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

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 subsequ...

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Published inBiochimica 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
LanguageEnglish
Published Netherlands Elsevier B.V 15.06.2001
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Summary: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.
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ISSN:0304-4165
0006-3002
1872-8006
DOI:10.1016/S0304-4165(01)00137-4