Studies toward the biosynthesis of vasoactive intestinal peptide (VIP)

Abstract

In view of the potential biological importance of VIP, we have begun to examine the regulation of its biosynthesis. For this purpose we have, as a first step, searched for an enriched source of VIP biosynthesis. By a combination of chromatographic procedures and radioimmunoassays we discovered an as yet unknown source for VIP production, namely a human buccal tumor, containing 0.67 +/- 0.05 ng VIP/micrograms protein which is greater than the richest source in brain (the cerebral cortex). Thus, we decided to use the tumor tissue for VIP-mRNA purification and characterization. To identify VIP-mRNA we are using as hybridization probes, synthetic oligodeoxynucleotides with relatively unambiguous nucleotide sequence complementary to the predicted VIP-mRNA sequence. These probes are synthesized, using the deoxynucleoside phosphoramidite approach, to a length of 17 bases each, and contain all the possible DNA sequences according to the genetic code. These specific probes are then radioactively labelled using the reaction catalyzed by the enzyme polynucleotide kinase and afterwards hybridized to mRNA, which had been resolved on denaturing agarose gels. Employing this approach, we identified a single putative VIP-mRNA band which was then partially purified by sucrose gradient centrifugation. Upon in vitro translation in a rabbit reticulocyte lysate cell free system, this mRNA was found to code for VIP immunoreactive proteins. In conclusion, our studies suggest the existence of high molecular weight precursors to VIP cross-reactive with anti-VIP antibodies, that are coded for by a partially purified mRNA containing VIP sequences.