FISHERY BULLETIN: VOL. 70. NO. 3 



MATERIALS AND METHODS 



DISC ELECTROPHORESIS 



Our use of the method has been described 

 (Lewis, Litteria, and Cheever, 1969), For rou- 

 tine analytical analyses the gel was made of 

 7.5% acrylamide and 0.25% N-N'-methylenebis- 

 acrylamide. For quantitation of the amount of 

 protein in a stained band, ethylene diacrylate 

 was substituted for the cross linking agent. 



MOLECULAR WEIGHT 



Molecular weights wer^ estimated by using 

 disc electrophoresis (Cheever and Lewis, 1969). 



PEPTIDE MAPPING AND 

 AMINO ACID ANALYSES 



Our procedure for peptide mapping has been 

 described (Seavey and Lewis, 1971). Total 

 amino acid analyses were done on performic acid 

 oxidized samples by means of automated anal- 

 ysis. Tryptophan was determined by the meth- 

 od of Beaven and Holiday (1952). 



IMMUNODIFFUSION 



Mouse growth hormone prepared by the meth- 

 od of Cheever et al. (1969) was used for im- 

 munization of monkeys; antiserum to mouse 

 prolactin was produced in rabbits by using hor- 

 mone prepared as described in the same publi- 

 cation. The antiserum to mouse growth hor- 

 mone was not contaminated with antibodies to 

 mouse serum proteins or mouse prolactin; the 

 antiserum to mouse prolactin, likewise, did not 

 give a precipitin line with serum proteins nor 

 did it cross react with mouse growth hormone. 

 Immunodiffusion was carried out by the method 

 of Ouchterlony. 



COLLECTION AND STORAGE OF GLANDS 



The pars distalis was removed within 30 min 

 after the animals were killed and immediately 

 homogenized in water (5 ml/g wet tissue) at 

 5°C. The homogenate was stored at — 20°C un- 

 til it could be fractionated. This procedure was 



used to minimize alteration of the proteins, as 

 judged by electrophoretic analysis. More rapid 

 changes occurred when the glands were stored 

 intact. 



ISOLATION PROCEDURE 



All processes were carried out at 5°C. The 

 pars distalis (7.6 g wet weight) from 87 blue 

 sharks were homogenized in 38 ml water. The 

 pH of the homogenate was adjusted to 10 with 

 dilute NaOH and made 10 -^ m in diisopropyl- 

 phosphofluoridate. After 4 hr of stirring the ho- 

 mogenate was centrifuged at 20,000 x g for 40 

 min. The supernatant fluid was decanted, its pH 

 lowered to 8 with dilute HCl and set aside. The 

 insoluble residue was suspended in 0.05 M 

 Na2C03-NaHC03 buflfer, pH 10 (2.5 ml/g ori- 

 ginal tissue) and stirred for 16 hr. After cen- 

 trifugation, the supernatant fluid was pooled 

 with the first extract and the pH of the mixture 

 adjusted to 8. The insoluble tissue was dis- 

 carded when electrophoresis showed that all the 

 desired components had been extracted. 



The crude extract was concentrated to about 

 15 ml on a Diaflo UM-10 membrane' (Amicon, 

 Lexington, Mass.) and placed on a column of 

 Sephadex G-150, 5 x 90 cm and developed with 

 0.01 M NH4HCO3. Every fifth tube was an- 

 alyzed by disc electrophoresis (100 /u,liter ali- 

 quots) and the tubes that contained the two 

 proteins that were assumed to be gro\\i;h hor- 

 mone and prolactin were combined and concen- 

 trated to about 10 ml on a UM-10 Diaflo mem- 

 brane. Figure 1 shows the elution pattern of 

 the Sephadex column; the stippled area con- 

 tained the desired components, S and F. 



The concentrated solution from the Sephadex 

 chromatography was applied to a column of 

 DEAE-cellulose (Whatman 32) with dimensions 

 of 0.9 X 10 cm and which had been equilibrated 

 with 0.01 M NH4HCO3. The flow rate was 

 38 ml/hr. Once the sample had been applied, 

 the column was washed with 25 ml of 0.01 M 

 NH4HC03. No appreciable amount of protein 



° Reference to trade names in this publication does 

 not imply endorsement of commercial products by the 

 National Marine Fisheries Service, 



934 



