Intrahepatic assembly of very low density lipoproteins. Phosphorylation of small molecular weight apolipoprotein B.
The possibility that apo-B is phosphorylated was examined using cultured rat hepatocytes. Rabbit antiserum prepared against rat apo-B was found to specifically react with both large and small molecular weight apo-B (by electroblotting assay and by immunoprecipitation of [35S]methionine-labeled proteins synthesized and secreted by hepatocytes). Following a 4-h incubation with [35P]orthophosphate, immunoprecipitation, and sodium dodecyl sulfate electrophoresis, an autoradiographic band corresponding to small molecular weight apo-B was obtained from cells and medium. Compared to the relative abundance of 32P which was associated with secreted small molecular weight apo-B, there was little (if any) detected in large molecular weight apo-B. Addition of excess unlabeled apo-B (obtained from rat serum) totally competed with the specific antiserum for this radioactive protein, indicating it was antigenically related to apo-B. Moreover, isolation of the 32P-labeled apo-B electrophoretic band, followed by acid hydrolysis and phosphoamino acid analysis, showed that at least 20% of the 32P originally associated with small molecular weight apo-B was in the form of phosphoserine. Control experiments ruled out the possible contamination of apo-B with phospholipid as well as the possibility that the phosphoserine produced by acid hydrolysis could have been derived from phosphatidylserine. To examine the relevance of these data to the in vivo state, rats were injected with [32P]orthophosphate. Immunoprecipitation of their livers followed by autoradiographic analysis showed the presence of 32P in small molecular weight apo-B. These data show for the first time that small molecular weight apo-B is synthesized as a phosphoserine containing protein.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001053:Apolipoproteins / Q000096:biosynthesis*; D001055:Apolipoproteins B; D066298:In Vitro Techniques; D008079:Lipoproteins, VLDL / Q000096:biosynthesis*; D008099:Liver / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D013462:Sulfur Radioisotopes
D000596:Amino Acids; D001053:Apolipoproteins; D001055:Apolipoproteins B; D008079:Lipoproteins, VLDL; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes