JPH03279396A - Rhodophyceae lectin and production thereof - Google Patents

Abstract

NEW MATERIAL:Lectin as an extract from algae belonging to genus Gracilaria of Rhodophyceae having about 52000 molecular weight and exhibiting strong activity to rabbit erythrocyte whereas substantially not exhibiting activity to erythrocyte of other animals. USE:Said lectin has sugar-bonding specificity exhibiting strong activity to rabbit erythrocyte and not exhibiting activity to erythrocyte of other animals. Therefore, said lectin is useful in a curing field or a field of biochemical industry, etc., with utilizing the above-mentioned characteristics. PREPARATION:A solution obtained from algae belonging to genus Gracilaria of Rhodophyceae by extraction with a phosphoric acid buffer is subjected to salting-out and the resultant precipitate is dissolved in water, then the resultant aqueous solution is dialyzed, thus subjected to gel filtration and a solid component is isolated from the resultant active fraction.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is a novel lectin extracted from red algae, and more specifically, it exhibits extremely strong activity against rabbit erythrocytes, but has virtually no activity against other animal erythrocytes. The present invention relates to a lectin derived from algae of the genus Ogonori that has sugar-binding specificity that does not exhibit superior activity, and a method for producing the same.

Conventional technology Lectins are sugar-binding proteins that are not immunological products (such as sugar-binding antibodies produced by immunity) found in animals, plants, or bacteria, and have the ability to aggregate animal and plant cells,
It has sugar binding specificity to precipitate polysaccharides and complex carbohydrates.

In recent years, in the medical field and the biochemical industry, attempts have been made to utilize lectins for various purposes by utilizing their properties such as sugar binding specificity. For example, applications to affinity chromatography to purify specific sugars and glycoproteins using the ability of lectins to recognize sugars, methods for searching for cell surface sugar chains using lectins with clear sugar binding specificity, and changes in sugar chains due to diseases. A clinical diagnostic method that recognizes the sugar chain portion of glycoprotein structures in human blood at the enzyme level using lectins, application to blood type determination tests used for paternity testing, and use as specific markers for cancer cells, etc. Research is being conducted on how to use lectins.

As described above, lectins are extremely important substances in the medical field, biochemical industry, etc., but until now, the functions of lectins derived from terrestrial animals and plants have mainly been elucidated and applied research has been conducted.

For example, concanavalin A, a lectin purified from jack beans, and kidney bean lectin (PHA) are known to act on peripheral blood lymphocytes and induce blast formation.
It is also known that lectins derived from Captomyces have properties such as facilitating the determination of blood type used in paternity tests, activating 1928 cells, and promoting the production of interleukins.

In contrast, not much research has been done on lectins derived from marine organisms, which have only recently begun to be studied, and lectins derived from algae are generally more complex than the sugars bound to those derived from land plants. It has been found that it binds to sugars that have a certain structure. However, the role of this algae-derived lectin in vivo is still insufficient.
Furthermore, applications in the fields of medicine, biochemistry, and others remain completely undeveloped. Therefore, it has been desired to search for lectins derived from marine organisms, elucidate their in vivo functions, and develop technologies to apply these functions.

Regarding this algae-derived lectin, a lectin with a molecular weight of 41,000 obtained from Gracilaria verrucosa has been reported [[Buretin of the Japanese Society of Scientific Fisheries] Leeds (
Bulletin of the Japanese
5ociety of 5cien-tific Fi
J, Volume 47, Page 1079 (1
981)). This lectin shows the strongest activity against horse red blood cells, but it also has a strong activity against rabbits, guinea pigs, and sheep, which is 1/2 to 1/8 of the activity against horse red blood cells, and there is little difference depending on the type of red blood cells. unacceptable.

Problems to be Solved by the Invention In order to utilize lectins in various fields of biochemistry and medicine as described above, it is desirable that lectins exhibit sugar binding specificity to as limited a target as possible. The purpose of the present invention is to provide a lectin that is unique in nature and is active only against red blood cells of a specific animal, but not against red blood cells of other animals. It is something.

Means for Solving the Problems As a result of intensive research to develop new lectins derived from marine organisms, the present inventors found that a lectin with a molecular weight of approximately 52,000 obtained from algae of the genus Ogonori, which had not been previously found, was found. Unlike the lectin, which has a molecular weight of 41,000 and is derived from algae of the genus Ogonori, it has a sugar-binding specificity that shows extremely strong activity against rabbit red blood cells, but virtually no activity against other animal red blood cells. Based on this finding, we have completed the present invention.

That is, the present invention is an extract from red algae of the genus Ogonori, which has a molecular weight of approximately 52,000 and exhibits strong activity against rabbit red blood cells, but substantially no activity against red blood cells of other animals. The present invention provides a lectin characterized by exhibiting no activity.

Such a lectin can be obtained by salting out a solution extracted from Rhodophyta algae with a phosphate buffer, dissolving the resulting precipitate in water, dialysis treatment of this aqueous solution, and gel filtration. It can be obtained by isolating the solid content from the obtained active fraction.

The algae used as the raw material in this process belong to the red algae family, and although they exist in cold seas, they are particularly abundant in warm seas, and are distributed in almost all coastal areas in Japan.
It is mixed into agar or used as tsuma for sashimi.

According to a preferred method for obtaining this lectin, first, a phosphate buffer is added to algae of the genus Ogonori and homogenized, followed by centrifugation, and the resulting supernatant is salted out to form a precipitate. An active fraction is obtained by dialysis and gel filtration, which is then sequentially subjected to treatments such as ultrafiltration and high performance liquid chromatography to isolate it as a solid lectin.

Effects of the Invention The lectin derived from algae of the genus Ogonori of the present invention is novel and shows extremely strong activity against rabbit red blood cells, but has virtually no activity against other animals. It has sugar binding specificity, and this property has a wide range of applications in the medical field and biochemical industry.

Examples Next, the present invention will be explained in more detail with reference to examples.
The present invention is not limited in any way by these examples.

Example 10 hours of dried samples obtained by washing Gracilaria sp. (scientific name: Gracilaria sp, produced in Aji-cho, Kida-gun, Kagawa Prefecture) with water and drying them at 37°C were added with 5 mM of ascorbic acid and Na.
10+1M at pH 7.0 containing Ca10.15M
After adding 500 mQ of phosphate buffer and performing extraction treatment by homogenization, this was centrifuged, and ammonium sulfate was added to the obtained supernatant to make a 70% saturated solution by salting out to form a precipitate. Ta.

Next, this precipitate was dissolved in distilled water, dialyzed against distilled water, and separated using a gel filtration column to obtain an active fraction. Next, this was concentrated by ultrafiltration, and then purified by gel filtration using high-speed liquid filtration fJ=z*] to obtain a lectin.

The molecular weight of this lectin was determined by gel filtration high performance liquid chromatography and was found to be 52,000.

Regarding sugar binding specificity, various animal erythrocytes 50
50 μQ of a lectin sample was added to μQ, incubated at 37° C. for 1 hour, and left at room temperature for 2 hours, after which the agglutination activity was observed. The results are shown in the table.

Reference Example Instead of the algae of the genus Ogonori in the example, Ogonori species (
Purified lectin was obtained in the same manner as in Example except that Gracilaria verrucosa (produced in Kominato, Chiba Prefecture) was used, and the hemagglutination activity was determined. The results are shown in the table.

The hemagglutination activity of the crude extract obtained by salting out the supernatant after centrifugation was also determined, and the results are also shown in the table.

[Note] The numerical value is the minimum protein concentration (μ9/mQ) that shows hemagglutination activity.

As can be seen from this table, the lectin of the present invention has extremely strong activity against rabbit red blood cells, but does not have very strong activity against other animal red blood cells.

Claims (1)

[Scope of Claims] 1. A substance extracted from red algae of the genus Ogonori, which has a molecular weight of approximately 52,000 and exhibits strong activity against rabbit erythrocytes, but substantially no activity against erythrocytes of other animals. A lectin characterized by not showing superactivity. 2. Salting out the liquid extracted from red algae of the genus Ogonori using a phosphate buffer, dissolving the resulting precipitate in water, dialysis treatment of this aqueous solution, gel filtration, and removing the solid content from the resulting active fraction. The method for producing a lectin according to claim 1, which comprises separating the lectin.