LEWIS ET AL. : GROWTH HORMONE- AND PROLACTIN-LIKE PROTEINS 



30 



40 



50 



70 80 90 

 TUBE NUMBER 



100 



120 



130 



Figure 1. — Elution pattern obtained by chromatography of an alkaline extract of the 

 pars distalis on Sephadex G-150. Each tube had a volume of 14 ml. The slow and fast 

 electrophoretic components (S and F) were found in the stippled area. A = albumin; 

 Hb = hemoglobin. 



was eluted by the process. A gradient was next 

 begun and was made by mixing equal portions 

 (100 ml each) of 0.01 M and 0.1 M NH4HCO3. 

 This eluted a major peak. A second gradient 

 of equal volumes (150 ml each) of 0.1 M and 

 0.2 M NH4HCO3 was begun immediately. This 

 gradient eluted a second peak. The results ob- 

 tained with the DEAE-cellulose chromatography 

 are shown in Figure 2. 



The two major peaks eluted during chroma- 

 tography on DEAE-cellulose were lyophilized. 

 The yield of component F was 43 mg; that of 

 component S, 22 mg. 



Figure 2. — Elution pattern obtained by chromatography 

 of the S + F fraction of Figure 1 on DEAE-cellulose. 

 The first gradient was begun at 1 and a second at 2. 

 The S and F components were resolved as indicated in 

 the drawing. 



RESULTS 



ELECTROPHORETIC ANALYSES 

 Pituitary Extracts 



The water homogenate of the pars distalis 

 showed two principal electrophoretic compo- 

 nents when analyzed within 30 min after the 

 sharks were taken from the sea. An alkaline 

 extract of the glands, made about a week later, 

 gave an almost identical pattern except that a 

 definite band could be seen just ahead of each 

 of the major components S and F. This is shown 

 in Figure 3. The S and F components were as- 

 sumed to be growth hormone and prolactin, re- 

 spectively, and were the components for which 

 we devised an isolation procedure. The bands 

 designated as A, B, and C were minor compo- 

 nents seen in both the water homogenate and 

 the alkaline extract. A fast component also 

 migrated near the buffer front. 



— 1 



^^^^S 



B 

 C 





I 



F 

 F 



v. 



FVTRarx INCUBATED 

 EXTRACT EXTRACT 



S F 



PURIFIED 



SERUM 



Figure 3. — Electrophoretic patterns (pH 9.5) of various 

 shark materials. The pattern for the incubated sample 

 was obtained after heating the alkaline extract at 37 °C 

 for 2 hr. S and F are the slow and fast components; 

 S' and F' are the bands that formed during isolation 

 of the proteins. Components A through E are discussed 

 in the text. A thread marks the original buffer front 

 (bromphenol blue) in two of the gels. 



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