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Subfractionation of cardiac sarcolemma with wheat-germ agglutinin
J H Charuk1, S Howlett, M Michalak
1Department of Pediatrics, University of Alberta, Edmonton, Canada.
Insights
Highly purified bovine cardiac sarcolemma was separated using wheat germ agglutinin (WGA). This method isolated distinct sarcolemmal and T-tubule membrane fractions with differing functional properties.
Area of Science:
- Cardiology
- Cell Biology
- Membrane Biochemistry
Background:
- Sarcolemma and T-tubules are critical for cardiac function.
- Understanding their distinct properties requires effective isolation techniques.
Purpose of the Study:
- To subfractionate bovine cardiac sarcolemma using wheat germ agglutinin (WGA).
- To characterize the biochemical and morphological properties of the isolated fractions.
Main Methods:
- Subfractionation of cardiac sarcolemma using WGA.
- Enzyme activity assays (Na+/K+-ATPase, Na+/Ca2+ exchange).
- Ligand-binding studies ([3H]quinuclidinyl benzilate, [3H]nitrendipine).
- Electron microscopy and freeze-fracture analysis.
Main Results:
- Two fractions were isolated: WGA+ (agglutinated) and WGA- (non-agglutinated).
- WGA+ vesicles showed typical sarcolemmal morphology and enzyme activities.
- WGA- vesicles contained fewer specific binding sites and had tubular structures, suggesting T-tubule origin.
- Protein composition was similar, indicating plasma membrane origin for both.
Conclusions:
- WGA effectively separates bovine cardiac sarcolemma into distinct fractions.
- The WGA+ fraction represents purified sarcolemma.
- The WGA- fraction is likely derived from T-tubule membranes, offering insights into their unique properties.
Abstract:
The properties of highly purified bovine cardiac sarcolemma subfractionated with the lectin, wheat-germ agglutinin (WGA) were studied. Two different membrane subfractions were isolated, one which was agglutinated in the presence of 1.0 mg of WGA/mg of protein (WGA+ vesicles) and a second fraction which failed to agglutinate (WGA- vesicles). These two membrane fractions had quantitatively different rates of Na+/K+-dependent, ouabain-sensitive ATPase and Na+/Ca2+ exchange activities, yet a similar protein composition, which suggests that they were both derived from the plasma membrane. WGA- vesicles had a decreased number of [3H]quinuclidinyl benzilate-binding sites and no detectable [3H]nitrendipine-binding sites. Electron-microscopic and freeze-fracture analysis showed that the WGA+ fraction was composed of typical spherical sarcolemmal vesicles, whereas the WGA- fraction primarily contained elongated tubular structures suggestive of the T-tubule vesicles which were previously isolated from skeletal muscle. Assays of marker enzymes revealed that these fractions were neither sarcoplasmic reticulum nor plasma membrane from endothelial cells. Moreover, WGA agglutination did not result in the separation of right-side-out and inside-out vesicles. On the basis of these findings we propose that the WGA+ fraction corresponds to highly purified sarcolemma, whereas the WGA- fraction may be derived from T-tubule membranes.