Purification of lutropin and follitropin in high yield from horse pituitary glands.
Abstract: A method has been developed for the purification of equine lutropin (eLH) and equine follitropin (eFSH) from horse pituitary glands which attains high yields of both hormones in contrast to previous methods that were devoted to one or the other with inferior recovery of the hormones. Two-pass chromatography over CM-Sephadex was used to separate eLH from eFSH. Subsequent steps employing QAE (quaternary amino-ethyl)-Sephadex chromatography and gel filtration on Sephacryl S-200 produced highly purified hormone preparations. Yields of purified eLH and eFSH were 110 and 60 mg/kg of frozen pituitaries, respectively. Subunits were prepared by dissociation in 8 M guanidine HCl followed by either gel filtration (eLH) or gel filtration followed by QAE-Sephadex chromatography (eFSH). The hormones and their subunits were characterized by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, NH2-terminal analysis, and by LH and FSH radioligand receptor assays.
Publication Date: 1984-02-10 PubMed ID: 6420415
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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The researchers developed a novel method for extracting and purifying equine lutropin (eLH) and equine follitropin (eFSH) from horse pituitary glands which resulted in high quality and quantity of both hormones, significantly improving on previous methods.
Methodology
- The researchers used a twice-pass chromatography over CM-Sephadex to separate eLH from eFSH. The process involved passing the mix of hormones through a chromatography column filled with CM-Sephadex, which is a substance that binds with certain proteins, allowing others to be filtered out.
- The separated hormones underwent further steps of purification. This involved QAE (quaternary amino-ethyl)-Sephadex chromatography and gel filtration on Sephacryl S-200. The QAE-Sephadex chromatography is another method of separation using different materials while the gel filtration method removes impurities from the hormone preparation.
- As the final step, the subunits of these hormones were prepared by dissociating them using 8M guanidine HCl. For the eLH hormone, only gel filtration was needed after this, while eFSH required another round of a QAE-Sephadex chromatography.
Results
- The purification process resulted in yields of 110 mg/kg and 60 mg/kg of purified eLH and eFSH respectively from frozen pituitaries. These figures represent the amount of hormones that were successfully extracted and purified from the pituitary glands.
- The hormones and their subunits were then characterized/analyzed in many ways. Sodium dodecyl sulfate-gel electrophoresis was used to see the proteins’ patterns after they are subjected to an electric field. Amino acid analysis was used to determine which types of amino acids make up these hormones. An NH2-terminal analysis was performed to get a comprehensive look of the amino acids at the beginning of the protein. Finally, the LH and FSH radioligand receptor assays were used to determine how well the hormones can bind to receptors.
Cite This Article
APA
Bousfield GR, Ward DN.
(1984).
Purification of lutropin and follitropin in high yield from horse pituitary glands.
J Biol Chem, 259(3), 1911-1921.
Publication
Researcher Affiliations
MeSH Terms
- Amino Acids / analysis
- Animals
- Biological Assay
- Chromatography, Gel / methods
- Chromatography, Ion Exchange / methods
- Ethanol
- Follicle Stimulating Hormone / isolation & purification
- Horses
- Luteinizing Hormone / isolation & purification
- Macromolecular Substances
- Pituitary Gland / analysis
Grant Funding
- AM 09801 / NIADDK NIH HHS
- HD 8338 / NICHD NIH HHS
Citations
This article has been cited 10 times.- Butnev VY, May JV, Brown AR, Sharma T, Butnev VY, White WK, Harvey DJ, Bousfield GR. Human FSH Glycoform α-Subunit Asparagine(52) Glycans: Major Glycan Structural Consistency, Minor Glycan Variation in Abundance. Front Endocrinol (Lausanne) 2022;13:767661.
- Zariñán T, Butnev VY, Gutiérrez-Sagal R, Maravillas-Montero JL, Martínez-Luis I, Mejía-Domínguez NR, Juárez-Vega G, Bousfield GR, Ulloa-Aguirre A. In Vitro Impact of FSH Glycosylation Variants on FSH Receptor-stimulated Signal Transduction and Functional Selectivity. J Endocr Soc 2020 May 1;4(5):bvaa019.
- Bousfield GR, Harvey DJ. Follicle-Stimulating Hormone Glycobiology. Endocrinology 2019 Jun 1;160(6):1515-1535.
- Bousfield GR, Butnev VY, White WK, Hall AS, Harvey DJ. Comparison of Follicle-Stimulating Hormone Glycosylation Microheterogenity by Quantitative Negative Mode Nano-Electrospray Mass Spectrometry of Peptide-N Glycanase-Released Oligosaccharides. J Glycomics Lipidomics 2015;5(1).
- Bousfield GR, Butnev VY, Butnev VY, Hiromasa Y, Harvey DJ, May JV. Hypo-glycosylated human follicle-stimulating hormone (hFSH(21/18)) is much more active in vitro than fully-glycosylated hFSH (hFSH(24)). Mol Cell Endocrinol 2014 Feb 15;382(2):989-97.
- Wehbi V, Tranchant T, Durand G, Musnier A, Decourtye J, Piketty V, Butnev VY, Bousfield GR, Crépieux P, Maurel MC, Reiter E. Partially deglycosylated equine LH preferentially activates beta-arrestin-dependent signaling at the follicle-stimulating hormone receptor. Mol Endocrinol 2010 Mar;24(3):561-73.
- Butnev VY, Gotschall RR, Baker VL, Moore WT, Gout PW, Bousfield GR. Glycosylated equine prolactin and its carbohydrate moiety. J Protein Chem 1996 Jul;15(5):413-26.
- Gordon WL, Bousfield GR, Ward DN. Comparative binding of FSH to chicken and rat testis. J Endocrinol Invest 1989 Jun;12(6):383-92.
- Zariñán T, Espinal-Enriquez J, De Anda-Jáuregui G, Lira-Albarrán S, Hernández-Montes G, Gutiérrez-Sagal R, Rebollar-Vega RG, Bousfield GR, Butnev VY, Hernández-Lemus E, Ulloa-Aguirre A. Differential effects of follicle-stimulating hormone glycoforms on the transcriptome profile of cultured rat granulosa cells as disclosed by RNA-seq. PLoS One 2024;19(6):e0293688.
- Zariñán T, Espinal-Enriquez J, De Anda-Jáuregui G, Lira-Albarrán S, Hernández-Montes G, Gutiérrez-Sagal R, Rebollar-Vega RG, Bousfield GR, Butnev VY, Hernández-Lemus E, Ulloa-Aguirre A. Differential effects of follicle-stimulating hormone glycoforms on the transcriptome profile of cultured rat granulosa cells as disclosed by RNA-seq. bioRxiv 2023 Oct 20;.
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