Saponin permeabilization of rough microsomes from rat liver reveals a novel prothrombin pool

• Published in: Biochimica et biophysica acta Vol. 1526; no. 3; pp. 249 - 256
• Main Authors: Tollefsen, Stig, Wierød, Lene, Skotte, Anne, Rob, Jan A, Helgeland, Liv
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.06.2001
• Subjects: Animals; Anticoagulants; Buffers; Carbon Dioxide - chemistry; Carbon Radioisotopes; Chemical Fractionation; Endoplasmic Reticulum - metabolism; Intracellular Membranes - drug effects; Intracellular Membranes - metabolism; Male; Microsome; Microsomes, Liver - drug effects; Microsomes, Liver - metabolism; Novel prothrombin pool; Permeability; Permeabilization; Polyethylene Glycols; Protein Precursors - metabolism; Prothrombin - chemistry; Prothrombin - metabolism; Rat; Rats; Rats, Wistar; Saponin; Saponins - pharmacology; Vitamin K - metabolism; Warfarin; Permeabilization; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Rat; Microsome; Tris, tris(hydroxymethyl)aminomethane; Novel prothrombin pool; ER, endoplasmic reticulum; Saponin
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(01)00137-4

Saponin permeabilization of rough microsomes in the presence of high salt revealed a novel pool of prothrombin associated by ionic interactions to the microsomal membrane. The lumenal content was obtained by treating rough microsomes with 0.32% saponin in a low salt (0.05 M KCl) buffer. By a subsequent treatment with 0.32% saponin in a slightly alkaline high salt buffer a fraction of peripherally associated membrane prothrombin was released from rough microsomes. Finally, the membrane-bound fraction was solubilized with 2.5% Triton X-100. The lumenal content fraction, the peripherally membrane-associated and the membrane-bound fraction from normal rats contained 55%, 29% and 16% of the total rough microsomal prothrombin, respectively. The corresponding fractions from warfarin-treated rats contained 86%, 5% and 9% of the total prothrombin. Following 14C-γ-carboxylation of intact microsomes for 30 min, the novel membrane-associated and the membrane-bound pool contained 42% and 33%, respectively, of labeled prothrombin. A similar distribution was found with warfarin-treated rats.