JPH01245810A - Ion permeating membrane and its control by light irradiation - Google Patents

Abstract

PURPOSE:To control ion permeability of a membrane by light irradiation by incorporating >=2 ion active transporting material groups in an ion impermeable lipid membrane by irradiation of light of different wavelength. CONSTITUTION:Equal to or larger than 2 kinds of ion active transporting material groups such as rhodopsins, iodopsins, bacteriorhodopsins, etc., are incorporated in an ion impermeable lipid membrane of glycerolinlipids, sphingoglycolipids, etc., or in a membrane having the same function by irradiation of light of different wavelength to give an ion permeable membrane. By light irradiation to the membrane, the kinds of ions to be transported and the direction of permeation are controlled according to the wavelength of the irradiated light. Meanwhile, the difference of ion concn. between the ion permeating membranes can be transformed into an electrical signal, therefore, the membrane is expected to use for a photoelectric devices transforming electric signals into photo signals.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a selective ion-permeable membrane by light irradiation using a group of substances that absorb light and transport ions, and a method for controlling the same. .

(Prior art) It is well known that one of the functions of biological membranes is the selective permeation of ions, etc., and it is possible to create structures similar to biological membranes by, for example, retaining substances with active ion transport properties in thin membranes. There are high expectations for the construction of dialysis membranes and various sensors by imparting these functions.

Furthermore, since the difference in ion concentration across the membrane can be easily converted into an electrical signal as a so-called membrane potential by combining it with an ion-sensitive electrode, it can also be applied as a chemical element that converts chemical signals into electrical signals. As an example of a device using a protein as an ion active transporting substance, a construction of a biochemical device such as a sensor for implantation in a living body is disclosed in Japanese Patent Laid-Open No. 11158/1983.

[Problem to be solved by the invention]

If optical input is used to control ion permeability, it not only facilitates external control methods, but also enables the realization of chemical devices that can perform photoelectric conversion with a small amount of heat, for example. Although there is no example to date in which the directionality and reversibility of ion transmission is controlled by light irradiation as in the present invention, ion permeable membranes with excellent volumetric efficiency and energy efficiency as well as good controllability are useful for optical communication. It is also promising as a conversion element in the field.

In order to control membrane ion permeability by light incidence, it is desirable to be able to arbitrarily set the wavelength of the incident light. In addition, membrane ion permeability is not controlled only in one direction;
Needless to say, it is desirable to be able to select the direction of transparency, and to be able to select it in accordance with the wavelength of incident light. The present inventors focused on the fact that the selectivity (specificity) and reaction efficiency of biochemical reactions observed in the living body are extremely high compared to physical or chemical reactions, and the irradiation wavelength range can be adjusted arbitrarily. The ion permeability of the membrane can be controlled with high sensitivity by adjusting the light irradiation energy.
Moreover, we have been studying an ion-permeable membrane that can select the direction of ion transmission depending on the incident wavelength, and a method for controlling it.

In the process, the present inventors focused on photoreceptor proteins, which are substances that exist in the retina of animals and control light sensing by transporting substances with extremely high sensitivity and high resolution to visible light in living organisms. However, we have discovered that the above-mentioned functions can be achieved by using a photoreceptor protein, which has a similar structure and function and can exist relatively stably at room temperature, in a lipid membrane, thereby achieving the present invention.

That is, one of the objects of the present invention is to provide an ion-permeable membrane that can be produced by light irradiation using a biochemical reaction.
Another object of the present invention is to provide a method for controlling the ion permeability of the ion permeable membrane by light irradiation.

[Means to solve the problem]

That is, the present invention is characterized in that two or more types of substances that actively transport ions when irradiated with light in different wavelength ranges are incorporated and retained in an ion-impermeable lipid membrane or a thin film having an equivalent function. Another invention is an ion-permeable membrane characterized in that the membrane ion permeability of the ion-permeable A membrane is controlled in accordance with the incident wavelength range, including the ion species to be transported and the direction of their transmission. This is a method of controlling transparency.

Photoreceptor proteins can be mentioned as substances with the function of absorbing light and transporting various ions as referred to in the present invention, but as long as they have such functions, various photoreceptor proteins or their analogues may be used. can be used, and the types are not limited.

A typical example of a photoreceptor protein is a pigment protein that exists in the animal retina, so-called photoreceptor. Receive light at the nodes;
It has the effect of replacing this with some kind of change in membrane ion permeability. Examples of such substances include rhodopsin, polyphyllopsin, and iodopsin, which have been extracted and purified. In addition, there are bacteriorhodopsin and herorhodobuscin, which are present in the cell membrane of halophilic bacteria, which have the same function as visual substances, and these are preferable because they are relatively easy to handle.

Bacteriorhodopsin is derived from Helobatatherium (Ila
Halobacterium halobium (tlalobac, Leri) belonging to the genus lobacterium
It is a main component of proteins in cell membranes (purple membranes) such as um halobium, contains retinal as a chromophore, and has the function of transporting hydrogen ions (proton pumping ability) in response to visible light [8, Danon, W. 5toecken
ius; Proc, Natl, Acad.

Sci., USA, 71. 1234 (197
4) ].

For example, [0°0este]
rhelt, and W, St, oeckcnius
;Method inEnzymology,
31.667 (1974)] as a purple membrane from highly halophilic bacteria, and
uang, 11. l1ayley and Il,G
, Khorana; Proc, Natl, Acad, S
It can be obtained by removing lipids from the purple membrane obtained using the method described in [Ci., USA, 77, 323 (+980)].

Herorhodobuscin is also used in highly halophilic bacteria such as RlmR,
A retinal protein discovered in bacteriorhodopsin-deficient strains such as L33, which has the property of transporting sodium ions in response to visible light [A, Y, Matsuno
and Y, Mukohata: Biochem, B
iophys.

Res, Commun, 78.237 (1977)
: R.E., Mac Donald.

R,U,Greene, R,D,Glark,E,
V, Lindley: J, Biol.

Chem, 254.11831 (1979)].

This halorodopusin is derived from highly halophilic bacteria such as [Y,
Mukohata, Y., Sugiyama and
Y., Niji, J. Usukura and E. Yam.
ada; Photochem, Photobiol,
, 33, 539 (1981)].

Further, the structure of a natural photoreceptor protein isolated from a living body can be changed without impairing its function to form a derivative with a changed photosensitive wavelength, which can be used in the present invention.

Typically, the retinal moiety can be substituted to change the optical absorption wavelength. To give a specific example of the formation of such a derivative in rhodopsin, for example, bacteriorhodopsin with a maximum absorption wavelength of 570 nm by changing the retinal moiety to a) all-trans-retinal [
P.Townor, W.Gaerther, ct.
al., Eur, J.

Biochem,, 117.353(198])],
b) Bacteriorhodopsin with maximum absorption wavelength of 550 nm by using 13-cis-retinal [
c) bacteriorhodopsin with maximum absorption wavelength of 475 nm by using 5,6-dihydroretinal [
R, Mao, R, Govindjee, et al.
,Biochemistry, 428 (198
1)], d) Bacteriorhodopsin with maximum absorption wavelength of 430 nm by retro-γ-retinal [S, Huang, H, Baylay, et
,al, ,Fed,Proc.

40, 1659 (1981)], e) Bacteriorhodopsis with maximum absorption wavelength of 593 nm by using 3,4-dihydroretinal: /
[F, Tokunaga, T, Ebrey, Bi
ochemistry.

17, 1915 (1978)], etc.

Furthermore, the amino acid sequence of bacteriorhodopsin has already been revealed [Yu, A., Ovchinnikov.

N., G., Abdullaev, et. al., Bio
org, Khim, i, 1573 (+978)]
, and the nucleotide sequence of the basophilic bacteriorhodopsin gene [R, J, Dunn, J, M-Mc (: oy et al.

Proc, Natl, Acad, Sci, 78.6
744 (1981)]. Based on these findings, it has become possible to synthesize protein analogs in which the amino acid sequence of bacteriorhodopsin is substituted by constructing recombinant DNA [N., R. Hackett,
L, J, 5tern, etc.

al,, J, Biol, hem,, 262.92
77 (1987)], such photoreceptor protein-like substances can also be used in the present invention. Depending on the configuration of the membrane ion-permeable membrane in which two or more proteins with different light absorption wavelength regions are desired, the above-mentioned photoreceptive proteins may be selected and used.

In the present invention, known amphipathic compounds capable of forming a monomolecular film or a multimolecular film can be used as the material for the lipid film that retains the photoreceptor protein.

These lipid molecules with membrane-forming ability are composed of a long-chain alkyl group with 8 or more carbon atoms and a parent group, and the parent group is a cation such as (20nN"2(+), for example CI+3 (CII2). Anions such as -, -QC-GH2, e.g. (2, -glycero-(glycidoxy)
It may be either a nonion such as x) or a zwitterion such as (2C+a N''CIC2PO4-).

Among the lipid materials of Kowara, glycerophospholipids such as phosphatidylcholine (lecithin), phosphatidylethanolamine, and diphosphatidylglycerol; sphingophospholipids such as sphingomyelin and ceramide syriatin; sphingoglycolipids such as cerebroside, sulfatide, and ceramide oligohexoside and glyceroglycolipids such as glycosyldiacylglycerol that contain carbohydrates as hydrophilic groups are lipids that constitute biological membranes, so they incorporate the photoreceptive proteins mentioned above to form lipid membranes that retain photoreceptive proteins. It can be said that it is a particularly suitable material for making the protein function efficiently.

In addition, the lipid membrane referred to in the present invention is one formed from the above-mentioned lipid materials and consists of a monolayer of lipids, or a membrane composed of two monolayers of lipids (lipid bilayer). (membrane) or a structure in which three or more lipid monomolecular films are laminated (lipid multilayer film) can be used.

Among these, retaining photoreceptive proteins within a lipid bilayer membrane is advantageous because the photosensitive pigment proteins can be reconstituted into a structure similar to that in vivo, and their functions can be effectively utilized. Furthermore, the bacteriorhodopsin described above exists in halophilic bacteria as a complex with a lipid layer called the purple membrane, and it is convenient because it is possible to extract fragments of this lipid-protein complex.

To form a lipid complex with a photoreceptor protein such as bacteriorhodopsin, for example [E, Packerand
W, 5toeckenius, J, Biol,
Chem, 249,662(+974)] and [K-5, Iluang, H, Bayley and
H,G.

Khorana, Proc, Natl, Acad, S
ci, USA, 77, 323 (1980)], the above-mentioned lipids are suspended in a solution with an appropriate salt concentration, and liposomes are formed while being sonicated as necessary. At this time, the desired photosensitive pigment protein can be added to the solution and incorporated into the formed lipid membrane.

The product thus obtained can be processed, for example, by column chromatography, [G, Lind.

B, ll-1ojeber and tl, G, Kho
rana, J., Biol, Chem.

Proteoliposomes incorporating photosensitive pigment proteins can be separated and purified using the ultracentrifugation method using the sucrose concentration gradient method described in J.D., 8298 (1981)].

Proteoliposomes purified in this way are produced by immersing a lipid bilayer membrane layer previously formed on a porous substrate in an appropriate solvent and adsorbing and fusing it into this membrane to form a photoresponsive ion-permeable membrane. It is possible to do so. Collagen, cellulose, porous glass, etc. can be used as the base.

Furthermore, as is well known for purple membranes, a protein-lipid bimolecular composite membrane can be formed by developing proteoliposomes in a Langmuir tank and attaching them to the substrate surface. In this case, a planar film can be obtained in which a hydrophilic surface is formed on the substrate side by horizontal deposition, and a hydrophobic surface is formed on the substrate side by vertical dipping. By immersing the thus constructed composite membrane in an appropriate solvent, it is possible to adsorb and fuse proteoliposomes holding a protein different from the protein in the membrane. By this method, it is possible to form a composite membrane (photoresponsive ion permeable membrane) in which a photoreceptive protein that reverses the direction of ion permeation is incorporated into a single layer of membrane.

Next, a method for controlling the photoresponsive ion-permeable membrane configured as described above will be explained with reference to the drawings.

FIG. 1 is a schematic cross-sectional view showing an example of the structure of the permeable A membrane of the present invention.

By the method described above, a photoresponsive permeable membrane 1 in which two different photoreceptor proteins 1a and 1b are oriented within two lipid molecules is formed on a porous support base 2.

The two different photoreceptor proteins 1a and 1b are selected such that their absorption maximum wavelengths are located, for example, in such a way that 1a is located on the short wavelength side λ1, and lb is located on the long wavelength side λ2. In the figure, a and b indicate incident light on the short wavelength side and incident light on the long wavelength side, respectively. The ion permeability of this membrane is achieved over a wider wavelength range than when 1a or lb is present alone.

FIG. 2 is a schematic cross-sectional view showing another configuration example of the present invention.

The permeable membrane 1 in this example is formed on a porous support base 2 in the same manner as in the case shown in FIG. has been done. In this film, the directionality of ion transport can be arbitrarily selected by, for example, switching the wavelength of incident light from λ1 to λ2. In addition, by utilizing this difference in directionality and selecting photoreceptor proteins 1a and lb so that the light absorption bands of la and 1b suitably overlap, wavelength selection for light with wavelengths near λ or λ2 can be achieved. It can also significantly improve gender.

〔Example〕

The present invention will be described in detail below with reference to Examples, but the scope of the present invention is not limited thereto.

The purple membrane extracted from Halobacterium halobium, strain R1 by the method of Oesterhelt et al. described above, was treated with a surfactant Trit according to the method of Huang et al.
on X-100 (manufactured by Wako Pure Chemical Industries, Ltd.) to remove lipids from the obtained purple membrane to obtain bacteriorhodopsin, which is a protein component. Using a portion of bacteriorhodopsin purified in this way, the chromophore was
The method of a et al. [F, Tokunaga and T.

Iwasa, Membrane, '4.73 (+
984)] was substituted with naphthyl retinal, which is a type of retinal analog. While the maximum absorption wavelength of bacteriorhodopsin is around 560 nm, the maximum absorption wavelength of bacteriorhodopsin analogs having naphthyl retinal as a chromophore is distributed from 442 nm to 503 nm depending on the mixing ratio. Like bacteriorhodopsin, this bacteriorhodopsin analog exhibits active proton transport activity upon irradiation with light.

The thus prepared bacteriorhodopsin and its analog were mixed in equal amounts, and the method of Kagawa et al. [Y,
Kagawa and E., Rucker, J., Bi.
ol, Ghem, 246°5477 (1971)
:] Using the soybean phospholipid azo lectin purified by
Mixed proteoliposomes were constructed based on the method of Huang et al. described above. This proteoliposome is 0.1
In a C1 solution with a concentration of 5 mol/It, irradiation with monochromatic visible light from a mercury lamp was carried out through a monochromator,
The pH change at that time was examined and compared with the pH change of proteoliposomes produced only by normal bacteriorhodopsin without any bacteriorhodopsin analog. As a result, in the mixed proteoliposomes, from 450 nm to 5
It was shown that the active proton transport properties were comparable over the incident light range of 70 nm, and the range of use of controlled incident light could be greatly expanded.

A nitrocellulose filter obtained by immersion in a solution of lOmg azolectin/mJZ decane was used as a dialysis membrane, and an azolectin layer was formed on the surface of the filter. Proteoliposomes having retinal and naphthyl retinal as chromophores prepared in Example 1 were mixed, and 0.15 mol MCI/ffi and 20 mmol C were mixed.
aCl2/J! The internal solution is an aqueous solution containing
Dialysis treatment was carried out until H settled to 7.0. By this operation, a membrane 1 with proteins fused and adsorbed on the surface of the nitrocellulose filter as schematically shown in FIG. 1 is obtained. A chamber was constructed using this membrane as a diaphragm to separate two layers, and was filled with a 0.15 molar KCI/4 solution. Next, when the wavelength of incident light and the concentration of protons transmitted through the diaphragm were measured using a pH electrode, the proton permeability of this membrane was approximately 44.
It was confirmed that it functions for incident light of 0 nm to about 570 nm. In other words, a planar film was constructed that greatly expanded the range of controlled incident light that is the object of the present invention.

Fire side 1 Purple membrane was extracted in the same manner as in Example 1, dissolved in a 25% by weight solution of dimethylformamide to obtain a developing solution, and developed in a Langmuir tank according to a conventional method. Using a nitrocellulose filter tightly attached to a glass substrate, a monomolecular film of purple membrane was attached to the filter surface by the vertical dipping method. The above filter was incubated in the solution of proteoliposomes prepared in Examples 1 and 2 in which the chromophore was replaced with naphthyl retinal, and was adsorbed and fused.

As schematically shown in FIG. 2, the membrane 1 constructed in this manner allows two different types of proteins to coexist. When a nitrocellulose filter with a purple membrane monolayer and proteoliposomes fused thereon was examined for proton permeability by light irradiation in the same manner as in Example 2, it was found that hydrogen ion permeability was reduced when the wavelength of incident light was 450 nm and 550 nm. It was confirmed that the direction was reversed.

〔Effect of the invention〕

According to the present invention, it is possible to construct an ion-permeable membrane that has selective ion permeability by light irradiation by using a group of substances that absorb light and transport ions, and to improve the membrane ion permeability by light irradiation. When controlling by irradiation, the wavelength range of incident visible light can be set over a wide range, and the effective wavelength range can also be expanded. Roughly speaking, it is also possible to switch the direction of ion transmission through the transmission film of the present invention depending on the incident wavelength and to change it reversibly. This also makes it possible to significantly improve the wavelength selectivity of the ion-permeability.

Since the ion concentration difference between the ion-permeable A membranes can be easily converted into an electrical signal using an ion electrode or the like, the present invention is suitable for optical information processing, such as when converting an optical signal into an electrical signal. It is highly expected that it will contribute to the construction of photoelectric conversion elements in industry and the optoelectronics field.

[Brief explanation of the drawing]

FIG. 1 is a schematic cross-sectional partial view showing an example of the structure of the photoresponsive ion-permeable membrane of the present invention, and FIG. 2 is a schematic cross-sectional view showing another example of the structure of the photoresponsive ion-permeable membrane of the present invention. It is a diagram. DESCRIPTION OF SYMBOLS 1... Photoresponsive ion permeable membrane, 2... Porous support, a... Short wavelength personal incident light, b... Long wavelength side incident light, la-... Short wavelength photoreceptive protein, I b -...Long wavelength photoreceptor protein. Patent applicant Canon Co., Ltd.

Claims (1)

[Claims] 1. Two or more types of substances that actively transport ions when irradiated with light in different wavelength ranges are incorporated and retained in an ion-impermeable lipid membrane or a thin film with an equivalent function. An ion-permeable membrane characterized by 2. The ion-permeable membrane according to claim 1, wherein the substance group that performs ion active transport is a photoreceptor protein or a derivative having an equivalent function. 3. Membrane ion permeation of an ion-permeable membrane in which two or more types of substances that actively transport ions when irradiated with light in different wavelength ranges are incorporated into an ion-impermeable lipid membrane or a thin film with an equivalent function. A method for controlling ion permeability using light irradiation, which is characterized by changing the properties of ions. 4. The method for controlling ion permeability by light irradiation according to claim 3, wherein the substance group that performs ion active transport is a photoreceptor protein or a derivative having an equivalent function.