Pulse-chase studies of the synthesis of apolipoprotein B in a human hepatoma cell line, Hep G2

Abstract

Pulse-chase methodology was used 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 apoB 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 chase. The apolipoprotein B molecules that were labeled 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. Apparently, apolipoprotein B is synthesized as 1 polypeptide with a large molecular mass, newly synthesized apoliprotein B molecules are secreted after a delay of 30-35 min, no intracellular accumulation of apolipoprotein B occurs and that apolipoprotein B is recovered in the density fraction < 1.21 g/ml of the medium suggesting that it is secreted in lipoprotein form.