Pulse-chase radiolabeling experiments

Several experiments have indicated that the ER is the site of addition of the bulk of TG to apo B. First, the size distribution of lipoproteins is the same in the lumen of the ER and Golgi of rat liver, and in newly secreted lipoproteins (J.E. Vance, 1993). Furthermore, a series of pulse-chase radiolabeling experiments in rat hepatoma cells indicate that the majority of TG associates with apo B in the ER (H.N. Ginsberg, 2003). On the other hand, immuno-electron microscopy studies in rat liver (R.J. Havel, 1976), and pulse-chase radiolabeling experiments in chicken hepatocytes (M.D. Lane, 1990) and rat hepatoma cells (E.A. Fisher, 2003), have suggested that the majority of TG assembles with apo B in the Golgi apparatus. The reasons for these conflicting conclusions remain unclear. 

Pulse-chase experiments are particularly useful for tracing changes in the intracellular location of proteins or the transformation of a metabolite Into others over time. In this experimental protocol, a cell sample Is exposed to a radiolabeled compound—the pulse —for a brief period of time, then washed with buffer to remove the labeled pulse, and finally incubated with a nonlabeled form of the compound— the chase (Figure 3-36). Samples taken periodically are assayed to determine the location or chemical form of the radiolabel. A classic use of the pulse-chase technique was In studies to elucidate the pathway traversed by secreted proteins from their site of synthesis in the endoplasmic reticulum to the cell surface (Chapter 17). 

To check if RNA turnover was influencing our assays, pulse-chase experiments were carried out (Table I, column II). When 5 min pulselabeling periods were followed by a 5 min chase, the amount of radiolabel in psbD-psbC transcripts declines significantly (13% to 53%). PsbD-psbC RNAs transcribed in plastids of 8 day-old dark-grown plants were less... 

---