Effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes. Studies on transferrin, very low density lipoprotein, and serum albumin

D.K Struck,P.B Siuta,M.D Lane,W.J Lennarz

doi:10.1016/s0021-9258(17)30374-5

Abstract

Using rat or chick hepatocyte monolayers, we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the synthesis and secretion of serum proteins. Tunicamycin inhibited glucosamine incorporation into rat liver transferrin and the apoprotein B chain of chick liver very low density lipoprotein (VLDL) by 75 to 90%. In contrasts, amino acid incorporation into these two glycoproteins, as well as into the normally unglycosylated proteins, rat serum albumin and apoprotein A of chick liver VLDL, was decreased by only 10 to 25% in the presence of the antibiotic. Despite the inhibitory effect of tunicamycin on glycosylation, secretion of all four proteins was virtually unimpaired. Thus, the carbohydrate moieties of rat liver transferrin or apoprotein B of chick liver VLDL do not appear to play an essential role in the secretion process.

Highlights

Using rat or chick hepatocyte monolayers, we have studied tht effect of tunicamycin, a specific inhibitor of protein glycosylation, on the synthesis and secretion of serum proteins
To explore the possible role of carbohydrate in the secretion of serum proteins, we have studied the effects of tunicamycin on the synthesis and secretion of the unglycosylated protein, serum albumin [3], and the glycoprotein, transferrin [6, 7], by
The data presented in this report suggest that the oligosaccharide units of transferrin and the apoprotein B chain of very low density lipoprotein (VLDL) do not play an essential role in regulating the secretion of these proteins

Summary

PROCEDURES

Materials and Routine Methods-“H-Amino-acid mix (algal profile), L-[4,5-“Hlleucine (5 Ci/mmol), and ]‘H]glucosamine The standard culture medium contained Eagles basal medium supplemented with 5% rooster serum, 0.22% NaHCO. mM glucose (total concentration, 25 mM), streptomycin (0.1 mg/ml), penicillin (100 units/ml), insulin (1 pg/ml), and amino acids [8]. Aliquots (0.2 ml) of this supernatant or incubation medium were mixed in Beckman microfuge tubes with 10 ~1 of carrier chicken serum VLDL (35 pg of protein) [8]. 24 h, tubes were centrifuged for 2.5 min in a Beckman microfuge and the pellets were washed four times with 0.15 M NaCl. Immunoprecipitates were dissolved in 1%~ sodium dodecyl sulfate and subjected to SDS-polyacrylamide gel electrophoresis (3.3% acrylamide gels) as previously described [8, 23]. The slices were incubated overnight at 37°C in 90% NCS tissue solubilizer and counted in toluene scintillator

RESULTS

D MEDIA lobaled proteu

DISCUSSION

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