Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemistry

Faculty/School

Faculty of Science

First Advisor

Dr. Lillian DeBruin

Advisor Role

Thesis Supervisor

Abstract

The wrapping of multiple layers of myelin membrane sheets around an axon plays an important role in normal neuronal function. In the central nervous system (CNS), oligodendrocytes are responsible for forming multiple myelin internodes which consist of lipids and proteins. Within these regions, there are various membrane microdomains including the radial component/tight junctions. The radial component is a junctional complex believed to stabilize the myelin membranes in the CNS myelin. In this study, the radial component from bovine and mouse myelin was isolated using differential detergent extraction. A highly detergent resistant fraction, containing previously identified proteins of the radial component (oligodendrocyte specific protein (OSP) and myelin oligodendrocyte basic protein (MOBP)), was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2-D electrophoresis (with immobilized pH gradient (IPG) strip or liquid-phase isoelectric focusing (IEF) followed by SDS-PAGE), traditional Western blotting and multiplex fluorescent Western blotting, and mass spectrometry. The fraction was found to be significantly and consistently enriched in some high and low abundant proteins. The most abundant proteins identified are myelin basic protein (MBP), proteolipid protein, 2, 3-cyclic nucleotide 3 phosphodiesterase, cytoskeletal proteins (actin, tubulin, spectrin, neurofilament triplet M), glial fibrilary acidic protein, mitochondrial-type proteins (ATP synthase, ADP/ATP translocase) and ion transport sodium-potassium ATPase. A large-scale analysis of radial component myelin proteome was performed using a shot-gun approach. A total of 472 proteins were identified in bovine and 881 proteins in mouse. Furthermore, evidence implicates the presence of plasmalemmal voltage dependent anion channel (VDAC) proteins in the myelin membrane. A select group of proteins (MBP, actin, tubulin, OSP, MOBP, VDAC, Na+/K+ ATPase, and ATP synthase) identified were confirmed by Western blotting with protein specific antibodies. This study indicates that radial component of myelin can be isolated successfully and proteomic study reveals crucial information about high-abundance cytoskeletal and transport proteins and low-abundance signalling proteins. This information will aid in deciphering the roles of various proteins of the radial component in health and disease.

Convocation Year

2012

Convocation Season

Fall

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Included in

Biochemistry Commons

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