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

Biochim Biophys Acta. 2001 Jun 15;1526(3):249-56. doi: 10.1016/s0304-4165(01)00137-4.

Abstract

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.

MeSH terms

  • Animals
  • Anticoagulants
  • Buffers
  • Carbon Dioxide / chemistry
  • Carbon Radioisotopes
  • Chemical Fractionation
  • Endoplasmic Reticulum / metabolism
  • Intracellular Membranes / drug effects
  • Intracellular Membranes / metabolism
  • Male
  • Microsomes, Liver / drug effects*
  • Microsomes, Liver / metabolism
  • Permeability
  • Polyethylene Glycols
  • Protein Precursors / metabolism
  • Prothrombin / chemistry
  • Prothrombin / metabolism
  • Rats
  • Rats, Wistar
  • Saponins / pharmacology*
  • Vitamin K / metabolism
  • Warfarin

Substances

  • Anticoagulants
  • Buffers
  • Carbon Radioisotopes
  • Protein Precursors
  • Saponins
  • Vitamin K
  • Carbon Dioxide
  • Polyethylene Glycols
  • Warfarin
  • Prothrombin
  • Nonidet P-40