Vitamin E Photoaffinity Label

Vitamin E (or a-tocopherol) is now accepted as the premier lipid soluble antioxidant in mammals. The absorption of vitamin E into tissues after ingestion largely parallels that of other lipids. A number of different lipoproteins serve as carriers of bulk lipids through the blood stream and the lymph. Eventually these aggregates reach the liver where phospholipids, triglycerides and fatty acids are 'repackaged' into new structures for transport back to specific tissues in the body.

Alpha-Tocopherol Binding Protein

Alpha-tocopherol contains three stereogenic centres and thus exists as a mixture of eight stereoisomers in the all-racemic form that is provided in most commercially available vitamin preparations. Recent work at the NRC using differentially deuterated optical isomers of a-tocopherol has shown that the natural isomer (D or R,R,R) disappears more quickly from the liver after dosing than the enantiomer (L or S,S,S). This implies that these two stereoisomers of a-tocopherol are discriminated from each other and suggests that there is an interaction with another chiral entity, likely a macromolecule. Indeed, two groups of researchers have now identified a protein that acts as a binding or transfer protein for a-tocopherol. The purification and structural identification of this protein would be greatly simplified if there existed a form of vitamin E that could be selectively and irreversibly bound to the protein. The synthesis has been designed such that one intermediate can be used to complete the photoaffinity label or be attached to an inert matrix for the preparation of a protein affinty-purification column.

The synthesis of such a label would provide an opportunity for the student to work with chiral materials, perform carbanion chemistry and several interesting oxidations. In addition it offers the chance for a chemist to 'cross over' into the wet chemsitry of protein labeling and purification. All compounds synthesized will have to be tested for their potential binding affinity to a tocopherol binding protein. Depending on the success of synthetic work this may also be performed by a student on this project. Such tests would involve the preparation of rat liver protein isolates, and filter based binding assays wiht tritiated tocopherol. The synthetic design provides a key internediate that can be used to generate a ligand for an affinity gel. This would allow the specific isolation of proteins with the abaility to bind to tocopherol. It is likely that most of the time on this project would be for synthesis and for characterization of the photochemical properties of any produced photoaffinity labels.

Protein Kinase C In a recent development, Swiss workers have published accounts of their work investigating the inhibition of smooth muscle cell proliferation by a-tocopherol and have discussed their results in terms of their initial observation that a-tocopherol inhibits protein kinase C (PKC). They have proposed a model where a-tocopherol binds to the soluble cytosolic form of PKC, thus inhibiting is membrane translocation and consequent activation. They have supported this model by demonstrating the in vitro inhibition of PKC. No correlation appears to exist between the antioxidant activity of various tocopherol analogues and their ability to inhibit PKC. PKC is an important component of the secondary messenger system and has been linked to several pathological conditions that encompass gene regulation, cell differentiation and cancer.

Phospholipase A2 It has been demonstrated that a-tocopherol inhibits phospholipase A2 activity in platelets as well as in rat cardiac tissue, and thus has a role in modulating the arachidonic acid cascade. This is understood to occur wihtout recourse to tocopherol's antioxidant activity. While a-tocopherol attenuates the activity phospholipase A2 and thus the release of arachidonate from platelets, it appears to stimulate this enzyme's activity in cultured endothelial cells stimulating prostacyclin synthesis. There is currently no explanation for the differing modulation of phospholipase A2 activities seen in these experiments.

CoA-Independent Acyltransferase Investigations of phospholipid metabolism in endothelial cells has provided further details of tocopherol's modifying effects on enzyme activity. Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; alkylacetyl-GPC) is synthesized from 1-O-alkyl-2-arachidonyl-GPC by the phospholipase A2 mediated release of archidonate to give alkyl-GPC, followed by acetylation at the 2-position by an acetyltransferase. Experiments performed with cultured endothelial cells showed that a CoA-independent acyltransferase is capable of reacylating alkyl-GPC. This is important insofar as it limits the accumulation of lysophospholipids such as alkyl-GPC which are cytotoxic at high concentrations. This activity was enhanced by a-tocopherol in a dose dependent manner at concentrations below 23 mM.

This project would be in collaboration with Dr. Patrick Choy, Dept. Biochemistry and Molecular Biology, University of Manitoba, Winnipeg and Dr. Maret Traber, Department of Molecular and Cell Biology, University of California, Berkeley.