| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1987: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1986: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
The present studies were conducted to develop an HPLC method for determination of halocynine, a new betaine isolated from the muscle of an ascidian Halocynthia roretzi, and to survey its distribution in the tissues of the ascidian and in the related species. Biosynthesis of halocynine in H. roretzi was also investigated with tracer technics. Results obtained were summarized as follows.
1. The authentic halocynine was esterified with UV-absorbing ,p-dibromoacetophenone and subjected to an HPLC using Shim-pack FLC-CN column and 5 mM KH_2PO_4 buffer containing 5 % CH_3CN (pH 2.0) with an ultraviolet detector operater at 262 nm. Halocynine p-bromophenacyl ester was well separated from the ester of glycinbetaine and other UV-absorbing substances which were abundant in the ascidian muscle. The linear relationship was obtained between the peak areas of halocynine ester and the amounts of halocynine up to 300 nmol.
2. More than 50 mg of halocynine per 100 g was detected in the muscle of H. roretzi throughout the year, but its content was high in winter and low in summer. Small amounts of halocynine were found in the liver and gills but not in the genital gland and tunica. Halocynine ranging from a trace amount to 30 mg per 100 g was also detected in the muscle, liver, gonad and gills of Styela plicata and in the whole bodies of Botrylloides violaceus, Pseudoistoma antinboja and Ciona intestinalis.
3. After ^<14>C-lysine was injected into the muscle of H. roretzi, the animals were kept in a seawater tank for 10 days. Then, a picric acid extract was prepared from the muscle and subjected to an ion exchange column chromatography to isolate halocynine, which showed a radioactivity. The radioactivity migrated with the spots of isolated halocynine on TLC with four different solvent systems. These results clearly show that halocynine is biosynthesized from lysine in H. roretzi.
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