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UBC Theses and Dissertations

Investigations into the physiological significance of the brain enzyme 2', 3'-cyclic nucleotide-3'-phosophohydrolase.. Olafson, Robert W.


Preliminary observations of the restricted regionalization of the enzyme 2' ,3'-cyclic nucleotide-3’ -phosphohydrolase led to an investigation of the subcellular regionalization of this enzyme in cerebral white matter. Since bovine corpus callosum contained eighteen times as much enzyme activity as grey matter, an association of the enzyme with myelin was suggested. Subsequent fractionation of bovine cerebral white matter by sucrose density gradient according to the procedure of Autilio, Norton, and Terry for the purification of myelin (1), showed that greater than 60% of the total activity was associated with the myelin rich fractions. In order to fractionate cerebral white matter more thoroughly, a modified De Robertis fractionation procedure was utilized allowing for separation of nuclear, mitochondrial, and microsomal pellets by differential centrffugation (2). Phosphohydrolase activity was distributed in all fractions, and electron microscopy demonstrated the presence of myelin in all of these fractions. Subsequent fractionation of these primary fractions on a discontinuous sucrose density gradient, showed essentially all of the phosphohydrolase activity in the lightest fraction at the top of each gradient. This band was comprised primarily of myelin figures as verified by electron microscopy. These studies indicated that the enzyme was associated with myelin. The foregoing result was further supported by a study of the increase in enzyme activity during myelination in rats. Myelination is known to occur early in the life of the rat, being initiated a few days after birth, entering a rapid phase of onset at about 10 days and being essentially complete after 50 days (3). Cholesterol was shown to increase in a corresponding manner indicating that myelination was indeed proceeding. Further evidence that the enzyme is associated with myelin came from an investigation of mutant mice. Quaking mice have been shown to be deficient in myelin, containing, according to Bauman and co-workers (4), only 62% of the normal galactolipid levels. Since galactolipids are presently accepted markers for myelin, and since adult quaking mice had 50% of the control enzyme activity, in agreement with the published galactolipid values, it was thought not unlikely that the two phenomena were related. This result also inferred an association of the enzyme with myelin. In attempt to further uncover the physiological role of the 2’,3'-cyclic nucleotide-3'-phosphqhydrolase, investigations have been directed towards elucidation of the substrate specificity of the enzyme. Uridine and guanosine-2’,3'-cyclic phosphothioates, kindly donated by Dr. Fritz Eckstein of the Max Planck Institut für Experimentelle Medizin, were hydrolyzed at rates of l.4 and l4.3% that of adenosine-2’,3'-cyclic phosphate. Cyclic inositol phosphate, synthesized from inositol-2-phosphate in the presence of dicyclohexylcarbodi-imide and pyridine, and glucose-l,2-cyclic phosphate, synthesized from (formula omitted)-D-glucose-l-phosphate, in a similar manner, were not hydrolyzed td any measureable extent. Preliminary results also show that ribose cyclic phosphates are not hydrolyzed, indicating a requirement for a purine or pyrimidine ring in the substrate molecule. These results are discussed with respect to their possible physiological significance.

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