Characterization and Localization of a Long‐Chain Isoprenyltransferase Activity in Porcine Brain: Proposed Role in the Biosynthesis of Dolichyl Phosphate

Characterization and Localization of a Long‐Chain Isoprenyltransferase Activity in Porcine... Pig brain microsomes catalyzed the enzymatic transfer of radiolabeled isoprenyl groups from (1‐14C) isopentenyl pyrophosphate ((1‐MC) I‐P‐P) into long‐chain polyisoprenyl pyrophosphates (Poly‐P‐P) and unidentified neutral lipids. The brain isoprenyltransferase activity synthesizing the Poly‐P‐P (1) required 5 mM Mg2+ and 10 mM vanadate ions for maximal activity; (2) exhibited an apparent Km of 8 μM I‐P‐P; (3) utilized exogenous farnesyl pyrophosphate and two stereoisomers of geranylgeranyl pyrophosphate as substrates; (4) was optimal at pH 8.5; and (S) was stimulated by dithiothreitol. The major products were identified as C90and Q95 allyhic Poly‐P‐P on the basis of the following chemical and chromatographic properties: (1) the intact product cochromatographed with authentic Poly‐P‐P on silica‐gel‐impregnated paper, (2) the major product was converted to a compound chromatographically identical to polyisoprenyl monophosphate (Poly‐P) by alkaline hydrolysis; (3) treatment of the labeled Poly‐P with wheat germ acid phosphatase or mild acid yielded neutral labeled products; (4) the KOH hydrolyzed product coeluted with authentic Poly‐P from lipophilic Sephadex LH‐20; and (5) the labeled lipids produced by enzymatic dephosphorylation had mobilities identical to fully unsaturated polyisoprenols containing 18 (C90) and 19 (Q95) isoprene units when analyzed by reverse‐phase chromatography. When subcellular fractions from rat brain gray matter were compared, the highest specific activity was found in the heavy microsomes. These results demonstrate that brain contains an isoprenyltransferase activity, associated with the rough endoplasmic reticulum, capable of synthesizing long‐chain Poly‐P‐P. The enzymatic reactions by which the Poly‐P‐P intermediate is converted to dolichyl phosphate remain to be elucidated. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Neurochemistry Wiley

Characterization and Localization of a Long‐Chain Isoprenyltransferase Activity in Porcine Brain: Proposed Role in the Biosynthesis of Dolichyl Phosphate

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Publisher
Wiley
Copyright
Copyright © 1991 Wiley Subscription Services, Inc., A Wiley Company
ISSN
0022-3042
eISSN
1471-4159
DOI
10.1111/j.1471-4159.1991.tb08301.x
Publisher site
See Article on Publisher Site

Abstract

Pig brain microsomes catalyzed the enzymatic transfer of radiolabeled isoprenyl groups from (1‐14C) isopentenyl pyrophosphate ((1‐MC) I‐P‐P) into long‐chain polyisoprenyl pyrophosphates (Poly‐P‐P) and unidentified neutral lipids. The brain isoprenyltransferase activity synthesizing the Poly‐P‐P (1) required 5 mM Mg2+ and 10 mM vanadate ions for maximal activity; (2) exhibited an apparent Km of 8 μM I‐P‐P; (3) utilized exogenous farnesyl pyrophosphate and two stereoisomers of geranylgeranyl pyrophosphate as substrates; (4) was optimal at pH 8.5; and (S) was stimulated by dithiothreitol. The major products were identified as C90and Q95 allyhic Poly‐P‐P on the basis of the following chemical and chromatographic properties: (1) the intact product cochromatographed with authentic Poly‐P‐P on silica‐gel‐impregnated paper, (2) the major product was converted to a compound chromatographically identical to polyisoprenyl monophosphate (Poly‐P) by alkaline hydrolysis; (3) treatment of the labeled Poly‐P with wheat germ acid phosphatase or mild acid yielded neutral labeled products; (4) the KOH hydrolyzed product coeluted with authentic Poly‐P from lipophilic Sephadex LH‐20; and (5) the labeled lipids produced by enzymatic dephosphorylation had mobilities identical to fully unsaturated polyisoprenols containing 18 (C90) and 19 (Q95) isoprene units when analyzed by reverse‐phase chromatography. When subcellular fractions from rat brain gray matter were compared, the highest specific activity was found in the heavy microsomes. These results demonstrate that brain contains an isoprenyltransferase activity, associated with the rough endoplasmic reticulum, capable of synthesizing long‐chain Poly‐P‐P. The enzymatic reactions by which the Poly‐P‐P intermediate is converted to dolichyl phosphate remain to be elucidated.

Journal

Journal of NeurochemistryWiley

Published: Oct 1, 1991

References

  • Developmental changes in enzymes involved in dolichyl phosphate metabolism in cultured embryonic rat brain cells
    Bhat, Bhat; Frank, Frank; Wolf, Wolf; Waechter, Waechter
  • The structure of brain polyisoprenols
    Breckenridge, Breckenridge; Wolfe, Wolfe; Ng Ying Kin, Ng Ying Kin
  • Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus
    Hirschberg, Hirschberg; Snider, Snider
  • Assembly of asparagine‐linked oligosaccharides
    Kornfeld, Kornfeld; Kornfeld, Kornfeld
  • High levels of brain dolichols in neuronal ceroid‐lipofuscinosis and senescence
    Ng Ying Kin, Ng Ying Kin; Palo, Palo; Haltia, Haltia; Wolfe, Wolfe

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