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Study of myelin purity in relation to axonal contaminants
Abstract:
Axonal remnants are considered a probable source of contamination of isolated myelin in view of the relatively tight axon-glial intercellular junction. Using the rabbit optic system to label specifically axonal components, we have found the levels of such contaminants to depend on the myelin isolation procedure, the tissue source, and the nature of the contaminant. A procedure employing repetitive treatments with EGTA was found to be highly effective in removing proline-labeled axonal proteins, the estimated upper limit of such contamination being approximately 0.6-1.2% of the myelin protein. The standard isolation procedure of Norton and Poduslo, supplemented with an additional discontinuous gradient step, proved equally effective in removing rapidly transported proteins from myelin isolated from the superior colliculus or lateral geniculate body. When the optic tract was the source, however, the EGTA procedure proved more effective in removing both rapidly and slowly transported proteins. Axonal gangliosides labeled with N-[3H] acetylmannosamine were efficiently removed by both procedures, adding support to the proposition that gangliosides detected in isolated myelin are intrinsic to that membrane.
Insights
Contaminants in isolated myelin, like axonal proteins and gangliosides, depend on isolation methods. Repetitive EGTA treatments effectively remove proline-labeled axonal proteins from myelin.
Area of Science:
- Neuroscience
- Biochemistry
- Cell Biology
Background:
- Isolated myelin is crucial for studying its composition and function.
- Axonal remnants pose a challenge as potential contaminants in myelin preparations.
- Understanding and minimizing axonal contamination is vital for accurate myelin research.
Purpose of the Study:
- To evaluate the effectiveness of different myelin isolation procedures in removing axonal contaminants.
- To determine the impact of tissue source and contaminant type on contamination levels.
- To confirm the intrinsic nature of gangliosides in isolated myelin.
Main Methods:
- Utilized the rabbit optic system for specific labeling of axonal components.
- Employed repetitive ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) treatments.
- Applied the standard Norton and Poduslo myelin isolation procedure with an additional gradient step.
- Labeled axonal proteins with proline and gangliosides with N-[3H] acetylmannosamine.
Main Results:
- Repetitive EGTA treatments reduced proline-labeled axonal protein contamination to an estimated 0.6-1.2% of myelin protein.
- Both EGTA and modified Norton/Poduslo procedures effectively removed rapidly transported proteins from myelin derived from the superior colliculus and lateral geniculate body.
- The EGTA procedure was superior for removing both rapidly and slowly transported proteins when the optic tract was the source.
- Both isolation methods efficiently removed N-[3H] acetylmannosamine-labeled axonal gangliosides.
Conclusions:
- Myelin isolation procedures significantly influence the level of axonal contamination.
- Repetitive EGTA treatment is a highly effective method for reducing axonal protein contamination in myelin.
- Gangliosides detected in isolated myelin are likely intrinsic components, not axonal contaminants.
- The choice of isolation technique and tissue source impacts the purity of isolated myelin.