral component. This was demonstrated on intact membrane and on Nonidet P-40 solubilized membrane protein using antitubulin antibodies in antibody retention and radioimmune assays. Membrane tubulin content varied from 2.2 to 4.4 micrograms/mg protein. The involvement of membrane tubulin in tubulin-membrane interactions remains questionable since erythrocyte membranes devoid of membrane tubulin exhibited a low (one-tenth of that of rat liver plasma membranes) but significant tubulin-binding activity. These results show that membranes specialized in different cell functions possess high- affinity, large-capacity tubulin-binding sites...     

Localization of light-harvesting complex II to the occluded surfaces of photosynthetic membranes

The photosynthetic membranes of green plants are organized into stacked regions interconnected by nonstacked regions that have been shown to be biochemically and structurally distinct. Because the stacking process occludes the surfaces of appressed membranes, it has been impossible to conduct structural or biochemical studies of the outer surfaces of the photosynthetic membrane in regions of membrane stacking. Although stacking is mediated at this surface, it has not been possible to determine whether membrane components implicated in the stacking process, including a major light-harvesting complex (LHC-II), are in fact exposed at the membrane surface. We have been able to expose this surface for study in the electron microscope and directly label it with antibodies to determine protein exposure. The appearance of the newly exposed outer stacked surface highlights the extreme lateral heterogeneity of the photosynthetic membrane. The surface is smooth in contrast to the neighboring nonstacked surface that is covered with distinct particles. Although some investigators have suggested the existence of a cytochrome b6/f-rich boundary region between stacked and nonstacked membranes, our results provide no structural support for this concept. To explore the biochemical nature of the occluded membrane surface, we have used an mAb against the amino terminal region of the LHC-II. This mAb clearly labels the newly exposed outer stacked surface but does not label the inner surface or the outer nonstacked surface. These experimental results confirm the presence of the amino terminal region of this complex at the outer surface of the membrane in stacked regions, and also show that this complex is largely absent from nonstacked membranes.     

Electrostatic interactions at charged lipid membranes. Hydrogen bonds in lipid membrane surfaces

Hydrogen-bonded structures within lipid membrane surfaces are not disrupted by water and are of thermodynamic and therefore potential structural importance in biological systems.     

Amphotericin B channels in phospholipid membrane-coated nanoporous silicon surfaces: implications for photovoltaic driving of ions across membranes

The antimycotic agent amphotericin B (AmB) functions by forming complexes with sterols to form ion channels that cause membrane leakage. When AmB and cholesterol mixed at 2:1 ratio were incorporated into phospholipid bilayer membranes formed on the tip of patch pipettes, ion channel current fluctuations with characteristic open and closed states were observed. These channels were also functional in phospholipid membranes formed on nanoporous silicon surfaces. Electrophysiological studies of AmB-cholesterol mixtures that were incorporated into phospholipid membranes formed on the surface of nanoporous (6.5 nm pore diameter) silicon plates revealed large conductance ion channels ( approximately 300 pS) with distinct open and closed states. Currents through the AmB-cholesterol channels on nanoporous silicon surfaces can be driven by voltage applied via conventional electrical circuits or by photovoltaic electrical potential entirely generated when the nanoporous silicon surface is illuminated with a narrow laser beam. Electrical recordings made during laser illumination of AmB-cholesterol containing membrane-coated nanoporous silicon surfaces revealed very large conductance ion channels with distinct open and closed states. Our findings indicate that nanoporous silicon surfaces can serve as mediums for ion-channel-based biosensors. The photovoltaic properties of nanoporous silicon surfaces show great promise for making such biosensors addressable via optical technologies.     

Plant membranes: Structure,assembly and function

Book Review

Plant membranes: Structure, assembly and functionJ.L. Harwood and T.J. Walton (Eds.), London: The Biochemical Society, 1988, 251 pages. £25. ISBN 0-904498-23-9