HETEROGENEITY IN BUFFALO PITUITARY PROLACTIN

The Ellis procedure of serial extraction of gonadotropins and growth h ormone (GH) followed by alkaline ethanol extraction was adopted to pro cess freshly frozen buffalo pituitaries. The procedure after slight mo dification was found very useful as more than 2 mg of GH free immunore active prolactin (PRL) could be isolated from each gram of wet pituita ry tissue. Further, the biochemical purity and immunobiological potenc y of the extracted PRL, designated as P-I, was comparable with that of the highly purified samples of homologous and heterologous PRLs. No n on-PRL protein was detectable in P-I. Micro-heterogeneity with regard to size, charge, co-and post-translational modifications was also inve stigated under different conditions of extraction and at different sta ges of purification. Immunological and biological potencies were compa red in homologous competitive enzyme linked immunosorbent assay (ELISA ) developed for buffalo PRL and in rat Nb-2 lymphoma proliferation ass ay respectively. Structural heterogeneity was observed in all the prep arations checked including fresh pituitary homogenate and highly purif ied hormone. Nevertheless a 25 K species corresponding to the hormone monomer was always the only paramount form comprising more than 90% of the total PRL protein in all the samples including P-I. Similar size forms were observed in all preparations and were found to be equivalen ts of monomers, dimers, covalent-and non-covalent multimers, disulphid e bridged forms and cleaved fragments. Other sibling species identifie d were glycosylated PRL, charge isoforms and forms that perhaps differ ed in their extractability from the pituitary tissue. Strong apparent size heterogeneity was displayed by the monomeric buffalo PRL. In ligh t of these observations and the information on the structural and func tional significance and the consequences of polymeric forms, the use o f a heterogeneous PRL (P-I) as a reference hormone is recommended for a valid assay.