Immunological identification of high molecular weight forms common to bovine neurophysin and vasopressin

Abstract

Extracts of bovine neurohypophysis made in acid/ethanol solution containing protease inhibitors were fractionated by two successive filtrations on Sephadex G-75 columns equilibrated in the presence and then in the absence of 4 M urea. Analysis of the pattern of neurophysin-like immunoreactivity in the eluate, with two different antibodies, indicated the presence of high M_r forms of neurophysin (apparent sizes, [unk]70,000 and 20,000-25,000, respectively) besides the M_r 10,000 neurophysin. [8-Arginine]vasopressin-like immunoreactivity was also detected, coeluting with the neurophysin-like species, in the material recovered in the exclusion and M_r 20,000-25,000 elution volumes of the same molecular sieve fractionation of neurohypophyseal extracts. Upon subsequent Sephadex G-150 filtration, the immunoreactive material recovered in the exclusion volume of the Sephadex G-75 filtration showed an apparent M_r of approximately 140,000. Both neurophysin-like and vasopressin-like immunoreactivities coeluted in the same volume. The elution profile of this M_r 140,000 material was unmodified when reanalyzed by the same molecular sieve filtration after exposure to 8 M urea. When these M_r 140,000 immunoreactive forms of vasopressin and neurophysin were submitted to affinity chromatography on anti-neurophysin antibodies immobilized on Sepharose, both immunoreactivities were selectively coadsorbed to the immunoadsorbent. Similarly, the neurophysin and vasopressin immunoreactivities associated with M_r≈25,000 were retained together on the same anti-neurophysin immunoadsorbent. The M_r 140,000 and M_r 25,000 species having both neurophysin and [8-arginine]vasopressin antigenic determinants generated the two neurosecretory components when exposed to proteolytic activities. This in vitro processing was inhibited in acid medium, at low temperature, and in the presence of a mixture of protease inhibitors. It is concluded that these two large forms of proteins containing both neurophysin and vasopressin may represent common biosynthetic precursors of these two neurohypophyseal components.