POSTTRANSLATIONAL PROCESSING OF THE NEUROTENSIN NEUROMEDIN-N PRECURSOR IN THE CENTRAL-NERVOUS-SYSTEM OF THE RAT .1. BIOCHEMICAL-CHARACTERIZATION OF MATURATION PRODUCTS

Neurotensin and neuromedin N are two biologically active related pepti des which are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C-te rminal region one copy each of neurotensin and neuromedin N. Four Lys- Arg sequences which are thought to represent putative processing sites occur in the precursor molecule. Of these sites, the three that are c losest to the C-terminus flank and separate neurotensin and neuromedin N. The fourth precedes a neuromedin N-like sequence. The present stud ies were aimed at determining the extent to which each of these four d ibasic sites is cleaved and at identifying and quantifying the interme diate and mature products to which this cleavage gives rise in extract s from whole rat brain, hippocampus and globus pallidus. This was achi eved by means of radioimmunoassays specific for sequences of the neuro tensin/neuromedin N precursor that are adjacent to the dibasic process ing sites used in combination with high pressure liquid chromatography and arginine-directed trypsin digestion of tissue extracts. In all ti ssue extracts, it was found that the three most C-terminal dibasic pro cessing sites in the neurotensin/neuromedin N precursor are processed to a similar extent, whereas the dibasic site that precedes the neurom edin N-like sequence is processed to a lesser extent. As reported prev iously, the globus pallidus was shown to contain proportionally lower levels of neuromedin N than other brain regions. This lower content wa s not the consequence of a lack of precursor processing at the dibasic site that flanks the N-terminus of neuromedin N but, rather, resulted from atypical cleavage of this site or from differential post-process ing disposal of neuromedin N. The high levels of precursor messenger R NA previously reported by others in the hippocampus relative to other brain regions were not matched by high concentrations of precursor-der ived products. In conclusion, the immunological and biochemical approa ches described here provide powerful means for establishing the proces sing pattern of the neurotensin/neuromedin N precursor in brain region s. The present data set the biochemical basis for the immunohistochemi cal studies on the regional and cellular brain distribution of neurote nsin/neuromedin N precursor-derived peptides, as reported in a compani on paper.