TEST-SPECIFIC PROTEIN AND PROTAMIN OF THE BULLFROG,Rana Catesbeiana

牛蛙的测试特异性蛋白质和蛋白质

• 批准号: 07640890
• 负责人: HAYASHI Hiroko
• 金额: $ 1.41万
• 依托单位: GUNMA PREFECTUARAL COLLEGE OF HEALTH SCIENCES
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07640890/
• 关键词: bullfrog; testis-specific protein; transition protein; protamin; gonadotropin

To clarify the relationship between the functions of gonadotropins and the maturation of sperm in testis gland of male bullfrog, Rana catesbeiana, we had isolated lutropin (LH) and follitropin (FSH), studied their biological activities such as ovulation activity and enhance the increase of the weight of testis gland. We also determined the amino acid sequence, and the sugar chain structures bound to the bullfrog LH and FSH.We attempted to isolate the testis-specific protein or transition protein (TP), which believed to appear and disappear during the maturation of sperm cell, and protamine. Testis glands kept in cold acetone or flesh, were delipidated by homogenizing with acetone. Basic proteins were extracted by two methods, one was directly extracted with 0.25M HCL,and other was after washing with Na-citrate buffer containing Na-bisulfite and p-amidinophenyl methanesullfonyl fluoride as the protease inhibitor. Two preparations were compared by 15% polyacrylamide SDS or acid urea gel electrophoresis system. As the references, liver, kidney, spleen glands were also treated with the same ways. One band observed only in the preparation from testis with histones seemed to be TP or protamine. This protein and histone H1s were completely precipitated with 73% acetone by volume, whereas other histones were with 90% acetone. Materials precipitated with 73% acetone were further fractionated by reversed phase HPLC using C4, C8, C10 and phenyl columns. Proteins were separated into 8 peaks. TP or protamine was eluted in 6 peaks having the same mobilities in SDS gel electrophoresis. It is not known how many spices of TP or protamine are present.

为了阐明雄性牛蛙睾丸腺中促性腺激素的功能与精子成熟的关系，本实验分离了促黄体生成素（LH）和促卵泡生成素（FSH），研究了其生物学活性，如促排卵活性和促进睾丸腺增重作用。我们还测定了牛蛙LH和FSH的氨基酸序列和糖链结构，并试图分离睾丸特异性蛋白或过渡蛋白（transition protein，TP）和鱼精蛋白。将睾丸腺置于冷丙酮或果肉中，用丙酮脱脂。碱性蛋白的提取方法有两种，一种是用0.25M盐酸直接提取，另一种是用含有亚硫酸氢钠和对脒基苯基甲磺酰氟作为蛋白酶抑制剂的柠檬酸钠缓冲液洗涤后提取。用15%聚丙烯酰胺SDS或酸性尿素凝胶电泳系统对两种制剂进行比较。以肝、肾、脾腺为对照。仅在睾丸组织蛋白中观察到一条带，似乎是TP或鱼精蛋白。该蛋白质和组蛋白H1用73%体积的丙酮完全沉淀，而其它组蛋白用90%体积的丙酮沉淀。用73%丙酮沉淀的物质通过反相HPLC使用C4、C8、C10和苯基柱进一步分馏。蛋白质分离为8个峰。在SDS凝胶电泳中，TP或鱼精蛋白以具有相同迁移率的6个峰洗脱。目前还不知道有多少香料的TP或鱼精蛋白存在。

项目成果

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Hiroaki Hayashi：“来自公猪晚期精子的过渡蛋白 4 的氨基酸序列及其与核体核心 DNA 的相互作用”Eur.J.Biochem. 233. 179-185 (1995)

Kikuyama, S.Toyoda, F., Ohmiya, Y., Matsuda, K., Tanaka, D.and Hayashi, H: "A female-attracting peptide pheromone in the newt cloacal glands." Science. 367. 1643-1645 (1995)

Kikuyama, S.Toyoda, F.、Ohmiya, Y.、Matsuda, K.、Tanaka, D. 和 Hayashi, H：“蝾螈泄殖腔腺中的雌性吸引肽信息素。”

林 宏昭: "Sodefrin A Female Attracting Peptidl Pheromone in Newt Cloaeal Glands" Science. 267. 1643-1645 (1995)

Hiroaki Hayashi：“Sodefrin 在蝾螈泄殖腔腺中吸引雌性肽信息素”《科学》267。1643-1645 (1995)

Kitu yama,S.: "A female-attracting peptlde pheromone in the neut cloacal glands" Science. 367. 1643-1645 (1995)

Kitu yama，S.：“中性泄殖腔腺中的女性吸引肽信息素”科学。

Toyoda, F., Hayashi, H., Tanaka, S., Motida, H., Matsuda, K.and Kikuyama, S.: "Novel female-attractant in the abdominal gland of the mama newt, Cynops pyrrhogaster." C.R.Soc. Biol.189. 1143-1148 (1995)

Toyoda, F.、Hayashi, H.、Tanaka, S.、Motida, H.、Matsuda, K. 和 Kikuyama, S.：“蝾螈妈妈腹腺中的新型雌性引诱剂。”