First Stage. the Tumor Knods on Omentum Majus of Rats Transplanted
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The Tohoku Journal o! Experimental Medicine, Vol. 65, Nos. 2-3, 1957 Biochemical Studies on Carbohydrates CXC‡Z. Glucidamins in Yoshida Sarcoma By Kohtaroh Hasizume (橋 爪 幸 太 郎) From the Medico-chemical Institute, Tohoku University, Sendai; Director : Prof. H. Masamune (Received for publication, June 26, 1956) Molisch-giving high-molecular components excepting the pentose- nucleic acid were sought in Yoshida sarcoma. Three glucidamins were isolated. Their yields, however, were very small probably because of the presence of large amounts of nucleic acids and a hexuronic acid-containing mucopolysaccharide or mucopolysaccharides from which they had to be liberated. No Molisch-giving mucopolysaccharide was detected . EXPERIMENTAL Fractionation was carried out, taking the result of Molisch test as the main indication. First stage. The tumor knods on omentum majus of rats transplanted with Yoshida sarcoma were collected and preserved in 4 volumes of methanol for various durations of time. They were squeezed and dried at first in a Faust apparatus, and after pulverizing in an iron mortar, further dried in a vacuum-desiccator containing CaCl2. The material given weighed 1,040 g. Second stage. A 250 g. portion of it was shaken with 10 volumes of water at 7-14•Ž for 72 hours, employing a few drops of toluene to avoid putrefaction. The residue was further treated in a similar manner with 7.5, 5 and 5 volumes of water in succession. The fourth solution was only slightly Molisch positive. The somewhat opalescent solutions were added to with 30% acetic acid to pH 4.2 and centrifuged. The centrifu gates which were Molisch negative or faintly Molisch positive were dis carded. The slightly turbid supernatant fluids were each distilled in vacuo to about 1/30 the volume and kept in a refrigerator. They were then com bined and dialyzed against running water for two days. The precipitate that occurred during the dialysis was rejected, and the clear fluid was condensed to a syrup, precipitated with an excess of ethanol, and after washing with ethanol (Sodium acetate was added to the ethanol when 195 196 K. Hasizume necessary), dried in a desiccator. 6.1 g. of a white powder with brownish hue were given. After repetition of this procedure, 25.8 g. of substance were yielded from the whole material-Fr. A. Third stage. 8.6 g. of it were taken for one run. It was dissolved in 100 volumes of water and acidified to pH 4.2 by means of 30% acetic acid as above to remove the part insoluble at this pH. The clear centri fuged supernatant was added to with 1/4 the volume of 20% trichloro- acetic acid under agitation. After standing in a refrigerator for several hours, the voluminous flocculent precipitate was centrifuged off, and the transparent supernatant here was dialyzed against running water for 2 days until the pH of the solution was raised to 5.6 and distilled in vacuo to a syrup, which was precipitated with excessive ethanol and dried after washing with several changes of ethanol. Yield 2.1 g. The products after three runs, which weighed 6.2 g. in total, were united-Fr. B. Attempts failed to free the trichloroacetic acid-precipitate from nucleic acids. Fourth stage. Fr. B was Neuberg-Saneyoshi positive and its solution showed strong absorption at the wave-length of 260 my characteristic of nucleic acids. Therefore an orientating experiment was made with a part of the substance to see if liberation from polyuronic and nucleic acids is achievable, as follows. 0.6 g. of it was taken up in 100 volumes of water, and crystalline barium acetate was added to 3%. The solution that became cloudy was placed in a refrigerator for several hours, and the centrifuged supernatant was added to with ice-chilled ethanol to 15% and again stood in the refrigerator for several hours. The precipitate was quickly separated by centrifugation, and the clear supernatant was further subjected to fractionations in a similar manner, adding additional volumes of ethanol. The precipitates were separately dissolved in 100 volumes of water and added to with the sat. Na2SO4 just sufficient to carry down all the barium, and the filtrates were each dialyzed until no more SO4-ions were detected, condensed and precipitated with alcohol. * The precipitate that occurred on addition of barium acetate. t The fluid containing 60% ethanol was dialyzed, condensed to a small volume and precipitated with a large volume of ethanol. All of the divisions showed the positive Molisch reaction and absorption at the wave-length of 260 mƒÊ. The absorption was strongest in Div. I and became weaker and weaker with in- crease of the Division number, but even Div. 6 which was not precipitated by alcohol at 60% Glucidamins in Yoshida Sarcoma 197 concentration showed the absorption though weak. Hence fractionation was continued in a similar manner on 220 mg. of that last division. The subdivisions were given with yields : * The part soluble in 60% ethanol was recovered. It was also found that the higher the ethanol concentration was, the subdivision precipitated showed the less strong absorption. Subdiv. 3•Œ was almost free from nucleic acids and gave Goldschmiedt reaction scarcely. Therefore, the balance of Fr. B was treated in the same way and 0.72 g. of a fraction corresponding to Subdiv. 3•Œ was obtained-Fr. C. Balances of Divs. 1-5 and Subdivs. l•Œ-2•Œ were combined and subjected to Kerr and Serai- darian1) procedure and treatments with cupric chloride and lead acetate. But the mixture could not be perfectly freed from nucleic acids. Fifth stage. Since biuret reaction of Fr. C was very strong in con trast to Molisch reaction, an orientating experiment was again processed in advance: 130 mg. of Fr. C were taken up in 10.3 cc. of 90% phenol and the centrifuged supernatant (transparent brownish) was fractioned with increasing volumes of ethanol at room temperature. The precipitates were separately washed with abs. ethanol and ether thoroughly, until they smelled no more of phenol. The substances given were white powders. The yields of them will be tabulated together with the results of their Molisch and biuret tests. * The insoluble part in 90% phenol . t The supernatant of Div. vii was pre cipitated with an excess of ethanol containing some sodium acetate. Divs. iv and v which were Molisch positive were combined and again taken up in 5.5 cc. of 90% phenol and the solution was further fractioned in a similar manner. The results here will be also tabulated : 198 K. Hasizume *The 90% phenol-insoluble (This subdivision will be referred to as Fr. Dl). t The supernatant of Subdiv. vii•Œ was precipitated with an excess of ethanol containing sodium acetate. Based on the finding, the balance of Fr. C (590 mg.) was treated as above. 382 mg. of a white powder were obtained after precipitation at ethanol concentration range of 0-50% in the primary fractionation, and 85 mg. of this fraction remained undissolved in 90 % phenol (Fr. D2) and 253 mg. were precipitated from the solution by addition of ethanol to 50% in the secondary fractionation. The latter fraction in the secondary frac tionation was again taken up in 90% phenol and after setting aside the 90% phenol-insoluble part (23.5 mg., Fr. D3), the portion which preci pitated on addition of ethanol to the solution up to 30 % (68 mg.; Fr. E) and that which precipitated on increasing the ethanol concentration to 45 % (112.1 mg.; Fr. F) were separated. Sixth stage. a) Frs. Dl-D3, which were insoluble in 90% phenol, (131.5 mg. in total ) were united, washed exhaustively with 90% phenol, alcohol and ether and dried. Then, the mixture was dissolved in 13 cc. of water and alkalinized with NaOH to pH 9.0, whereby a precipitate occurred. The centrifugate, which remained mostly as an ash on ignition, was discarded, and the clear supernatant was neutralized, condensed and precipitated with ethanol. 74 mg. was the substance given. A part of it was dissolved in 100 volumes of acetate buffer of pH 4.6 and I 0.2 and examined electrophoretically by sending 10 mA current at 14•Ž. The solution showed a main slowly-moving boundary with mobility of 3.24 •~ 1.0-5 cm2/volt/sec and an accessory boundary quickly moving with mobility of 29.0 •~ 10-5 cm2/volt/sec, so that Masamune and others' electrophoretic sedimentation method2) of fractionation was applied to the balance of the substance. Because of shortage of material, the fractionation was limited to a single run. The slowly-moving fraction separated (Prep. I) was 43.1 mg. The quickly-moving amounted to 14.9 mg. and proved Molisch faintly positive, direct-Osaki-Turumi negative, indirect-Osaki-Turumi faint- ly positive, Neuberg-Saneyoshi markedly positive and biuret negative. Glucidamins in Yoshida Sarcoma 199 b) Purification of Fr. F (Purification of Fr. E was postponed because of its small amount). Fr. F still showed several boundaries when its 1 solution in acetate buffer of pH 4.6 and I 0.2 was examined in a Tiselius apparatus. Hence, it was again fractioned by the procedure in the fourth stage. Namely, 98 mg. of it were dissolved in 10 cc. of water containing 0.3 g. of crystalline barium acetate, and ethanol was added to 60%. The precipitate was discarded, because it looked to be chiefly nucleic acids. The mother fluid was distilled in vacuo to dryness. The still-residue was dissolved in 100 cc. of water and passed through a 1.2 •~ 7 cm. column of Amberlite IR-120, then the effluent was condensed to a syrup, precipitated with ethanol and dried-Prep.