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

为了阐明雄性牛蛙睾丸中促性腺激素的功能与精子成熟的关系，我们分离了lutropin （LH）和卵泡激素（FSH），研究了它们的生物学活性，如排卵活性和促进睾丸重量的增加。我们还测定了牛蛙LH和FSH的氨基酸序列和糖链结构。我们试图分离精子细胞成熟过程中出现和消失的睾丸特异性蛋白或过渡蛋白（TP）和鱼精蛋白。睾丸保存在冷丙酮或肉中，用丙酮匀浆使其脱水。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氨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。采用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、脾腺治疗为参照。仅在用组蛋白制备的睾丸中观察到的一个条带似乎是TP或鱼精蛋白。该蛋白和组蛋白H1s用73%的丙酮完全沉淀，而其他组蛋白用90%的丙酮完全沉淀。以73%丙酮沉淀的物料，采用C4、C8、C10、苯基柱进行反相高效液相色谱分馏。蛋白质被分离成8个峰。在SDS凝胶电泳中，TP或鱼精蛋白被洗脱出具有相同迁移率的6个峰。目前尚不清楚有多少TP或鱼精蛋白香料存在。

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.