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Home / Equine Research Bank / J Protein Chem / Glycosylated equine prolactin and its carbohydrate moiety.
Journal of protein chemistry1996; 15(5); 413-426; doi: 10.1007/BF01886848

Glycosylated equine prolactin and its carbohydrate moiety.

Abstract: Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were purified to homogeneity from side fractions obtained during isolation of LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcine PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, NaCl and isoelectric precipitation, and gel filtration. Purification of G-ePRL required additional Con A chromatography. The N-terminal amino acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequences identical to the known primary structure of ePRL. Based on MALDI mass spectrometry analysis and SDS-PAGE mobilities, G-ePRL and ePRL had estimated molecular weights of 25,000 and 23,000 Da, respectively. G-ePRL displayed only 60% of the immunoreactivity of ePRL in homologous radioimmunoassay. Using the Nb2 lymphoma cell bioassay, ePRL was found to have about 1/30th the mitogenic activity of bovine PRL; G-ePRL was approximately 1/10th as active as ePRL. Glycosylation of G-ePRL at Asn31 was confirmed by isolation and sequence analysis of an enzymatically derived G-ePRL glycopeptide spanning residues 29-37. Monosaccharide compositions of intact G-ePRL and this glycopeptide were very similar (Man3, GlcNAc2, GalNAc1, Fuc0.6, Gal0.2, NeuAc0.15) and resembled that of G-pPRL. The glycopeptide contained one sulfate residue as determined by ion chromatography after acid hydrolysis, indicating the presence of a sulfated monosaccharide. Comparative carbohydrate analysis of G-ePRL and other G-PRL preparations suggests that the functionally significant Asn31 carbohydrate unit is a fucosylated complex mono- and /or biantennary oligosaccharide terminating with a sulfated GalNAc residue and two or three Man residues.
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Publication Date: 1996-07-01 PubMed ID: 8895086DOI: 10.1007/BF01886848Google Scholar: Lookup
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  • Comparative Study
  • Journal Article
  • Research Support
  • U.S. Gov't
  • P.H.S.

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article primarily discusses the purification, sequencing, and analysis of glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL from horse pituitaries. The investigation reveals information on the molecular weights, immunoreactivity, mitogenic activity, and the makeup of these proteins, underscoring differences attributable to the presence of a carbohydrate unit.

Purification of G-ePRL and ePRL

  • The researchers purified the glycosylated and non-glycosylated versions of equine prolactin (ePRL – a type of hormone) from horse pituitaries (a gland in the brain).
  • The process involved acetone extraction/precipitation, sodium chloride and isoelectric precipitation, and gel filtration.
  • For the purification of G-ePRL specifically, the researchers used additional Con A chromatography.

Sequencing and Molecular Analysis

  • They performed N-terminal amino acid sequencing for 32 cycles, which resulted in sequences identical to the known primary structure of ePRL.
  • Through MALDI mass spectrometry analysis and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) mobilities, the researchers estimated the molecular weights of G-ePRL and ePRL to be 25,000 Da and 23,000 Da, respectively.
  • These analyses allowed the researchers to compare the properties and biological activity of the glycosylated and non-glycosylated versions of ePRL

Immunoreactivity and Mitogenic Activity

  • A comparison of the immunoreactivity between the two revealed that G-ePRL displayed only 60% of the immunoreactivity of ePRL in a homologous radioimmunoassay, meaning it was less reactive to antibodies present in a given sample.
  • With the use of the Nb2 lymphoma cell bioassay, the mitogenic activity of ePRL was found to be much lower than that of bovine prolactin and G-ePRL was found to have approximately 1/10th the activity of ePRL.
  • This part of the analysis indicates the functional differences between glycosylated and non-glycosylated versions of the prolactin hormone.

Composition Analysis

  • A composition analysis of the G-ePRL confirmed that its glycosylation – the process where a carbohydrate is added to a protein – occurs at Asn31. The monosaccharide components of intact G-ePRL and this glycopeptide were found to resemble each other and that of glycosylated porcine prolactin (G-pPRL).
  • A comparative carbohydrate analysis of G-ePRL and other G-PRL preparations suggests that the functionally significant Asn31 carbohydrate unit is a fucosylated complex mono- and /or biantennary oligosaccharide, capped with a sulfated GalNAc residue and two or three Man residues.
  • This study reveals important details about how the structure of this hormone can fluctuate and how that impacts its functionality.

Cite This Article

APA
Butnev VY, Gotschall RR, Baker VL, Moore WT, Gout PW, Bousfield GR. (1996). Glycosylated equine prolactin and its carbohydrate moiety. J Protein Chem, 15(5), 413-426. https://doi.org/10.1007/BF01886848

Publication

Journal of protein chemistry
ISSN: 0277-8033
NlmUniqueID: 8217321
Country: United States
Language: English
Volume: 15
Issue: 5
Pages: 413-426

Researcher Affiliations

Butnev, V Y
  • Department of Biological Sciences, Wichita State University, Kansas 67260-0026, USA.
Gotschall, R R
    Baker, V L
      Moore, W T
        Gout, P W
          Bousfield, G R

            MeSH Terms

            • Amino Acids / analysis
            • Animals
            • Cell Division / drug effects
            • Chromatography, Gel
            • Electrophoresis, Polyacrylamide Gel
            • Glycosylation
            • Horses
            • Molecular Weight
            • Monosaccharides / chemistry
            • Pituitary Gland / chemistry
            • Prolactin / chemistry
            • Prolactin / isolation & purification
            • Prolactin / pharmacology
            • Tumor Cells, Cultured

            Grant Funding

            • HD 29047 / NICHD NIH HHS

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