JPH0488995A - Atp synthesizing device - Google Patents

Abstract

PURPOSE:To obtain an ATP synthesizing device useful for bio-element, etc., readily integrating a material to be integrated by blending liposome comprising phospholipid, bacteriorhodopsin and ATP synthase as constituent elements with a phospholipid antigen as a constituent element. CONSTITUTION:Soybean phospholipid Asolectin 43, etc., shown by formula (R is alkyl) is used as phospholipid, a halophilic bacteria derived bacteriorhodopsin as rhodopsin, ATP synthase 49 prepared from vituline heart mitochondrion as ATP synthase and further phospholipid antigen 45 shown by formula II as a constituent element and these components are dissolved in dried chloroform, irradiated by an ultrasonic grinding device under a condition of one minute irradiation and one minute stopping for 2 hours to give liposome 41. Then, the liposome is integrated through an antigen 51 in to glass substrate 55, a material to be integrated of ATP synthesizing device to give the objective ATP synthesizing device.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention is directed to ATP (adenosine 5°
-triphos-phate: Adenosine triphos M
).

(Prior Art) Current progress in electronic devices, for example, progress in electronic computers, is largely dependent on advances in high integration technology for semiconductor devices, which are the basic constituent devices of electronic computers. On average, 2 per year
The rate has increased by twice as much, and now the rate has quadrupled every three years, although the base is slightly down, it is expected to remain open for the time being.

However, the design rule for semiconductor devices is 0.5 to 0.2.
5 um and the degree of integration becomes 109 transistors/chip, it is predicted that the progress in highly integrated technology will reach a plateau, and therefore the progress of electronic computers will also be expected to reach its limits.

Therefore, as an element to replace a semiconductor device, for example, see Document ① (Japanese Science and Technology (1987)).

As disclosed in p. 69), biologically derived functional devices (hereinafter abbreviated as bio-devices) that combine biomolecules and electronics are attracting attention. The reason is that the ionic element has high tertiary nature and self-organization ability.

For example, in the above document ■, although it has not been realized yet, M
The concept of an ionic element proposed by John Calear is introduced. FIG. 5 is a conceptual diagram of the ionic element.

This biodevice is a monoclonal device that applies cell fusion.
It consists of an antibody 11, an antigen 13, an enzyme 15, a peptide interface 17, and a molecular electronic switch 19.
0, which takes in the conductor 21 and creates a circuit
The molecular electronic switch 19 has a structure in which a protein contributor molecule having porphyrin 1!131 is arranged between the main electrodes 33, as shown in FIGS. 6(A) and 6(B).

In this biodevice, as shown in FIGS. 6(A) and 6(B), when a signal is output from the control electrode 35, solitons propagate through the polyene portion 37, change the structure of the porphyrin ring 31, and cause the main electrode 33 to rotate. It is turned on (state CB in FIG. 6) or off (state WB in FIG. 6(A)). Furthermore, this bio-element is said to create its own circuit network using the amino acids, sugars, lipids, etc. present around the bio-element as raw materials through the action of enzymes such as phosphorase and ATP.

Therefore, just as living organisms require ATP, this type of biodevice also uses ATP as the energy to drive it.
becomes essential.

For example, literature ■ (Journal of Biological Chemistry)
, 249 (2), p. 662 (1974)),
ADP (adenosine 5-diphos)
phate adenosine diphosphate) and inorganic phosphoric acid (Pl)
In order to synthesize ATP from
This is called %ATP synthesis blindness. ) was disclosed.

In this conventional HeTP synthesis device, when exposed to light, bacteriorhodopsin takes H+ into the liposome and A
TP synthase transports + to the outside of the liposome.
At this time, ATP synthesis occurs by ATP synthase using the H1 concentration gradient generated inside and outside the liposome.

(Problems to be Solved by the Invention) However, the conventional HeTP synthesis device disclosed in Document ① does not have a structure that includes a means for incorporating it into a biodevice, so it does not include the biodevice explained using FIG. If various bio-devices are to be created in the future and it becomes necessary to incorporate an ATP synthesis device aL into the bio-devices to drive these bio-devices, there is a problem in that it will not be possible to satisfy the requirements.

The present invention has been made in view of these points, and therefore, an object of the present invention is to provide an ATP synthesis device that can be incorporated into a predetermined position of a biodevice.

(Means for Solving the Problems) In order to achieve this object, according to the present invention, in an ATP synthesis device using a liposome composed of phospholipids, bacteriorhodopsin, and ATP synthase, as constituent elements of the liposome, It is characterized by further containing a phospholipid antigen.

(Function) The ATP synthesizer of the present invention has a structure that includes a phospholipid antigen, so it is possible to incorporate the ATP synthesizer into an object such as a biodevice (hereinafter also referred to as an object to be incorporated). By placing an antigen suitable for the above-mentioned phospholipid antigen in advance at a predetermined position in the phospholipid antigen, the ATP synthesizer can be easily incorporated into the object by utilizing the antigen-antibody reaction.

(Example) Hereinafter, an example of the ATP synthesis apparatus of the present invention will be described with reference to the drawings. Each figure used in the following explanation shows the size, shape, and arrangement of each component to the extent that this invention can be understood. ! The relationship is schematically illustrated. In addition, the names of chemicals used, chemical concentrations, amounts of chemicals used, numerical conditions such as processing time, processing methods, equipment N, etc. used in the following explanation are only preferred examples within the scope of this invention. It should be understood that is not limited to only these conditions.

First, as explained below, the liposomes of Examples (A
TP synthesizer) is prepared. The soybean phospholipid azolectin (manufactured by Sigma) represented by the following formula 0 was used as the phospholipid, and Halobac was used as the bacteriorhodobuscin.
Bacteriorhodopsin prepared from Terlumhalobium (halophilic bacterium) is used, ATPase prepared from calf heart mitochondria is used as the ATP synthase, and an antigen represented by the following formula 0 is used as the phospholipid antigen. However, R in the formula (■) is an alkyl group (C-82-+1
).

0 CH2-QC-R The above mixed lipid of azolectin (phospholipid) and phospholipid antigen, bacteriorhodopsin, and ATPase are mixed, and the mixture is dissolved in dry chloroform in an eggplant-shaped flask.

Next, chloroform is distilled off using a rotary evaporator.

Next, place this flask in the VorteX mixer,
A predetermined pH-adjusted buffer solution is put into this flask to swell and peel off the contents in the flask.

Next, this flask was placed in an ultrasonic crusher (W-385 manufactured by Heat System Co., Ltd.) and kept cool at 4°C.
Ultrasonic waves of 475 W are irradiated for 2 hours under 2 air currents under irradiation conditions of 1 minute irradiation and 1 minute stop. The suspension obtained by this,
A liposome suspension is obtained by fractionation on a Sepharose 4B column (manufactured by Pharmacia, 1.8φ x 4Qcm) equilibrated with the above buffer solution.

Figure 1 shows the concentration of 1 in the liposome suspension thus obtained.
FIG. 3 is a diagram schematically showing the appearance of one liposome 41.

In Figure 1, 43 is azolectin, 45 is phospholipid antigen,
47 is bacteriorhodopsin and 49 is ATPase. This liposome 41 contains a phospholipid antigen 45 as a component of the liposome.

When this liposome 41 is irradiated with light (light containing light with a wavelength of 540 nm, the absorption peak of bacteriorhodopsin),
H” in bacteriorhodopsin 47 or liposome 41!
The uptake ATPase carries this H+ out of the liposome and ATP is synthesized. This ATP synthesis function is the same as that of the conventional ATP synthesis apparatus disclosed in Document (2).

However, the liposome 4 of this invention] (i.e., the liposome of this invention
TP synthesizer) injects phospholipid antigen 45 into liposome 41.
Therefore, the liposome 41 can be easily incorporated into the object by utilizing an antigen-antibody reaction. This will be explained in detail below.

A glass substrate is used as the object to be incorporated into ATPA, and the liposome 41 is incorporated into this glass substrate as described below.

First, the glass substrate surface is hydrophobized by immersing the glass substrate in octadecyltrichlorosilane in advance.

In addition, γ-globulin%, which is an antibody against phospholipid antigen 45 (particularly aroma 111 of the above-mentioned phospholipid antigen of formula 0)
The antibody is developed on a subphase aqueous solution in a water tank for the LB (Langmuir-Prodgett) method, and the aqueous solution is compressed to form a monomolecular film of the antibody on the aqueous solution.

Next, the above-mentioned hydrophobized glass substrate is immersed in this water tank in which the antibody monomolecular film has been formed, and the antibody monomolecular film is transferred onto the glass substrate by the horizontal deposition method of the LB method.

FIG. 2 shows the monomolecular film 5 of antibody 51 produced in this way.
FIG. 3 is a cross-sectional view schematically showing the appearance of a glass substrate 55 having a size of 3.

Next, a glass substrate 55 having a monomolecular film 53 of the antibody 51 is
by immersing it in the liposome suspension described above,
Allow antigen-antibody reaction to occur. As a result, as shown in a plan view in FIG.
1 (ATP synthesis system @) is installed in a two-dimensional array state.

Next, we investigated whether ATP could be synthesized by irradiating the glass substrate 55 in which the liposome 41 was incorporated with light.
Confirm by the method disclosed in Document ①.

For this purpose, first, 50 units of yeast hexokinase, 20 mM (mmol) of inorganic phosphoric acid 32P, 2 m
M9SO4, 1mM ATP, 32mM glucose, 5mM Tris sulfate, 0.3mM EDTA (ethylenediaminetetraacetic acid) and 1mg calf serum albumin! Prepare a test solution by dissolving O, IM in sucrose. Then, the above-described glass substrate 55 with liposomes 41 is immersed in this test liquid.

Next, the amount of radioactive glucose-6-phosphate produced when the glass substrate 55 is irradiated with white light in this state is analyzed.

Figure 4 is a characteristic diagram showing this result, with the horizontal axis representing the irradiation time (minutes) of white light and the vertical axis representing the amount of glucose-6-phosphate produced (micromoles (M)). FIG. 2 is a characteristic diagram showing the relationship between the two.

Since the main component of radioactive glucose-6-phosphate is associated with ATP synthesis from 32P, it is understood that ATP synthesis is performed by light in the glass substrate 55 incorporating ribo-worm 41 of the example. I can do it. In this manner, the ATP synthesis device of the present invention can perform ATP synthesis even after being incorporated into a glass substrate.

Although the embodiments of the ATP synthesis apparatus of the present invention have been described above, the present invention is not limited to the above embodiments.

For example, in the above embodiment, the object to be incorporated is a glass substrate, but it is expected that this ATP synthesis apparatus can be used by being incorporated into an object other than a glass substrate, such as a biodevice as shown in Fig. 5. Specifically, it is expected that the antibody against the phospholipid antigen of the example can be incorporated into the biodevice shown in FIG. 5, and the ATP synthesizer of the example can be incorporated and used by utilizing the antigen-antibody reaction.

It is also expected that it can be incorporated into various biochemical reaction systems that use ATP.

(Effect of the invention) As is clear from the above explanation, the ATP of this invention
Synthetic devices can be incorporated into predetermined positions of various objects such as bio-devices that use ATP as an energy source using antigen-antibody reactions, and ATP% can be synthesized in response to light at that position. Because it can do so, its utility value is higher than that of non-electronics.

[Brief explanation of the drawing]

FIG. 1 is a diagram schematically showing the liposome of the example which is formed in the ATP synthesis device tank. FIG. 2 is a diagram for explaining the material to be incorporated in the example. FIG. 3 is the liposome of the example Figure 4 is a diagram showing the results of a confirmation experiment for ATP synthesis. Figure 5 is a conceptual diagram of a conventionally proposed bio-device. Figures (A) and (B) are diagrams for explaining the bioelement shown in Figure 5. 41--Liposome (ATP synthesizer) 43--Azolectin (phospholipid) 45--One lipid antigen 47--Bacteriorhodopsin 49-ATPase, 51--Antibody 53--Antibody Monomolecular film 55 = -Glass substrate (object to be incorporated into ATP synthesis device) Patent applicant Oki Electric Industry Co., Ltd. ATP synthesis device Fig. 1 schematically showing the liposome of Tora Example constituting the ATP synthesis device. Fig. 2 shows the results of a confirmation experiment for ATP synthesis;

Claims (1)

[Claims] (1) An ATP synthesizer using a liposome composed of phospholipids, bacteriorhodopsin, and ATP synthase, characterized in that the liposome further contains a phospholipid antigen as a component.