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Updated: Aug 10, 2026
Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas
Published on: April 1, 2014
Isolation and characterization of membranes from normal and transformed tissue-culture cells
Abstract:
Homogenates of baby-hamster kidney cells and rat embryo fibroblasts prepared by nitrogen cavitation contain a small population of slowly sedimenting mitochondria or mitochondrial fragments, which contaminate the microsomal fraction. This appears to limit the resolution of surface membrane and endoplasmic reticulum on magnesium-containing dextran gradients. The microsomal material and mitochondria can, however, be completely separated on a 10-60% (w/w) sucrose zonal gradient containing a 30% sucrose plateau. On magnesium-containing dextran gradients this mitochondria-free microsomal material can be resolved into at least two surface membrane fractions and at least two endoplasmic reticulum fractions. Comparison of polyoma virus-transformed and normal baby-hamster kidney cells reveals some interesting differences in their microsomal fractionation patterns and the characteristics of the Na(+)/K(+)-Mg(2+) adenosine triphosphatase of their surface membranes, in particular a tenfold lower K(m) in the virus-transformed cells. The fractionation patterns of normal and spontaneously transformed rat embryo fibroblasts are also briefly discussed, particularly in relation to the significance of the observation that both the surface membrane and endoplasmic reticulum from these cells can be subfractionated.
Insights
Researchers improved cell fractionation by separating mitochondria from microsomes using sucrose gradients. This enhanced the resolution of surface membranes and endoplasmic reticulum, revealing differences in virus-transformed cells.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Mitochondria contaminate microsomal fractions, limiting the resolution of surface membranes and endoplasmic reticulum.
- Nitrogen cavitation homogenization produces slowly sedimenting mitochondria in cell homogenates.
Purpose of the Study:
- To develop a method for separating mitochondria from microsomal fractions.
- To improve the resolution of surface membrane and endoplasmic reticulum fractions.
- To investigate differences in microsomal fractionation and surface membrane Na(+)/K(+)-Mg(2+) ATPase between normal and polyoma virus-transformed cells.
Main Methods:
- Nitrogen cavitation for cell homogenization.
- Sucrose zonal gradient centrifugation (10-60% w/w) with a 30% sucrose plateau for separating microsomes and mitochondria.
- Magnesium-containing dextran gradients for resolving microsomal subfractions.
Main Results:
- Complete separation of mitochondria from microsomes was achieved using sucrose zonal gradients.
- Mitochondria-free microsomal material resolved into at least two surface membrane and two endoplasmic reticulum fractions.
- Polyoma virus-transformed cells showed altered microsomal fractionation patterns and a tenfold lower K(m) for surface membrane Na(+)/K(+)-Mg(2+) ATPase compared to normal cells.
Conclusions:
- Sucrose zonal gradients effectively separate mitochondria from microsomes, enabling better resolution of cellular membranes.
- Microsomal fractionation patterns and surface membrane enzyme characteristics differ between normal and virus-transformed cells, suggesting oncogenic transformation impacts cellular organization and function.
