Kinetics of translation and glycosylation of pituitary glycoproteins.

The present study determines the basal kinetics of synthesis of translation (by [14C] leucine incorporation, [14C]leu-PROT] and of proximal (by [3H]mannose incorporation, [3H]man-PROT) and distal (by [3H]galactose incorporation, [3H]gal-PROT) glycosylation of total adenohypophyseal glycoproteins, by rat pituitary cells in primary culture. In order to obtain more information regarding the role of both steps of glycosylation on the secretory process, the effects of cycloheximide (CH; translation inhibitor) and tunicamycin (TM;glycosylation inhibitor) on the kinetics of synthesis and release of pituitary glycoproteins were also studied. Cells were incubated in medium containing [14C]leu plus [3H]man or [14C]leu plus [3H]gal, for various time-intervals (from 0.5 to 5 h) in the absence (control) or presence of different doses of CH (1.0; 4.0 or 16.0 micrograms/ml) or TM (0.5; 1.0 or 2.0 micrograms/ml). The kinetics of synthesis (slope = 3488) and release (slope = 622) of [14C]le-PROT were higher than those of the sugar precursors (slopes: [3H]man-PROT = 1751 and 526; [3H]gal-PROT = 1231 and 506). Leucine or mannose-labeled protein was barely detectable in the medium after 2 h incubation, whereas galactose-labeled protein had already been released into the incubation medium by 30 min. Cycloheximide induced translation blockage and, concomitantly, produced a marked inhibition of [3H]man incorporation. On the other hand, TM inhibited the kinetics of synthesis and release of [3H]man-PROT without affecting those of [14C]leu-PROT. The kinetics of synthesis and release of [3H]gal-PROT, although diminished, maintained linearity and increased in function of time, even in the presence of the antibiotics. Thus, the present results on glycoproteins from the pituitary gland are consistent with the previous conclusion for other mammalian glycoproteins that carbohydrate attachment occurs in several steps to molecules destined to be secreted. Addition of mannose (proximal glycosylation) is a co-translational event and that of galactose (distal glycosylation) is post-translational and can be designated as final stages in carbohydrate assembly, occurring close to the time of release. Furthermore, it has been demonstrated that the absence of the carbohydrate side chains of the pituitary glycoprotein does not prevent the intracellular transport of the protein and its export from the cell.