Biochemical Studies on Halocynine, a New Betaine Isolated from the Muscle of an Ascidian Halocynthia roretzi.

本研究建立了一种从罗氏梭梭海鞘肌肉中分离出的新甜菜碱--卤草碱的高效液相色谱测定方法，并对其在海鞘组织和近缘物种中的分布进行了调查。用示踪技术研究了罗氏梭子藻对卤代嘌呤的生物合成。研究结果总结如下：1.研究结果。对二溴苯乙酮经紫外吸收酯化后，采用Shim-pack FLC-CN色谱柱，含5%CH3CN(pH 2.0)的5 mM KH_2PO_4缓冲液，检测波长为262 nm。卤代嘌呤对溴苯乙酸酯与甘氨酸甜菜碱的酯以及腹水肌肉中丰富的其他紫外线吸收物质有很好的分离。结果表明：1.卤代碱酯的峰面积与其量在300nmol时呈线性关系。罗氏沼虾肌肉中的卤代嘌呤含量全年在50 mg/100g以上，但冬季含量较高，夏季含量较低。在肝脏和鳃中发现少量的卤代嘌呤，而在生殖腺和生殖器膜中未发现。皱瘤海鞘的肌肉、肝脏、性腺和鳃以及紫背拟木鱼、假孔虫和肠囊的全身组织中也检测到微量至30毫克/100g的卤代嘌呤。将14&gt；C-赖氨酸注入罗氏沼虾肌肉后，放入海水中饲养10天。然后，从肌肉中提取苦味酸提取物，经离子交换柱层析分离出具有放射性的卤代嘌呤。在四种不同溶剂体系的薄层色谱上，放射性随分离的卤草碱斑点而迁移。这些结果清楚地表明，在H.roretzi中，从赖氨酸生物合成了卤代嘌呤。

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.