title: Noncoordinate synthesis of the fibrinogen subunits in hepatocytes cultured under hormone-deficient conditions.
abstract: Differential detergent gel electrophoresis conditions are described which enable the accurate quantitation of radiolabel incorporated into each of the closely migrating, constituent polypeptides of chicken fibrinogen: glycosylated and nonglycosylated A alpha, B beta, gamma', and gamma. These methods were applied to analysis of fibrinogen synthesis by monolayer cultures of chick embryo hepatocytes to determine whether the cells coordinate biosynthesis of the fibrinogen subunits under nonstimulated or basal conditions (i.e. in the absence of hormones) and in the presence of serum, which is a potent stimulator of fibrinogen production. Since secretion of the subunits apparently depends on their oligomeric assembly into the general structure (A alpha, B beta, gamma)2, it was thought that their synthesis might be stoichiometric. Incorporation of [35S]methionine into the subunit chains was determined for both cellular and secreted fibrinogen, immunoprecipitated from pulse-labeled and continuously labeled cultures. Molar ratios of subunit synthesis and the degree of serum-induced stimulation for each subunit were calculated. Specific subunit mRNA levels were also evaluated with a cell-free translation assay as well as microinjection of RNA into Xenopus oocytes. The results indicate, to the contrary, that in hormone-deprived hepatocytes there is a deficiency in A alpha chain synthesis, correlating with reduced A alpha-specific mRNA levels, which leads to hepatocellular degradation of surplus B beta and gamma chains. Addition of serum to the cellular environment, while increasing rates of subunit synthesis, also corrects the deficiency in A alpha chain synthesis, thereby restoring a measure of balance and preventing much of the degradation. The outcome of this serum-induced enhancement and coordination of fibrinogen subunit gene expression is a dramatic (more than 20-fold) stimulation of fibrinogen secretion.
mesh_terms: D000818:Animals; D001790:Blood Physiological Phenomena; D002417:Cattle; D002474:Cell-Free System; D002478:Cells, Cultured; D002642:Chick Embryo; D003470:Culture Media / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005260:Female; D005340:Fibrinogen / Q000096:biosynthesis*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006728:Hormones / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D014908:Triticum / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D003470:Culture Media; D006728:Hormones; D012333:RNA, Messenger; D012967:Sodium Dodecyl Sulfate; D005340:Fibrinogen; C028913:dodecyl sulfate