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Abstract
Primary cultures of estrogen-induced chick parenchymal liver cells have been used to study the assembly of the apolipoprotein and glycerolipid constituents of hepatic very low density lipoprotein (VLDL). Liver cell monolayers in serum-free medium were pulse-labeled for 2.5 min with either [3H]leucine or [3H]glycerol and were then chased for 4 h. The kinetics of secretion of VLDL 3H-apolipoproteins (labeled from [3H]leucine) and [3H]triacylglycerol and 3H-phosphoglycerides (labeled from [3H]glycerol) was determined through the chase. Maximal rates of VLDL [3H]triacylglycerol secretion were reached by 20-25 min into the chase, preceding the initial appearance of 3H-apolipoproteins in the medium at 30-35 min of chase time. The secretion of VLDL 3H-phosphoglycerides was maximal much earlier, between 5 and 15 min into the chase, plateaued for 15 min, and then began again at 30 min, thereby displaying a distinctive biphasic pattern. In the presence of cycloheximide or puromycin, such that apopeptide synthesis was halted from the start of the chase, the secretion of VLDL 3H-glycerolipid was depressed after 30 min of chase, without having influenced the temporal pattern of the newly synthesized VLDL 3H-apolipoprotein and 3H-glycerolipid secretion. As compared to the cycloheximide-treated cells in which apolipoprotein B nascent chains were arrested intracellularly, the puromycin-treated cells secreted discharged apolipoprotein B nascent chains as VLDL constituents assembled with triacylglycerol. The differential kinetics of 3H-apolipoprotein, [3H]triacylglycerol, and 3H-phosphoglyceride secretion as VLDL and the timing of the effects of protein synthesis inhibitors on their secretion indicate that VLDL constituents are assembled sequentially in the intact liver cell. Some VLDL phosphoglyceride and the VLDL triacylglycerol are assembled with apolipoprotein early in the secretory pathway, forming a triacylglycerol-rich lipid-protein particle into which further phosphoglyceride is introduced just prior to its secretion as mature VLDL.
Highlights
Inthepresentstudy, we employestrogen-induced chick having influenced the temporal pattern of the newly parenchymal liver cells in primary, nonproliferating monosynthesized VLDL 3H-apolipoprotein and 3H-glycero- layer culture [7] to address theseissues
Morphological Characteristics of Secreted V L D L and Electrophoretic Analysis and Kinetics of Synthesis and Secretion of Immunoprecipitable V L D L Apolipoproteins in the Absence and Presence of Cycloheximide or Puromycin-To verify that the VLDL secretedby the chick liver cell culture system has the expectedmorphological characteristics of particulate plasma VLDL, cell monolayers were extensively washed with lipid-free medium and thenallowed to secrete the VLDL components into fresh lipid-free medium for 240 min
I 1 munoprecipitable VLDL Triacylglycerol and the Effect of Cycloheximide or Puromycin on VLDL TriacylglycerolAssembly and Secretion-To determinethekinetics of secretion of VLDL triacylglycerol, liver cell monolayers were pulse-labeled for 2.5 min with [2-"Hlglycerol in lipid-free medium and were chased for 240 min with lipid-free medium containing an excess of unlabeled glycerol and one of the following: no inhibitor of protein synthesis, 10 ~ L Mcycloheximide, or 150 g~ puromycin.Media were removed during the chase and were subjected to two-step immunoprecipitation with anti
Summary
Morphological Characteristics of Secreted V L D L and Electrophoretic Analysis and Kinetics of Synthesis and Secretion of Immunoprecipitable V L D L Apolipoproteins in the Absence and Presence of Cycloheximide or Puromycin-To verify that the VLDL secretedby the chick liver cell culture system has the expectedmorphological characteristics of particulate plasma VLDL, cell monolayers were extensively washed with lipid-free (i.e. serum-free) medium and thenallowed to secrete the VLDL components into fresh lipid-free medium for 240 min. These concentrations of cycloheximide label associated with apolipoprotein B is recovered quantitaand puromycin inhibit [''Hlleucine incorporation into trichlo- tively whenpuromycin is present throughout the 240-min roacetic acid-precipitable cellular protein by >98% within 30 chase as a secretory product immunoprecipitable with antis (results not shown)C. ycloheximide efficiently inhibits poly- VLDL antibody (Fig. 2C). This 'H label is associatedwith peptide chainelongation and reinitiation [22], therebyblock- polypeptide chains of just below 350 kDa to -20 kDa which ing the completion of nascent polypeptide chains and trapping have been identified [2] as VLDL apolipoprotein B nascent them intracellularly opnolysomes in their steady state patternchains. Under our conditions of labeling and electrophoretic polypeptide resolution, significant levels of secreted VLDL'H-apolipoprotein I1 nascent chains were not detected when puromycin was present during the240-min chase
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