Figure 3

Glycosylation and sulphation of POMC in hyperactive and basally active Xenopus melanotrope cells. A, Neurointermediate lobes (NILs) from black-adapted (BA) and white-adapted (WA) animals were pulse labelled with [³⁵S]methionine/cysteine for 60 minutes and chased for two hours. The WA NILs were pulsed and chased in the presence of 10^-6 M apomorphine to retain their basally active characteristics. NIL proteins were control treated (C) or deglycosylated with Peptidyl N-glycosidase F (PNGaseF; F) or Endoglycosidase H (EndoH; H), and subsequently analysed by SDS-PAGE and autoradiography. B, Newly synthesised proteins in NILs from BA (n = 12) and WA (n = 4) animals were double-labelled with [³H]lysine and [³⁵S]sulphate for 15 minutes. The WA NIL proteins were labelled in the presence of 10^-6 M apomorphine. NIL proteins (40% of the cell lysate) were separated by 12.5% SDS-PAGE and the relative amount of each label incorporated in newly synthesised 37 K POMC was determined. C, NIL proteins from BA and WA animals were labelled as in B, control treated (C) or deglycosylated with PNGaseF (F) or EndoH (H), and analysed by 12.5% SDS-PAGE and autoradiography. Data are shown as means +/- s.e.m.; **, p < 0.01.