Purification and electrophoretic analysis of glycosylated chicken growth hormone (G-cGH): evidence of G-cGH isoforms.
It has been shown that chicken growth hormone (cGH) exhibits functional and molecular heterogeneity. Mass and charge variants have been described in fresh pituitary extracts and in pure preparations of the hormone. In an attempt to further study the molecular heterogeneity of cGH we have purified the glycosylated variant of this hormone by affinity chromatography and analyzed it by different electrophoretic methods. Purification was achieved by homogeneizing chicken pituitaries in a protease inhibitor solution (0.5 mM PMSF and aprotinin, 50 KIU/ml); the supernatant of the alkaline extract (pH 9.5) was precipitated with 0.15 M ammonium sulfate and metaphosphoric acid, pH 4.0. The supernatant from this step was further precipitated with 80% ammonium sulfate, pH 6.5. After dialysis and lyophilization, the extract was chromatographed in a Con A-Sepharose column. The fraction eluted with 10 mM alpha-methylmannoside (which contained the glycoproteins) was passed through an immunoaffinity column (anticGH). Glycosylated cGH (G-cGH) was obtained pure after this step. Pure G-cGH was analyzed by nondenaturing electrophoresis (ND-PAGE), SDS-PAGE, isoelectrofocusing (IEF), and bidimensional electrophoresis (2D-PAGE) followed by Western blot and staining either with a specific antibody or with peroxidated Con A. Results showed that monomeric G-cGH has a MW of 29 kDa (under reducing conditions) and is heterogeneous, showing at least three important charge variants with pIs 6.5, 6.7, and 7.2. Mass variants of G-cGH were also detected under nonreducing conditions. Bidimensional analysis revealed that the charge variants had a similar MW (29 kDa).
Arámburo
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Departamento de Fisiología, Universidad Nacional Autónoma de México, D.F.
Navarrette
S
S
Montiel
J L
JL
Sánchez
R
R
Berghman
L R
LR
D000818:Animals; D015153:Blotting, Western; D002645:Chickens; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D013006:Growth Hormone / Q000032:analysis* / Q000302:isolation & purification; D007525:Isoelectric Focusing; D010903:Pituitary Gland, Anterior / Q000737:chemistry
D013006:Growth Hormone