Pulse-chase studies of the synthesis of apolipoprotein B in a human hepatoma cell line, Hep G2.
We have used pulse-chase methodology to study the synthesis of apolipoprotein B in a human hepatoma-derived cell line, the Hep G2 cells. A 2-min pulse with [35S]methionine was followed by a chase period varying from 5-90 min. A protein of large molecular mass (estimated molecular mass: 312 +/- 41 kDa, mean +/- SD, n = 8) could be immunoprecipitated from the cells at all chase periods between 5 min and 60 min with both monoclonal antibodies to a narrow density cut of the low density lipoprotein LDL-2 (density: 1.030-1.055 g/ml) and polyclonal antibodies to the apolipoprotein B apo B 100 or to a narrow density cut of LDL-2 (density: 1.030-1.055 g/ml). In addition to this large molecular mass protein, nascent polypeptides could be precipitated after 5, 10 and 15 min of chase. The apolipoprotein B molecules that had been labelled during the pulse disappeared from the cells after 60-90 min of chase, while they started to appear in the medium after 30-35 min of chase. The results obtained indicate (a) that apolipoprotein B is synthesized as one polypeptide with a large molecular mass, (b) that newly synthesized apolipoprotein B molecules are secreted after a delay of 30-35 min, (c) that no intracellular accumulation of apolipoprotein B occurs, and (d) that apolipoprotein B is recovered in the density fraction less than 1.21 g/ml of the medium suggesting that it is secreted in lipoprotein form.
Wettesten
M
M
Boström
K
K
Bondjers
G
G
Jarfeldt
M
M
Norfeldt
P I
PI
Carrella
M
M
Wiklund
O
O
Borén
J
J
Olofsson
S O
SO
D001055:Apolipoproteins B / Q000096:biosynthesis*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002460:Cell Line; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007120:Immunochemistry; D008113:Liver Neoplasms; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010452:Peptide Biosynthesis; D013462:Sulfur Radioisotopes; D013997:Time Factors
D001055:Apolipoproteins B; D013462:Sulfur Radioisotopes; D008715:Methionine