Related Experiment Videos
Biochemical studies of rat liver Golgi apparatus. I. Isolation and preliminary characterization
Abstract:
A method is described for the isolation of morphologically well-preserved Golgi apparatus from rat liver. The method is essentially the same as that of Morré et al. (Morré, D.J., Hamilton, R.L., Mollenhauser, H.H., Mahley, R.W., Cunningham, W.P., Cheetham, R.D., & Lequire, V.S. (1970) J. Cell Biol. 44, 484-491) except that mild cell disruption is achieved by means of a stainless-steel sieve. The average recoveries of protein and galactosyltransferase in the isolated fraction are about 6 mg from 10 g of perfused liver and about 35% from the homogenate, respectively. The preparation is virtually free from succinate-cytochrome c reductase, glucose-6-phosphatase, acid phosphatase, and 5'-nucleotidase. The Golgi fraction as well as its vesicular fragments is homogeneous upon isopycnic centrifugation in both sucrose and dextran density gradients. Their buoyant densities in sucrose are significantly higher than those in dextran, indicating that both forms of the organelle are closed systems which are impermeable to macromolecules. The galactosyltransferase activity of a freshly prepared Golgi fraction, measured with ovalbumin as galactosyl acceptor, is activated 26-fold by the addition of Triton X-100, whereas those of homogenized, sonicated, and aged preparations are only activated 2- to 4-fold.
Insights
This study details a refined method for isolating intact Golgi apparatus from rat liver using a stainless-steel sieve. The improved technique yields a pure organelle fraction with high galactosyltransferase activity, crucial for cellular research.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- The Golgi apparatus is a vital organelle involved in modifying, sorting, and packaging proteins and lipids.
- Previous isolation methods often resulted in damaged or fragmented Golgi structures.
- Understanding Golgi function requires access to well-preserved organelles.
Purpose of the Study:
- To develop an improved method for isolating morphologically intact Golgi apparatus from rat liver.
- To characterize the purity and integrity of the isolated Golgi fraction.
- To assess the enzymatic activity of key Golgi markers in the isolated fraction.
Main Methods:
- Rat liver perfusion and homogenization.
- Mild cell disruption using a stainless-steel sieve.
- Isopycnic density gradient centrifugation in sucrose and dextran.
- Assay of galactosyltransferase activity and contaminant enzyme markers.
Main Results:
- The method successfully isolated morphologically well-preserved Golgi apparatus.
- The preparation showed high recovery of protein and galactosyltransferase, with minimal contamination from other organelles.
- Isolated Golgi fractions maintained integrity, evidenced by differential buoyant densities in sucrose and dextran gradients.
- Freshly prepared Golgi exhibited significantly higher galactosyltransferase activation compared to disrupted preparations.
Conclusions:
- The stainless-steel sieve method provides a superior approach for isolating intact Golgi apparatus.
- The isolated Golgi fraction is suitable for studying its enzymatic activities and structural properties.
- The integrity of the Golgi apparatus is critical for maintaining optimal enzyme function.