ji ä ' ^ é * 



*■ ^ i; -^ 



18] STRUCTURE OF PITUITARY HORMONES 317 



noted that the amount of material originally applied to the starch trough 

 could be accounted for by the protein recovered from the peak. 



Biological assays of prolactin — SCH2CONH2 or prolactin — SH accord- 

 ing to either its crop sac-stimulating or its luteotropic activity indicate that 

 reductive cleavage of the disulfide bridges causes the complete destruction 

 of the hormonal activity, indicating that the integrity of the disulfide bridges 

 is essential for the biological function of lactogenic hormones. 



Action of Corboxypeptidose on Sheep Proloctm-S-CHgCONHj 

 0.161- 



2 4 



Hours 



Fig. 7. Action of carboxypeptidase on prolactin — S — CH2CONH2; enzyme-substrate 

 tio, 1 :35;pH8 5at40°C. 



ratio 



When either prolactin — SCH2CONH2 or prolactin — SH was allowed to 

 react with carboxypeptidase, it was found that CyS — CH2CONH2 (or 

 CySH), asparagine, and leucine are released successively (Fig. 7). The libera- 

 tion of CyS — CH2CONH2 was nearly complete in 4 hours under the con- 

 ditions of digestion used. From these studies, it was concluded that prolactin 

 — SCH2CONH2 possesses the sequence . . .Leu.AspNH2.CyS — CH2CONH2 

 at the C-terminus. That this C-terminal sequence is derived from reductive 

 cleavage of a disulfide bridge which forms a loop at the C-terminus of the 

 untreated prolactin molecule is further supported by the identification^^ of 

 C-terminal cysteic acid in performic-acid-oxidized prolactins, by means of 

 the hydrazinolysis method of Akabori. Hence, it may be concluded that 

 sheep and beef prolactins consist of a single polypeptide chain with the 

 sequence Thr.Pro.Val.Thr.Pro at the A^-terminus and with an intrachain di- 

 sulfide loop at the C-terminus (Fig. 8). In this connection, it is of interest 

 to recall that both insulin^^ and the posterior pituitary hormones* have 

 similar intrachain disulfide loops, involving only 20-atom rings. There re- 

 mains to be investigated the size of the loop and the location of the remaining 

 two disulfide bridges in prolactin molecules. In the case of insulin, the only 

 differences between pig, sheep and beef hormones are found among the 

 amino acid residues in the ring. 2*^ So far no differences have been found in 



