In vitro biosynthesis of the beta-subunit of the Na+/K+-ATPase in developing brine shrimp: glycosylation and membrane insertion.
We demonstrate here translation, glycosylation, and membrane insertion of the beta-subunit of the Na+/K+-ATPase of the developing brine shrimp, Artemia, in a reticulocyte lysate translation system. The apparent molecular weight of the primary translation product as determined by SDS-PAGE is 33,000 +/- 1000 (n = 7). When microsomal membranes are present during the entire translation period, a new band with an apparent molecular weight of 37,000 +/- 1000 (n = 7) appears. This change in apparent molecular weight is due to the addition of about two N-linked oligosaccharides. The temporal relationship between protein synthesis and glycosylation have also been examined. Glycosylation and membrane insertion could be achieved if membranes were added after completion of about 70% of the peptide chain. However, glycosylation did not occur if membranes were added after the completion of translation of the beta-subunit. The beta-subunit was synthesized on membrane-bound polysomes, where about two N-linked oligosaccharides were added to the growing polypeptide chain. These studies demonstrate that in vitro translation systems will be useful for studying the biosynthesis of the beta-subunit of the brine shrimp, which is a good model system to examine the developmental regulation of the Na+/K+-ATPase.
Baxter-Lowe
L A
LA
Department of Pharmacology, University of Wisconsin Medical School, Madison.
Yohanan
J M
JM
Hokin
L E
LE
D000818:Animals; D001156:Artemia / Q000201:enzymology / Q000254:growth & development*; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007163:Immunosorbent Techniques; D007425:Intracellular Membranes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D011132:Polyribosomes / Q000201:enzymology; D014176:Protein Biosynthesis; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis*
D009844:Oligosaccharides; D000254:Sodium-Potassium-Exchanging ATPase