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 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 (14)C-gamma-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.
Tollefsen
S
S
Department of Biochemistry, University of Oslo, P.O. Box 1041 Blindern, 0316, Oslo, Norway.
Wierød
L
L
Skotte
A
A
Rob
J A
JA
Helgeland
L
L
D000818:Animals; D000925:Anticoagulants; D002021:Buffers; D002245:Carbon Dioxide / Q000737:chemistry; D002250:Carbon Radioisotopes; D005591:Chemical Fractionation; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects* / Q000378:metabolism; D017830:Octoxynol; D010539:Permeability; D011092:Polyethylene Glycols; D011498:Protein Precursors / Q000378:metabolism; D011516:Prothrombin / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012503:Saponins / Q000494:pharmacology*; D014812:Vitamin K / Q000378:metabolism; D014859:Warfarin
D000925:Anticoagulants; D002021:Buffers; D002250:Carbon Radioisotopes; D011498:Protein Precursors; D012503:Saponins; D014812:Vitamin K; D002245:Carbon Dioxide; D011092:Polyethylene Glycols; D014859:Warfarin; D011516:Prothrombin; D017830:Octoxynol; C010615:Nonidet P-40