Isolation of growth hormone and in vitro translation of mRNA isolated from pituitaries of the gilthead sea bream Sparus aurata.
Growth hormone (GH) polypeptide was purified from pituitary glands of the gilthead sea bream (Sparus aurata) by a two-step procedure involving gel filtration on Sephadex G-100 and reverse-phase high-performance liquid chromatography (rpHPLC). At each stage of purification, fractions were monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting using anti-bonito GH antiserum. The molecular weight of the sea bream GH was estimated by SDS-PAGE to be 21 kDa when electrophoresed in the absence of beta-mercaptoethanol (nonreduced conditions) and 22 kDa when electrophoresed under reduced conditions (in the presence of 1% beta-mercaptoethanol). Pituitary RNA was used to direct cell-free translation. When specific immunoisolation from 35S-labeled proteins was conducted, using antisera against Sparus or tilapia GH, a larger prehormone was immunoprecipitated. The size of the pre-GH was estimated to be 27-28 kDa under reduced conditions and 26-27 kDa under nonreduced conditions, in agreement with the calculated molecular weight of Sparus pre-GH of 26,296 based on the deduced amino acid sequence of Sparus GH cDNA. The specificity of the immunoprecipitation reaction was demonstrated by the ability of recombinant tilapia GH to compete with the radioactively labeled translation product. No such competition was found after the addition of BSA. Our results demonstrate that the sea bream GH is similar in its size to other purified fish GHs and provide direct evidence for the synthesis of GH as a prepeptide, thus supporting the conclusions presented earlier by GH cDNA cloning.
Cavari
B
B
National Institute of Oceanography, Israel Oceanographic & Limnological Research, Tel Shikmona, Haifa.
Le Bail
P Y
PY
Levavi-Sivan
B
B
Melamed
P
P
Kawauchi
H
H
Funkenstein
B
B
D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D013006:Growth Hormone / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010473:Perciformes / Q000378:metabolism*; D010902:Pituitary Gland / Q000737:chemistry* / Q000378:metabolism; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D013006:Growth Hormone