Expression of blood clotting factor VIII:C gene in capillary endothelial cells.
The essential role of Factor VIII:C (FVIII:C, anti-hemophilia factor A) as a cofactor for Factor IXa-dependent activation of Factor X has been established. In this paper, we describe that capillary endothelial cells from bovine adrenal medulla express active FVIII:C gene. Accumulation of FVIII:C in conditioned media from an 8-day-old culture is approximately twice as high as that stored in the cell when immunoprecipitated FVIII:C was analyzed for its ability to convert Factor X to Factor Xa. Analysis of [35S]methionine-labeled and immunoprecipitated FVIII:C from cells or conditioned media on SDS-PAGE under fully denatured conditions indicated that the newly synthesized FVIII:C consists of heavy chain of M(r) 200,000 and light chain of M(r) 46,000. The secreted FVIII:C in the non-reduced condition however, has a molecular weight of 270,000 which suggests that in native protein, the heavy and light chains are held together by S-S bonds. Furthermore, susceptibility of the immunoprecipitated FVIII:C to N-glycanase digestion establishes that the endothelial cells derived FVIII:C contains asparagine-linked carbohydrate side chains.
Banerjee
D K
DK
Department of Biochemistry and Nutrition, School of Medicine, University of Puerto Rico, San Juan 00936-5067.
Tavárez
J J
JJ
Oliveira
C M
CM
D000313:Adrenal Medulla / Q000098:blood supply; D000581:Amidohydrolases; D000818:Animals; D015153:Blotting, Western; D002196:Capillaries / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism*; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006596:Hexosaminidases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D014841:von Willebrand Factor / Q000378:metabolism
D014841:von Willebrand Factor; D005169:Factor VIII; D006596:Hexosaminidases; C060638:glycopeptide alpha-N-acetylgalactosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase