FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
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.
Get Full Text
Publication Date: 1996-07-01 PubMed ID: 8895086DOI: 10.1007/BF01886848Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • 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

            References

            This article includes 49 references
            1. Glycobiology. 1992 Oct;2(5):401-9
              pubmed: 1457969
            2. Endocrinology. 1988 Jun;122(6):2667-74
              pubmed: 3131120
            3. Mol Cell Endocrinol. 1991 Sep;80(1-3):203-13
              pubmed: 1955078
            4. Glycobiology. 1991 Mar;1(2):115-30
              pubmed: 1823155
            5. Nature. 1970 Aug 15;227(5259):680-5
              pubmed: 5432063
            6. J Clin Endocrinol Metab. 1980 Nov;51(5):1058-63
              pubmed: 7419681
            7. Int J Pept Protein Res. 1976;8(2):205-24
              pubmed: 1270193
            8. J Neuroendocrinol. 1991 Aug 1;3(4):375-81
              pubmed: 19215479
            9. Am J Physiol. 1978 Aug;235(2):E97-102
              pubmed: 686171
            10. Mol Cell Endocrinol. 1990 Jun 18;71(2):155-62
              pubmed: 2376286
            11. Glycobiology. 1991 Mar;1(2):139-47
              pubmed: 1823157
            12. Cancer Res. 1980 Jul;40(7):2433-6
              pubmed: 6992985
            13. Endocrinology. 1996 Jun;137(6):2530-42
              pubmed: 8641207
            14. Endocrinology. 1985 Jan;116(1):359-63
              pubmed: 2578035
            15. Proc Natl Acad Sci U S A. 1984 Jan;81(2):385-9
              pubmed: 6582495
            16. Biochem Biophys Res Commun. 1988 Sep 30;155(3):1187-93
              pubmed: 3178800
            17. Eur J Clin Chem Clin Biochem. 1993 Dec;31(12):815-27
              pubmed: 8136414
            18. J Clin Endocrinol Metab. 1990 Aug;71(2):379-83
              pubmed: 2380335
            19. Int J Pept Protein Res. 1992 Mar;39(3):250-7
              pubmed: 1399264
            20. Int J Pept Protein Res. 1986 Aug;28(2):113-23
              pubmed: 3771097
            21. Endocrinology. 1991 Nov;129(5):2639-46
              pubmed: 1935793
            22. Nature. 1962 May 5;194:495-6
              pubmed: 14450081
            23. Endocrinology. 1995 Aug;136(8):3325-30
              pubmed: 7628367
            24. J Biol Chem. 1982 Apr 25;257(8):4345-55
              pubmed: 7068639
            25. FEBS Lett. 1974 Aug 25;44(2):205-8
              pubmed: 4608931
            26. Int J Pept Protein Res. 1988 Jun;31(6):544-54
              pubmed: 3045032
            27. J Biol Chem. 1995 Sep 15;270(37):21665-71
              pubmed: 7545167
            28. Biochim Biophys Acta. 1991 Apr 29;1077(3):339-45
              pubmed: 2029533
            29. Endocr Rev. 1995 Jun;16(3):354-69
              pubmed: 7671851
            30. Endocrinology. 1988 Sep;123(3):1303-6
              pubmed: 3402386
            31. Endocrinology. 1992 Dec;131(6):2986-98
              pubmed: 1280209
            32. Endocrinology. 1989 Mar;124(3):1558-63
              pubmed: 2917524
            33. Anal Biochem. 1988 Apr;170(1):54-62
              pubmed: 3389518
            34. J Biol Chem. 1984 Feb 10;259(3):1911-21
              pubmed: 6420415
            35. FEBS Lett. 1987 Feb 23;212(2):220-4
              pubmed: 3817155
            36. Endocrinology. 1995 Nov;136(11):4827-33
              pubmed: 7588213
            37. J Endocrinol Invest. 1993 Nov;16(10):807-16
              pubmed: 8144855
            38. Arch Biochem Biophys. 1970 Dec;141(2):705-37
              pubmed: 5497153
            39. Arch Biochem Biophys. 1983 Jan;220(1):208-13
              pubmed: 6830233
            40. J Clin Endocrinol Metab. 1977 Nov;45(5):1112-5
              pubmed: 925136
            41. Biochem Biophys Res Commun. 1991 Aug 15;178(3):1194-201
              pubmed: 1908233
            42. J Endocrinol. 1989 Feb;120(2):201-6
              pubmed: 2926296
            43. Horm Metab Res. 1989 Dec;21(12):652-7
              pubmed: 2613179
            44. Biochem Biophys Res Commun. 1987 Oct 14;148(1):505-14
              pubmed: 3675595
            45. Comp Biochem Physiol B. 1991;99(3):563-70
              pubmed: 1769204
            46. J Chromatogr. 1987 Jul 10;398:255-64
              pubmed: 3654840
            47. J Endocrinol. 1991 Jul;130(1):43-51
              pubmed: 1880477
            48. Science. 1988 Nov 11;242(4880):930-3
              pubmed: 2460923
            49. Glycobiology. 1995 Feb;5(1):3-10
              pubmed: 7539645

            Citations

            This article has been cited 0 times.
            Subscribe to our newsletter
            Join 100,113 horse owners like you who receive our email updates on horse health and nutrition.