Characterization and localization of a long-chain isoprenyltransferase activity in porcine brain: proposed role in the biosynthesis of dolichyl phosphate.
Pig brain microsomes catalyzed the enzymatic transfer of radiolabeled isoprenyl groups from [1-14C]isopentenyl pyrophosphate [( 1-14C]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 microM for I-P-P; (3) utilized exogenous farnesyl pyrophosphate and two stereoisomers of geranylgeranyl pyrophosphate as substrates; (4) was optimal at pH 8.5; and (5) was stimulated by dithiothreitol. The major products were identified as C90 and C95 allylic Poly-P-P on the basis of the following chemical and chromatographic properties: (1) the intact product co-chromatographed 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 (C95) 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.
Crick
D C
DC
Department of Biochemistry, University of Kentucky College of Medicine, A. B. Chandler Medical Center, Lexington 40536.
Rush
J S
JS
Waechter
C J
CJ
D000818:Animals; D001921:Brain / Q000201:enzymology*; D004288:Dolichol Phosphates / Q000378:metabolism*; D045782:Hemiterpenes; D008861:Microsomes / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010487:Periaqueductal Gray / Q000201:enzymology; D011105:Polyisoprenyl Phosphate Sugars / Q000737:chemistry; D013347:Subcellular Fractions / Q000378:metabolism; D013552:Swine / Q000378:metabolism*
D004288:Dolichol Phosphates; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011105:Polyisoprenyl Phosphate Sugars; C028306:dolichol monophosphate; C004809:isopentenyl pyrophosphate