gamma MSH, a putative
hormone in the N-terminal region of the
ACTH/
beta-endorphin (beta-EP) precursor
protein, was studied by RIA with an antiserum against gamma 3MSH in
ACTH-producing mouse
pituitary tumor cells, AtT-20/D16v. Serial dilution of the culture medium or the
cell extract gave parallel lines to the standard curve in the RIA for
gamma MSH. Rat median eminence extracts enhanced the release of
gamma MSH-like immunoreactivity (
gamma MSH-LI) concomitant with
ACTH-like immunoreactivity (
ACTH-LI) and beta-EP-like immunoreactivity (beta-EP-LI).
Dexamethasone suppressed the release of
gamma MSH-LI as well as
ACTH-LI and beta-EP-LI. Gel exclusion chromatography of the culture medium and the
cell extract has revealed that
gamma MSH-LI consists of two peaks; one eluted near the elution position of
beta-lipotropin and the other near the elution position of beta-EP. There was no peak corresponding to the elution position of synthetic gamma 3MSH. However,
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) has demonstrated that
gamma MSH-LI migrated at five positions with molecular weights of 31K, 21-23K, 16-17K, 13-14K, and 3.8K, respectively. The 31K
gamma MSH coincided with the migration position of 31K
ACTH of 31K beta-EP, and 21-23K
gamma MSH coincided with the position of 21-23K
ACTH on SDS-PAGE. The 16-17K
gamma MSH coincided with the mouse 16K fragment (reported by Eipper and Mains) of
ACTH-beta-lipotropin precursor protein in the migration in SDS-PAGE and in immunoreactivity to anti-
gamma MSH antiserum. [3H]
Glucosamine was incorporated into 16K, 13K, and 3.8K
gamma MSH. These results suggest that AtT-20/D16v cells produce
gamma MSH-LIs with molecular weights of 31K, 21-23K, 16-17K, 13-14K, and 3.8K, and they are secreted concomitantly with
ACTH-LI and beta-EP-LI.