Physico-chemical and immunological characteristics of pituitary prolactin from water buffaloes (Bubalus bubalis)

Abstract

Prolactin (PRL) was purified from freshly frozen pituitary glands of water buffaloes (Bubalus bubalis) by a combination of existing procedures of Ellis and Jiang and Wilhelmi involving serial extraction of different pituitary proteins. The partially purified preparation was further fractionated on DEAF-Sephadex followed by Sephadex G-100 chromatography. This was finally purified on HPLC. This preparation was found to be homogeneous by SDS-PAGE and HPLC and had a single N-terminus amino acid (Threonine). The molecular size was estimated to be 24K ± 0.5 by SDS-PAGE and ~25K by GPC-HPLC. The buffalo PRL gave a dose dependent inhibition curve in a rat liver based radio receptor assay with a potency of 30-35 L U./mg and also in a partial homologous RIA using ¹²⁵I-buffalo PRL and rabbit anti-oPRL serum giving a potency of 30I.U./mg. Metabolic labelling studies using ³⁵SO₄^2- with buffalo pituitary minces showed the incorporation of radioactive sulfate into immunoprecipitable PRL-like material. Physico-chemical characterization of the site of the linkage between sulfate and PRL revealed the presence of Tyr-O-SO₄ in bu-PRL. A high affinity monoclonal antibody (MAB) with Ka of 10¹⁰ L/M, belonging to IgG₁ isotype, and capable of cross reacting with ovine and bovine PRL was generated. This MAB was conformation specific as reduced and carboxymethylated PRL did not react with it. A homologous RIA system using this MAB has been standardised.