Ampholyte displacement and high pressure liquid chromatographic separation of the estrogen-responsive neurophysin from human plasma.

Abstract

Estrogen-stimulated neurophysin (ESN) or oxytocin (OT)-neurophysin (Np) was measured in plasma of seven men before and after oral administration of 25 mg diethylstilbestrol (DES). Pre-DES levels of ESN averaged 0.93 +/- 0.3 (+/- SEM) ng/ml and increased to 29.8 +/- 6.5 and 25.4 +/- 5.1 ng/ml 24 and 48 h after DES treatment, respectively. To compare the estrogen-responsive Np in plasma with human OT-Np which is present in the posterior pituitary gland, the Np fraction of post-DES plasma was concentrated by double precipitation with ammonium sulfate and applied to ampholyte displacement and Sephadex G-75 columns. The Np fraction of this plasma extract contained ESN immunoreactivity (IR) but no nicotine-stimulated neurophysin-IR. ESN-IR of plasma and of an extract of human posterior pituitary eluted identically from a Sephadex G-75 column, indicating similar mol wt. The plasma extract containing ESN-IR eluted from the ampholyte displacement column at pH 4.3-4.2. No nicotine-stimulated Np (arginine vasopressin-Np)-IR was found in the plasma samples. ESN-IR in an extract of human posterior pituitary gland eluted from the ampholyte displacement column at the same pH as that of the ESN extracted from plasma. Peak ESN-IR-containing fractions from the ampholyte displacement were pooled, dialyzed, lyophilized, and reconstituted in appropriate carrier buffer for reverse phase high pressure liquid chromatography. The ESN-IR was resolved into two distinct ESN-IR peaks by high pressure liquid chromatography. Plasma and posterior pituitary gave identical pairs of peaks. Thus, the Np that is increased in human plasma in response to estrogen is identical to pituitary OT-Np, providing strong evidence that estrogen stimulates the human neurohypophysis.