Occurrence of higher molecular forms of LHRH in fractionated extracts from rat hypothalamus, cortex and placenta

Separation of higher molecular forms of LHRH-like immunoreactive material was attempted in homogenates and subcellular fractions of hypothalamus, cerebral cortex and placenta. Physiological activity was checked by means of antibodies directed against different sequences of LHRH and shown to recognize synthetic LHRH analogs extended on either C- or N-terminal portions of the molecule. After molecular-sieve filtration, 2 peaks of immunoreactive material corresponding apparently to sequences of LHRH extended on the C terminus were recovered. Peak I, with a molecular weight of about 26 000 dalton, was found exclusively in a cytoplasmic and axoplasmic supernatant (S 2), where it migrated alone with microsomes. Peak II(1800 dalton) was present both in S 2 and in a synaptosomal fraction (P 2) corresponding to nerve endings. Native LHRH was almost exclusively recovered from the synaptosomal fraction. Extracts from placenta or cerebral cortex contained little or no native LHRH; in contrast, a small amount of immunoreactive material corresponding to peak II was detected in the cerebral cortex, and fairly large amounts of both putative precursors were found in the placenta. Chromatography of tissues containing no LHRH, such as cerebellum or liver, yielded no immunoreactive material at either elution site, thus suggesting specific detection of LHRH-like material under our experimental conditions. The present data suggest that 2 higher molecular forms, one slightly heavier than the native peptide itself, and another corresponding to a much larger protein, could represent LHRH precursors and are present in the hypothalamus as well as the cerebral cortex and the placenta.