BIOSYNTHESIS OF THYROTROPIN RELEASING FACTOR BY NEWT (<i>TRITURUS VIRIDESCENS</i>) BRAIN <i>IN VITRO.</i> ISOLATION AND CHARACTERIZATION OF THYROTROPIN RELEASING FACTOR

Abstract

Abstract— The in vitro biosynthesis of thyrotropin releasing factor (TRF) by hypothalamic and forebrain fragments from adult newts ( Triturus viridescens ) was studied by incubating the fragments in the presence of the radioactive amino acid precursors of thyrotropin releasing factor (TRF). The synthesized product was identified by successive chromatography and electrophoresis. A purification scheme was developed which can be applied to the processing of multiple samples labelled with l ‐[ 3 H]proline and yields a TRF peak which, when derivatized with 1‐fluoro‐2,4‐dinitrobenzene and applied to electrophoresis in acid buffer, yields a single radioactive peak corresponding to the migration of the N im ‐Dnp derivative made from standard synthetic [ 3 H]TRF. Both forebrain and hypothalamic fragments of the adult newt are capable of synthesizing a product which migrates with standard TRF after application of this purification scheme. Hypothalamic and forebrain fragments were also assayed for biological activity of TRF and it was determined that one newt hypothalamus contains approximately 200 pg TRF and that an equal amount of TRF is present in a forebrain piece of comparable size. It is concluded that newt TRF is identical to mammalian TRF (pGlu‐His‐Pro‐NH 2 ) and that the storage and synthesis of this substance is not confined to the hypothalamus in this species.