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Saponin permeabilization of rough microsomes from rat liver reveals a novel prothrombin pool
S Tollefsen1, L Wierød, A Skotte
1Department of Biochemistry, University of Oslo, P.O. Box 1041 Blindern, 0316, Oslo, Norway.
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.
Insights
Researchers discovered a new pool of prothrombin attached to the microsomal membrane via ionic interactions. This finding sheds light on prothrombin
Area of Science:
- Biochemistry
- Cell Biology
- Membrane Biology
Background:
- Prothrombin is a key protein in the blood coagulation cascade.
- Its localization within cellular compartments, particularly the endoplasmic reticulum, is crucial for its function.
- Understanding prothrombin's association with membranes is vital for comprehending its synthesis and secretion.
Purpose of the Study:
- To investigate the localization and association of prothrombin with rough microsomal membranes.
- To identify and characterize different pools of prothrombin associated with the membrane.
- To compare prothrombin distribution in normal and warfarin-treated rats.
Main Methods:
- Differential extraction of prothrombin from rough microsomes using saponin and high salt concentrations.
- Fractionation of microsomes to isolate lumenal, peripherally associated, and membrane-bound prothrombin.
- Quantification of prothrombin in different fractions.
- In vitro labeling of prothrombin using (14)C-gamma-carboxylation.
Main Results:
- A novel pool of prothrombin, associated ionically with the microsomal membrane, was identified.
- In normal rats, 55% of prothrombin was lumenal, 29% peripherally associated, and 16% membrane-bound.
- Warfarin treatment significantly altered distribution, with 86% lumenal, 5% peripherally associated, and 9% membrane-bound prothrombin.
- Newly synthesized prothrombin (labeled) showed significant association with membrane-associated and membrane-bound pools (42% and 33%, respectively).
Conclusions:
- Rough microsomes contain distinct pools of prothrombin, including a novel membrane-associated fraction.
- Prothrombin's association with the microsomal membrane is dynamic and influenced by factors like warfarin treatment.
- These findings provide new insights into the intracellular trafficking and membrane interactions of prothrombin during its synthesis.