甲状腺刺戟ホルモンの活性と含有糖の量的関係
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概要
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The thyroid stimulating hormone TSH is one of the proteohormones secreted from the anterior lobe of the pituitary. TSH retaining active biological property of proteohormone, however, has not been fully known and as yet it has to be further purified. The true nature of TSH can be ascertained only when successfully purified to a minimum unit.<BR>The present investigation is an attempt made on purifying TSH by means of ion exchange cellulose column chromatography by taking a crude TSH active substance Pretiron (Schering), and studying the relationship between TSH activity and carbohydrate content of TSH.<BR>1. Purification of TSH by ion exchange cellulose column chromatography<BR>For ion exchange cellulose, CM-cellulose and DEAE-cellulose were prepared from powdered cellulose available, and batch technique combined with chromatography was tried. For an optimum condition of purifying TSH, a column of DEAE cellulose was best used by dissolving with tris-buffer at pH 7.6. With this purification method, it was possible to obtain 60 jsu/mg of TSH active substances of which a unit was found to be twice as high as the original Pretiron. This fraction was examined by paper electrophoretically under various pH buffers and we succeeded in isolating a fraction which would present a single pattern at any pH. This supposedly paper electrophoretically identified purified fraction has shown not only content of 60 jsu/mg TSH active substance but it was also some minute quantity of gonadotropic active substance. Although it is possible to obtain and isolate a higher active unit per weight of purified TSH by means of DEAE-cellulose column chromatography, there still remains a further problem of separation of gonadotropic active substance which was dissolved in a fraction near ion concentration 0.4. However, a higher TSH active substance could be obtained in the fraction in the vicinity of 1.4.<BR>2. Purification of TSH and its carbohydrate content, br>Purified TSH active substance was initially obtained through DEAE-cellulose column chromatography and with continuous paperelectrophoresis. The relationship between biological activity and its carbohydrate content was studied. Quantitative measurement of hexose, and hexosamine and sialic acid were made. Hexose was measured by the method of orcinolsulfuric acid using galactose as a standard substance. Hexosamine was determined by the modified Elson-Morgan method using d-glucosamine hydrochloride as the standard substance. Estimation of sialic acid level was done by thiobarbituric acid method, using N-acetyl neuraminic acid originally devised by Yamamoto of Teikoku Hormone Manufacturing Company. For TSH activity Kishimoto's modified Bates method was applied.<BR>(a) Carbohydrate content in purified TSH, with cellulose column chromatography<BR>Pretiron was examined for TSH active substance and its carbohydrate content, and the fraction was extracted by DEAE cellulose column chromatography. It was found that purified TSH active fraction contained a higher unit of hexose than that of unpurified Pretiron, while an amount of hexose increased in direct proportion to further purification of TSH. It was revealed that a large amount of hexosamine was found in low TSH active fraction but hardly any was found in a higher active substance demonstrating the fact that hexosamine is unrelated with TSH. Sialic acid was found to be low compared with Pretiron in which TSH activity was less purified. When the TSH substance was low hardly any sialic acid could be found, though hexose and hexosamine could be detected. It seems that sialic acid is concerned with TSH in some respect.<BR>(b) Carbohydrate content in purified TSH, with continuous paperelectrophoresis.<BR>It was found that the quantity of hexose increased parallel with increased purification of TSH.<BR>3. Variation of TSH activity and carbohydrate content with reference to pH.
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