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Characterization of the membrane matrix derived from the microsomal fraction of rat hepatocytes
Abstract:
A highly purified membrane preparation derived from the microsomal fraction of rat hepatocytes has been chemically characterized and fractionated by means of gel filtration. The preparation has been freed of ribosomes and intravesicular protein and has a composition on a w/w basis of 52.1% protein, 45.0% phospholipid, 2.9% carbohydrate and no RNA. 97 +/- 2% of the total membrane phosphorus is accounted for as phospholipid phosphorus. Determination of the molecular weight distribution of the constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave values ranging from 171 000 to 16 000 for the major classes of proteins. Although several membrane glycoproteins have been indentified, the most prominent species has an apparent molecular weight of 171 000, 40% of the total microsomal protein is present in the 49 000-60 000 molecular weight region. Examination of the intrinsic polypeptide composition of membranes obtained from smooth and degranulated rough endoplasmic reticulum revealed no detectable qualitative differences. Sodium dodecyl sulfate-solubilized microsomal membrane proteins were separated by gel filtration into much simplified molecular weight classes, some of which showed predominantly a single electrophoretic component. Amino acid analysis of individual fractions showed a noticeable trend toward a decreasing ratio of acidic to basic residues with decreasing molecular weight. Membrane phosphorus was distributed between two chromatographic fractions: one containing membrane phospholipid (97% of the total) as well as essentially all the cholesterol, the other, at the inclusion volume of the gel filtration system, containing small molecular weight species (3% of the total phosphorus). The absence of a ribonuclease-resistant RNA component eluting near the void volume clearly distinguishes the microsomal membrane from the nuclear envelope.
Insights
This study chemically characterized rat hepatocyte microsomal membranes, revealing their protein and phospholipid composition. The research identified major protein molecular weights and distinguished microsomal membranes from nuclear envelopes.
Area of Science:
- Cell Biology
- Biochemistry
- Membrane Biology
Background:
- Microsomal fractions from rat hepatocytes are complex cellular components.
- Understanding their precise biochemical composition is crucial for cell biology research.
Purpose of the Study:
- To chemically characterize and fractionate highly purified rat hepatocyte microsomal membranes.
- To determine the molecular weight distribution and composition of microsomal membrane proteins and lipids.
Main Methods:
- Gel filtration chromatography for fractionation.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for molecular weight determination.
- Amino acid analysis for polypeptide characterization.
- Phosphorus analysis for lipid quantification.
Main Results:
- The purified membrane preparation consisted of 52.1% protein, 45.0% phospholipid, and 2.9% carbohydrate, with no detectable RNA.
- Major polypeptides ranged from 171,000 to 16,000 molecular weight, with a prominent glycoprotein at 171,000.
- 40% of microsomal protein was found in the 49,000-60,000 molecular weight range.
- No qualitative differences were observed between smooth and degranulated rough endoplasmic reticulum membranes.
- Phosphorus was primarily associated with phospholipids (97%), with a minor fraction containing small molecular weight species.
Conclusions:
- The study provides a detailed chemical characterization of rat hepatocyte microsomal membranes.
- Fractionation revealed distinct molecular weight classes of proteins and their compositional trends.
- The absence of specific RNA components differentiates microsomal membranes from nuclear envelopes.