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The enzymatic hydrolysis of ribonucleoside 2', 3'-cyclic phospates by a diesterase in nerve tissue Lee, Jack Foo


During the past 50 years, a number of nucleophosphodiesterases have been studied. Of recent interest, is a brain phosphodiesterase which converts ribonucleoside 2',3'-cyclic phosphates to the corresponding ribonucleoside 2'-phosphates. The physiological role of this enzyme is entirely unknown. The present studies were designed to learn something of its intracellular distribution, properties and mechanism of action. The assay of the enzyme was based on the hydrolysis of adenosine 2', 3'-cyclic phosphate by the diesterase followed by the hydrolysis of the reaction product with alkaline phosphatase and subsequent analysis for inorganic phosphate by a modified method of Fiske and SubbaRow. Rabbit brain homogenate was fractionated into nuclear, mitochondrial, microsomal and 100,000 x g supernatant fluid fractions by differential centrifugation. 2',3'-Cyclic phosphodiesterase activity was found in all the fractions with the greatest activity in the mitochondrial fraction. Since the mitochondrial fraction was a heterogenous mixture as revealed by electron microscopy, its components were separated by sucrose density gradient centrifugation by established methods. Five subtractions were obtained (A, B, C, D, E). The lightest subfraction (A) contained most of the diesterase activity and electron microscopy revealed that this subfraction consisted of fragments of myelin of various sizes. The microsomal and nuclear fractions were also subjected to sucrose density gradient centrifugation. Again, the least dense, myelin-containing sub-fraction, contained most of the diesterase activity. It was also found that rabbit brain white matter contained greater diesterase activity than grey matter. The data provide strong evidence that the diesterase is associated with myelin. It was found that acetone extraction of the myelin fraction from a homogenate of white matter of beef brain resulted in a doubling of diesterase activity with a five-fold purification. Efforts directed toward solubilization of the enzyme were unsuccessful. Some properties of the enzyme were also determined. The diesterase opened the cyclic diester bond of cyclic-ended oligonucleotides, at least as large as (A₈) and did so, without cleavage of internucleotide bonds. It was inactivated by the sulfhydryl reagent, para-hydroxymercuribenzoate, and was unaffected by the presence of a wide variety of purine and pyrimidine compounds or derivatives and various nucleoside mono-, di- and triphosphates. The Km of the diesterase was determined to be 1.9 x 10⁻³ M.

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