Localization of 2',3'-cyclic nucleotide-3'-phosphohydrolase of rabbit brain by sedimentation in a continuous sucrose gradient

Shapira, R.; Mobley, W.C.; Thiele, S.B.; Wilhelmi, M.R.; Wallace, A.; Kibler, R.F.

Journal of Neurochemistry 30(4): 735-744

1978


ISSN/ISBN: 0022-3042
PMID: 206668
DOI: 10.1111/j.1471-4159.1978.tb10779.x
Accession: 068524062

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Abstract
The enzyme 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNP, EC 3.1.4.37) was assayed in fractions from a continuous sucrose density gradient zoneal centrifugation of rabbit brain homogenates. Basic protein (BP) was also assayed by a radioimmunomethod. Fractions were examined by SDS-polyacrylamide gel electrophoresis and by EM. These studies show the major membrane fractions in the gradient differ greatly in the content of CNP and BP, and of high MW proteins (HMW). The lightest membrane fractions contained numerous multilamellae, the highest content of BP and the lowest content of CNP and HMW, while the heaviest membrane fractions contained single membrane fragments and vesicles of unknown origin, the lowest content of BP and the highest content of CNP and HMW. The fraction containing the largest amount of membrane measured by turbidity, protein content and water-washed dry weight contained only half the CNP specific activity of a denser fraction in the gradient. CNP specific activity in the lightest fractions was insignificant compared to that of denser fractions. This enzyme may be absent from the typical multilamellar myelin structures but present in the single-membrane structures associated with myelin, such as the glial membrane and the paranodal segments of myelin adjacent to the axon. BP appears to occupy the opposite positions, highest in the multilamellae and lowest in the single-membrane structures of myelin. CNP may not possibly be bound to myelin membranes, but rather to a membrane of different origin. Evidence that this enzyme is a myelin-marker enzyme is circumstantial. The enzyme could be present either in a unique portion of myelin membranes or in another membrane structure.