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. 1987 Feb 1;241(3):685–692. doi: 10.1042/bj2410685

Purification and biochemical characterization of three major acidic proteins (BSP-A1, BSP-A2 and BSP-A3) from bovine seminal plasma.

P Manjunath, M R Sairam
PMCID: PMC1147618  PMID: 3593217

Abstract

Three major acidic proteins of bovine seminal plasma, BSP-A1, BSP-A2 and BSP-A3, were purified to homogeneity, by employing fast protein liquid chromatography, gel filtration and h.p.l.c. The proteins were purified on the basis of their stimulatory effect on the basal release of gonadotropins by rat anterior-pituitary cells in culture. All three proteins migrated as distinct single bands in the presence or absence of 2-mercaptoethanol in SDS/polyacrylamide-gel electrophoresis. Their Mr values were estimated to be between 15,000 and 16,500 by SDS/polyacrylamide-gel electrophoresis. Similar Mr estimates were obtained when they were subjected to gel filtration on a calibrated column of Sephadex G-75 equilibrated in 0.05 M-acetic acid, pH 3.0. However, BSP-A1 and BSP-A2 were eluted as aggregated molecules (Mr 60,000-120,000) during gel filtration on Sephadex G-200 equilibrated in 0.05 M-NH4HCO3, pH 8.5, or phosphate buffer, pH 7.0, containing 0.15 M-NaCl. In the presence of 8 M-urea both BSP-A1 and BSP-A2 were eluted at positions corresponding to Mr values of 17,000-20,000. BSP-A1 and BSP-A2 had an identical amino acid composition, which differed largely from that of BSP-A3. All three proteins contained aspartic acid as the N-terminal residue, and cysteine was identified as the C-terminal residue. BSP-A1 and BSP-A2 are glycoproteins containing galactosamine, sialic acid and neutral sugars, but BSP-A3 did not contain any covalently attached sugars. Whereas BSP-A2 and BSP-A3 were eluted unadsorbed, BSP-A1 bound to wheat-germ lectin-Sepharose 6MB and could be eluted by the competing sugar N-acetyl-D-glucosamine. Treatment of BSP-A1 and BSP-A2 with trypsin resulted in complete loss of gonadotropin-release activity, but BSP-A3 retained full activity. Antibody raised against BSP-A1 did not cross-react with BSP-A3, or vice versa. All these properties indicated marked structural differences between BSP-A3 and BSP-A1 (or BSP-A2). On the basis of amino acid composition it was concluded that BSP-A1, BSP-A2 and BSP-A3 are the same as the gonadostatins [Esch, Ling, Bohlen, Ying & Guillemin (1983) Biochem. Biophys. Res. Commun. 113, 861-867].

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akiyama K., Yoshioka Y., Schmid K., Offner G. D., Troxler R. F., Tsuda R., Hara M. The amino acid sequence of human beta-microseminoprotein. Biochim Biophys Acta. 1985 Jun 10;829(2):288–294. doi: 10.1016/0167-4838(85)90200-6. [DOI] [PubMed] [Google Scholar]
  2. Chen C., Schilling K., Hiipakka R. A., Huang I. Y., Liao S. Prostate alpha-protein. Isolation and characterization of the polypeptide components and cholesterol binding. J Biol Chem. 1982 Jan 10;257(1):116–121. [PubMed] [Google Scholar]
  3. Esch F. S., Ling N. C., Böhlen P., Ying S. Y., Guillemin R. Primary structure of PDC-109, a major protein constituent of bovine seminal plasma. Biochem Biophys Res Commun. 1983 Jun 29;113(3):861–867. doi: 10.1016/0006-291x(83)91078-1. [DOI] [PubMed] [Google Scholar]
  4. Fauconnet M., Rochemont J. A single-column amino acid analysis method which resolves hexosamines and several cysteine derivatives. Anal Biochem. 1978 Dec;91(2):403–409. doi: 10.1016/0003-2697(78)90525-0. [DOI] [PubMed] [Google Scholar]
  5. Frachimont P., Chari S., Hagelstein M. T., Duraiswami S. Existence of a follicle-stimulating hormone inhibiting factor 'inhibin' in bull seminal plasma. Nature. 1975 Oct 2;257(5525):402–404. doi: 10.1038/257402a0. [DOI] [PubMed] [Google Scholar]
  6. Hammond K. S., Papermaster D. S. Fluorometric assay of sialic acid in the picomole range: a modification of the thiobarbituric acid assay. Anal Biochem. 1976 Aug;74(2):292–297. doi: 10.1016/0003-2697(76)90210-4. [DOI] [PubMed] [Google Scholar]
  7. Hayashi R. Carboxypeptidase Y in sequence determination of peptides. Methods Enzymol. 1977;47:84–93. doi: 10.1016/0076-6879(77)47010-1. [DOI] [PubMed] [Google Scholar]
  8. Heyns W., Peeters B., Mous J., Rombauts W., De Moor P. Purification and characterisation of prostatic binding protein and its subunits. Eur J Biochem. 1978 Aug 15;89(1):181–186. doi: 10.1111/j.1432-1033.1978.tb20910.x. [DOI] [PubMed] [Google Scholar]
  9. Isaacs W. B., Shaper J. H. Isolation and characterization of the major androgen-dependent glycoprotein of canine prostatic fluid. J Biol Chem. 1983 May 25;258(10):6610–6615. [PubMed] [Google Scholar]
  10. Kornfeld R., Kornfeld S. Comparative aspects of glycoprotein structure. Annu Rev Biochem. 1976;45:217–237. doi: 10.1146/annurev.bi.45.070176.001245. [DOI] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Lea O. A., Petrusz P., French F. S. Prostatein. A major secretory protein of the rat ventral prostate. J Biol Chem. 1979 Jul 10;254(13):6196–6202. [PubMed] [Google Scholar]
  13. Li C. H., Hammonds R. G., Jr, Ramasharma K., Chung D. Human seminal alpha inhibins: isolation, characterization, and structure. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4041–4044. doi: 10.1073/pnas.82.12.4041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Manjunath P., Sairam M. R. Biochemical, biological, and immunological properties of chemically deglycosylated human choriogonadotropin. J Biol Chem. 1982 Jun 25;257(12):7109–7115. [PubMed] [Google Scholar]
  15. Matsubara H., Sasaki R. M. High recovery of tryptophan from acid hydrolysates of proteins. Biochem Biophys Res Commun. 1969 Apr 29;35(2):175–181. doi: 10.1016/0006-291x(69)90263-0. [DOI] [PubMed] [Google Scholar]
  16. McKelvy J. F., Lee Y. C. Microheterogeneity of the carbohydrate group of aspergillus oryzae alpha-amylase. Arch Biochem Biophys. 1969 Jun;132(1):99–110. doi: 10.1016/0003-9861(69)90341-5. [DOI] [PubMed] [Google Scholar]
  17. Ostrowski M. C., Kistler M. K., Kistler W. S. Purification and cell-free synthesis of a major protein from rat seminal vesicle secretion. A potential marker for androgen action. J Biol Chem. 1979 Jan 25;254(2):383–390. [PubMed] [Google Scholar]
  18. Pan Y. C., Silverberg A. B., Harris S. E., Li S. S. Complete amino acid sequence of a major secretory protein from rat seminal vesicle. Int J Pept Protein Res. 1980 Aug;16(2):143–146. doi: 10.1111/j.1399-3011.1980.tb02947.x. [DOI] [PubMed] [Google Scholar]
  19. Sairam M. R., Ranganathan M. R., Ramasharma K., Lamothe P. Isolation and characterization of a bovine seminal plasma protein inhibiting pituitary FSH secretion. Mol Cell Endocrinol. 1981 May;22(2):231–250. doi: 10.1016/0303-7207(81)90094-0. [DOI] [PubMed] [Google Scholar]
  20. Seidah N. G., Arbatti N. J., Rochemont J., Sheth A. R., Chrétien M. Complete amino acid sequence of human seminal plasma beta-inhibin. Prediction of post Gln-Arg cleavage as a maturation site. FEBS Lett. 1984 Oct 1;175(2):349–355. doi: 10.1016/0014-5793(84)80766-8. [DOI] [PubMed] [Google Scholar]
  21. Seidah N. G., Ramasharma K., Sairam M. R., Chrétien M. Partial amino acid sequence of a human seminal plasma peptide with inhibin-like activity. FEBS Lett. 1984 Feb 13;167(1):98–102. doi: 10.1016/0014-5793(84)80840-6. [DOI] [PubMed] [Google Scholar]
  22. Wilson E. M., French F. S. Biochemical homology between rat dorsal prostate and coagulating gland. Purification of a major androgen-induced protein. J Biol Chem. 1980 Nov 25;255(22):10946–10953. [PubMed] [Google Scholar]

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