Abstract
To investigate biosynthesis and intracellular transport of 5'-nucleotidase, we purified this enzyme from rat liver and prepared antibodies. In immunoblot analysis, 5'-nucleotidase from the plasmalemmal, Golgi, and tritosomal fractions migrated as a single band of 72 kDa. The immunoreactive 68-kDa band was detected in the rough microsomal fraction and only the 72-kDa polypeptide contained sialic acids. The single polypeptide of 61 kDa immunoprecipitated from the translation products with the membrane-bound polysomal RNAs by the cell-free system converted to the 68-kDa form associated with membranes, when translated in the presence of microsomal vesicles. 5'-Nucleotidase from cultured primary hepatocytes pulse-labeled with [35S]methionine for 20 min migrated as a 68-kDa polypeptide. Within 45 min of chase, the 68-kDa form was converted to the 72-kDa form. Upon treatment with tunicamycin, a new immunoreactive polypeptide of 59 kDa was obtained. These results suggest that 5'-nucleotidase is translated on the membrane-bound polysomes as a 61-kDa precursor and that the cleavage of the 2-kDa signal peptide and the core glycosylation co-translationally convert the precursor to the 68-kDa form, which is subsequently processed in the Golgi complex to the 72-kDa form.
Highlights
The plasma membrane has ahighly differentiated structure and continues to exchange membranes with a pool of cytoplasmic membranes [9, 10] so that various cell surface phenomena can be expressed
Is low on the lateral surface, and tha5t ’-nucleotidase is almost exclusively located on the microvilli, in large clusters, both on the bile canalicular and sinusoidal surface
We investigated the biosynthesis of 5’-nucleotidase, using an in vitro translation system and primary hepatocyte cultures
Summary
Immunoblotting of 5’-Nucleotidase-To characterize the size of 5’-nucleotidase in subcellular fractions, we used the technique of immunoblotting. 5”Nucleotidase was mainly localized in theplasma membrane fraction and had amolecular weight of000 (mature form) (Fig. 3, lune 1 ). The 5”nucleotidase in the endoplasmic reticulum would be a 68-kDa polypeptide bearing high mannose oligosaccharides.Inthe Golgi fraction,onlythematureform, which was sialylated, was obtained (Fig. 4, lanes 3, 4, and 5). The detection of the 72-kDa form in the GF3 fraction for 30 min in the presence of this drug This treatmentof the may reflect contamination of the trans Golgi vesicles in this cells with tunicamycin inhibited the formation of the 68-kDa fraction. We consider that about a 2-kDa nucleotidase would generally follow a route taken by other signal polypeptide was cleaved off from the preform (61 kDa) secretory or integral membrane proteins [39,40].The primary and that the apparenmt olecular mass of the sugar moiety in translation product of 5’-nucleotidase had a slightly higher the microsomal 5’-nucleotidase was 9 kDa. apparent molecular weight than did the unglycosylated polypeptide immunoprecipitated from cultured primary hepato-
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