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A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels
Published on: May 7, 2018
Protein and enzyme distribution in microsomal and myelin fractions from rat and Jimpy mouse brain
Abstract:
The protein, glycoprotein and enzyme composition of myelin and myelin-related fraction (SN 4) from rat forebrain was compared with that of microsomal fractions. Acetylcholinesterase was largely confined to the microsomal fractions, wheras 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNP) showed a high specific activity in myelin and SN 4 fractions. Nevertheless, the total specific activities of CNP present in microsomal membranes and in a water-soluble form were not negligible, and suggest that this enzyme has a wide distribution among subcellular particles. A high molecular weight protein was identified in myelin and all the other fractions studied. This protein (X), which co-migrates with the major myelin glycoprotein, was present in myelin and in fractions lacking typical myelin components as well as in fractions from a myelin deficient mutant, the Jimpy mouse. The results suggest that the X protein is probably a contaminant in isolated myelin, although the occurrence of this protein as an intrinsic component of several different membranes cannot be ruled out. Despite substantial overlap in density upon zonal centrifugation between SN 4 and microsomal fractions, the enzyme patterns of the fractions were different.
Insights
Researchers compared myelin and microsomal fractions from rat forebrain. They found 2
Area of Science:
- Neuroscience
- Biochemistry
- Cell Biology
Background:
- Myelin is a crucial lipid-rich sheath insulating nerve axons.
- Understanding myelin composition is vital for neurological research.
- Microsomal fractions contain various cellular components, including membrane systems.
Purpose of the Study:
- To compare the protein, glycoprotein, and enzyme composition of rat forebrain myelin and related fractions (SN 4) with microsomal fractions.
- To investigate the distribution and activity of specific enzymes like acetylcholinesterase and 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNP).
- To identify and characterize a high molecular weight protein (Protein X) in myelin and other cellular fractions.
Main Methods:
- Differential centrifugation and zonal centrifugation to isolate myelin, SN 4, and microsomal fractions from rat forebrain.
- Enzyme assays to determine the specific activity of acetylcholinesterase and CNP.
- Protein analysis, including co-migration studies, to identify and characterize Protein X.
Main Results:
- Acetylcholinesterase was predominantly found in microsomal fractions.
- 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNP) exhibited high specific activity in myelin and SN 4 fractions, but also significant activity in microsomal and soluble fractions, indicating a wider distribution.
- A high molecular weight Protein X, co-migrating with a major myelin glycoprotein, was detected in myelin, other fractions, and even in myelin-deficient Jimpy mouse fractions, suggesting it might be a contaminant or present in multiple membrane types.
Conclusions:
- CNP enzyme is more widely distributed across subcellular particles than previously thought.
- Protein X's presence across various fractions and in myelin-deficient mutants suggests it is likely a contaminant in isolated myelin, though its intrinsic presence in other membranes cannot be excluded.
- Despite density overlaps, distinct enzyme profiles differentiate SN 4 and microsomal fractions.
