UBC Theses and Dissertations
Isolation and amino acid sequence of neurohypophysial hormones or Pacific chinook salmon (Oncorhynchus tschawytscha). Wilson, Nadine
The neurohypophysial hormones of Pacific chinook salmon (Oncorhynchus tschawytscha) were purified and the amino acid sequence of both hormones determined. The extraction and purification procedure was developed in an effort to maximize the yields of pure hormones. Pituitary glands were extracted at 4°C using 0.2 M acetic acid. Purification consisted of gel filtration, ultrafiltration, and ion exchange. Gel filtration on Sephadex G-15 columns was used to separate neurohypophysial peptides from high molecular weight material. Separation of the two hormones was accomplished on one of three cation exchangers: carboxymethylcellulose, phosphocellulose or sulfoethyl-Sephadex. The hormones were eluted from cation exchange columns using a sodium or ammonium ion concentration gradient at constant pH; pH 5 was used for chromatography on carboxymethylcellulose and on phosphocellulose; pH 2.45 was used for chromatography on sulfoethyl-Sephadex. The two hormones were purified further by rechromatography of individual hormones on another cation exchange medium. The material obtained by rechromatography was pure as determined by amino acid analyses. The yields of pure hormones at the end of purification procedure was 48% of the starting material. The specific activities of the two purified hormones were 145 and 229 oxytocic units per milligram for Hormone I and Hormone II respectively. The amino acid sequence of Hormone I was determined by a combination of three methods: subtractive Edman degradation, partial acid hydrolysis, and Dansyl-Edman technique. The amino acid sequence of Hormone I was found to be that of 4-serine, 8-isoleucine oxytocin. The amino acid sequence of Hormone II was determined by the Edman subtractive method, the Dansyl-Edman technique, and the mobility of the C-terminal residue on high voltage electrophoresis. The amino acid sequence of Hormone II was found to be that of 8-arginine oxytocin. The two neurohypophysial hormones described from salmon have amino acid sequences identical with those described from four species of Gadiformes and one species of Cypriniformes by other workers. The position of Salmoniformes on the evolutionary tree of teleost fishes suggests that these structures are characteristic of a wide range of teleosts.
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