Abstract
The Ellis procedure of serial extraction of gonadotropins and growthhormone (GH) followed by alkaline ethanol extraction was adopted to processfreshly frozen buffalo pituitaries. The procedure after slight modificationwas found very useful as more than 2 mg of GH free immunoreactive prolactin(PRL) could be isolated from each gram of wet pituitary tissue. Further, thebiochemical purity and immunobiological potency of the extracted PRL,designated as P-I, was comparable with that of the highly purified samplesof homologous and heterologous PRLs. No non-PRL protein was detectable inP-I. Micro-heterogeneity with regard to size, charge, co- andpost-translational modifications was also investigated under differentconditions of extraction and at different stages of purification.Immunological and biological potencies were compared in homologouscompetitive enzyme linked immunosorbent assay (ELISA) developed for buffaloPRL and in rat Nb2 lymphoma proliferation assay respectively. Structuralheterogeneity was observed in all the preparations checked including freshpituitary homogenate and highly purified hormone. Nevertheless a 25 Kspecies corresponding to the hormone monomer was always the only paramountform comprising more than 90% of the total PRL protein in all thesamples including P-I. Similar size forms were observed in all preparationsand were found to be equivalents of monomers, dimers, covalent-andnon-covalent multimers, disulphide bridged forms and cleaved fragments.Other sibling species identified were glycosylated PRL, charge isoforms andforms that perhaps differed in their extractability from the pituitarytissue. Strong apparent size heterogeneity was displayed by the monomericbuffalo PRL. In light of these observations and the information on thestructural and functional significance and the consequences of polymericforms, the use of a heterogeneous PRL (P-I) as a reference hormone isrecommended for a valid assay.
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References
Lewis UJ: Variants of growth hormone and prolactin and their posttranslational modifications. Ann Rev Physiol 46: 33–42, 1984
Sinha YN: Structural variants of prolactin: Occurrence and physiological significance. Endocr Rev 16(3): 354–369, 1995
Kohli R, Chadha N, Muralidhar K: Are sheep and buffalo prolactins sulfated? Biochem Biophys Res Commun 149: 515–522, 1987
Kohli R, Chadha N, Muralidhar K: Presence of tyrosine-O-sulfate in sheep pituitary prolactin. FEBS Lett 242: 139–143, 1988
Stroud CM, Deaver DR, Peters JL, Loeper DC, Toth BE, Darr JA, Hymer WC: Prolactin variants in Ram adenohypophysis vary with season. Endocrinology 130(2): 811–818, 1992
Lohrke B, Kunkel S, Kowitz J, Viergutz T, Tiemann U, Alm H, Kruger B: Prolactin heterogeneity: A limitation on the evaluation of results from prolactin assays due to differences in immunoassays and the different bioactivities of prolactin forms. Eur J Clin Chem Clin Biochem 31(12): 815–828, 1994
Kacsóh B, Veress Z, Tóth BE, Avery LM, Grosvenor CE: Bioactive and immunoreactive variants of prolactin in milk and serum of lactating rats and their pups. J Endocrinol 138(2): 243–258, 1993
Ellis S: Studies on the serial extraction of pituitary proteins. Endocrinology 69: 554–570, 1961
Chadha N, Kohli R, Kumari GL, Muralidhar K: Physicochemical and immunological characterization of pituitary prolactin from water buffaloes (Bubalus bubalis). Mol Cell Biochem 105: 61–71, 1991
Woods KR, Wang KT: Separation of dansyl-amino acids by polyamide layer chromatography. Biochim Biophys Acta 133: 369–370, 1967
Heinrikson RL, Meredith SC: Amino acid analysis by reverse-phase high-performance liquid chromatography: Pre-column derivatization with phenylisothiocyanate. Anal Biochem 136: 65–74, 1984
Vaitukaitis JL, Robbins JB, Nieschlag E, Ross GT: A method of producing specific antisera with small doses of immunogen. J Clin Endocrinol Metab 33: 988–991, 1971
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970
Davis BJ: Disc electrophoresis-ll. Method and application to human serum proteins. Ann NY Acad Sci 121: 404–427, 1964
Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76(9): 4350–4354, 1979
Veress Z, Kacsóh B, Grosvenor CE: A rapid colorimetric assay for prolactin-induced proliferation of Nb2 lymphoma cells: Evaluation as a bioassay for prolactin and comparison with the [3H] thymidine uptake assay. Abstract 3883 FASEB J 7: A671, 1993
Muralidhar K, Kumar TR, Chadha N, Khurana S, Khanna T, Sharma HP: Strategies for purification of four reproductive hormones from the same batch of buffalo (Bubalus bubalis) pituitaries. Indian J Exp Biol 32: 73–80, 1994
Ellis S, Grindland RE, Nucnke JM, Collahan PX: Purification and properties of rat prolactin. Endocrinology 85: 886–894, 1969
Cheng KW, Etches RJ: Isolation and characterization of turkey prolactin. J Reprod Fert 62: 407–415, 1981
Khurana S, Kunchaparty S, Muralidhar K: A possible urease-based homologous ELISA for buffalo prolactin. Indian J Exp Biol 32: 81–85, 1994
Aston R, Ivanyi J: Monoclonal antibodies to growth hormone and prolactin. Pharmac Ther 27: 403–424, 1985
Cole ES, Nichols EH, Lauziere K, Edmunds T, McPherson JM: Characterization of microheterogeneity of recombinant primate prolactin implications for posttranslational modifications of the hormone in vivo. Endocrinology 129(5): 2639–2646, 1991
Sinha YN, Baxter SR: Identification of non-immunoreactive but highly bioactive form of prolactin in the mouse pituitary by gel electrophoresis. Biochem Biophys Res Commun 86: 325–330, 1979
Meuris S, Svoboda M, Christophe J, Robyn C: Cleaving of disulphide bridges and apparent molecular weight of human prolactin variants as revealed by immunoperoxidase electrophoresis. Anal Biochem 143: 163–169, 1984
Randweaver M, Noso T, Kawauchi H: The complete amino acid sequences of two variants of growth hormone from Atlantic cod (Gadus morhua). Gen Comp Endocrinol 81(1): 39–50, 1991
Fukuoka H, Hanamoto R, Higurashi M: Heterogeneity of serum and amniotic fluid prolactin in humans. Horm Res 35 (Suppl): 58–63, 1991
Curlewis JD, McNeilly AS: Purification, partial characterization and radioimmunoassay of prolactin and growth hormone from the Bennetts Wallaby. Gen Comp Endocrinol 88: 341–350, 1992
Sinha YN, Gilligan TA: A cleaved form of prolactin in the mouse pituitary gland: Identification and comparison of in vitro synthesis and release in strains with high and low incidences of mammary tumors. Endocrinology 114: 2046–2053, 1984
Doneen BA, Bewley TA, Li CH: Studies on prolactin: Selective reduction of the disulphide bonds of the ovine prolactin. Biochemistry 18(22): 4851–4860, 1979
Jeffcoate SL: Analytical and clinical significance of peptide hormone heterogeneity with particular reference to growth hormone and luteinizing hormone in serum. Clin Endocrinol 38: 113–121, 1993
Ekins RP: Immunoassay standardization. Scand J Clin Lab Invest (Suppl. 205) 51: 33–46, 1991
Lowry OM, Rosenbrough NJ, Faar AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275, 1951
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Khurana, S., Muralidhar, K. Heterogeneity in buffalo pituitary prolactin. Mol Cell Biochem 173, 1–15 (1997). https://doi.org/10.1023/A:1006834113682
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DOI: https://doi.org/10.1023/A:1006834113682