Generation of Met-Enkephalin Arg6Phe7Immunoreactivity by Proteolytic Cleavage of Mammalian Plasma Precursors by Pepsin

Abstract

A region-specific antiserum raised against the C-terminal heptapeptide of proenkephalin A (Met-enk Arg6Phe7) was used in RIA studies to show that rat, human, and ovine plasma contain substrates (mol wt, 68K) that yield nanomolar amounts of Met-enk Arg6Phe7 (ME-RF) after treatment with pepsin under acid conditions. This ovine plasma-derived immunoreactivity diluted in parallel to the ME-RF standard in RIA and chromatographed as two low mol wt species (approximately 1K) which were less hydrophobic than the standard on size exclusion and reverse phase chromatography. The pepsin-generated material displaced [3H]naloxone from rat brain binding sites; its potency was about 1000-fold that of ME-RF, assuming near 100% cross-reactivity with the antiserum. Taken together these observations suggest that the pepsin-generated material is of similar mol wt and amino acid sequence to ME-RF, but differs with respect to opiate-binding efficacy, and that the plasma precursor is distinct from proenkephalin in both size and processing sites.