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Two-dimensional gel electrophoretic resolution of the polypeptides of rat liver mitochondria and the outer membrane
R J Pavlica1, C B Hesler, L Lipfert
1Department of Biological Sciences, St. John's University, Jamaica, NY 11439.
Abstract:
The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein.
Insights
Silver staining detects more mitochondrial proteins than Coomassie blue, especially smaller ones. A purer outer membrane preparation revealed distinct VDAC protein isoforms.
Area of Science:
- Biochemistry
- Cell Biology
- Proteomics
Background:
- Mitochondria are crucial organelles with complex protein compositions.
- Accurate protein profiling is essential for understanding mitochondrial function.
- Previous methods had limitations in detecting low-abundance or small proteins.
Purpose of the Study:
- To compare silver staining and Coomassie blue for detecting mitochondrial proteins.
- To characterize the proteome of the rat liver mitochondrial outer membrane.
- To identify isoforms of the voltage-dependent anion channel (VDAC) protein.
Main Methods:
- Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of rat liver mitochondria and microsomes.
- Protein detection using Coomassie blue and silver staining.
- Isolation of mitochondrial outer membrane using hypotonic swelling and digitonin methods.
- Analysis of outer membrane proteins by 2D-PAGE and non-equilibrium pH gradient electrophoresis.
Main Results:
- Silver staining detected 2-3 times more polypeptides than Coomassie blue, particularly those under 50,000 daltons.
- Hypotonic swelling yielded a 20-fold purer outer membrane fraction compared to digitonin treatment.
- Approximately 50 polypeptides were identified in the purified outer membrane, including three isoelectric forms of VDAC.
- Further analysis revealed at least six VDAC isoforms in the outer membrane.
Conclusions:
- Silver staining offers superior sensitivity for mitochondrial protein analysis.
- The hypotonic swelling method is highly effective for isolating pure mitochondrial outer membranes.
- The rat liver mitochondrial outer membrane proteome is complex, with multiple VDAC isoforms identified.