Heterogeneity in buffalo pituitary prolactin

Area of Science:

• Endocrinology
• Biochemistry
• Protein Chemistry

Background:

• Pituitary hormones, including prolactin (PRL), play crucial roles in mammalian physiology.
• Understanding the structural characteristics and purity of isolated PRL is essential for accurate assays and further research.
• Buffalo pituitary tissue is a potential source for isolating and characterizing PRL.

Purpose of the Study:

• To isolate and characterize growth hormone (GH)-free immunoreactive prolactin (PRL) from buffalo pituitaries.
• To assess the biochemical purity and immunobiological potency of the isolated PRL.
• To investigate the micro-heterogeneity of buffalo PRL and its implications for assay development.

Main Methods:

• Modified Ellis procedure involving serial extraction of gonadotropins and GH, followed by alkaline ethanol extraction.
• Biochemical purity assessed by detecting non-PRL proteins.
• Immunobiological potency evaluated using homologous competitive enzyme-linked immunosorbent assay (ELISA) and rat Nb2 lymphoma proliferation assay.
• Structural heterogeneity analyzed under various extraction and purification conditions.

Main Results:

• Successfully isolated over 2 mg of GH-free immunoreactive PRL per gram of wet pituitary tissue.
• The isolated buffalo PRL (P-I) exhibited biochemical purity and immunobiological potency comparable to homologous and heterologous PRL standards.
• No non-PRL proteins were detected in the P-I preparation.
• Significant micro-heterogeneity in size, charge, and post-translational modifications was observed across all PRL preparations, including P-I.
• A 25 kDa species, representing the PRL monomer, constituted over 90% of the total PRL protein in all samples.
• Various forms including monomers, multimers, glycosylated PRL, and charge isoforms were identified.

Conclusions:

• The modified Ellis procedure is effective for isolating high-purity buffalo PRL.
• The isolated buffalo PRL (P-I) is biochemically pure and immunobiologically potent.
• Buffalo PRL exhibits significant structural heterogeneity, with the monomer being the predominant form.
• The heterogeneous buffalo PRL (P-I) is recommended as a reference hormone for valid immunoassays.