Subfractionation of smooth microsomes from rat liver

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Smooth microsomes are crucial cellular components involved in various metabolic processes.
• Understanding the heterogeneity of smooth microsomes is essential for elucidating their diverse functions.
• Previous fractionation methods often faced challenges with enzyme inactivation and vesicle aggregation.

Purpose of the Study:

• To develop and apply an effective fractionation procedure for rat liver smooth microsomes.
• To investigate the compositional heterogeneity of smooth microsomes based on density and sedimentation velocity.
• To analyze the distribution of key enzymes and lipids within different smooth microsomal subfractions.

Main Methods:

• Isolation and concentration of total smooth microsomes from rat liver using Cs(+)-containing sucrose gradients.
• Fractionation of nonaggregated smooth microsomes by zone centrifugation on stabilizing sucrose gradients.
• Analysis of phospholipid/protein ratio, cholesterol content, and enzyme activities (NADH- and NADPH-oxidizing enzymes, G6Pase, Mg(++)-ATPase, AMPase) in subfractions.

Main Results:

• Smooth microsomes were successfully fractionated into subfractions with median equilibrium densities ranging from 1.10 to 1.18.
• Cholesterol was enriched in subfractions with higher sedimentation velocities, while the phospholipid/protein ratio remained constant.
• Distinct enzyme distribution patterns were observed, with some enzymes concentrated in upper gradient fractions and others in lower fractions.

Conclusions:

• Rat liver smooth microsomes exhibit significant compositional heterogeneity.
• The applied fractionation procedure effectively separates smooth microsomal vesicles based on their properties.
• These findings provide insights into the distinct roles and compositions of different smooth microsomal populations.