The subcellular distribution of polyisoprenyl pyrophosphate phosphatase activity has been examined in rat brain by assaying the release of 32Pi from [β-32P]dolichyl pyrophosphate (Dol-P-P) as described previously (Scher, M.G. and Waechter, C.J. (1984) J. Biol. Chem., 259, 14580-14585). The highest specific activities of Dol-P-P phosphatase in rat brain were found in the Golgi-enriched light microsomal, synaptic plasma membrane and heavy microsomal fractions. A comparative analysis of the distribution of galactosyltransferase and dolichol kinase reveals that Dol-P-P phosphatase activity co-fractionates with galactosyltransferase activity, and that the high level found in the Golgi-enriched fraction is not due to cross-contamination with heavy microsomes. When β-labelled C95 Dol-P-P and the C95 allylic polyisoprenyl pyrophosphate (Poly-P-P) were compared as substrates for the Golgi-enriched light microsomal and heavy microsomal fractions, similar Km values were calculated for the two pyrophosphorylated substrates for each membrane fraction. Based on these kinetic analyses, the enzyme(s) catalysing this reaction do not distinguish between substrates containing saturated or allylic α-isoprene units. When Dol-P-P phosphatase activity was assessed in submicrosomal fractions obtained from rat liver by two separate procedures, the highest specific activity was also detected in the Golgi-enriched fraction. While the specific activities for Dol-P-P phosphatase and sialyltransferase were in the relative order of Golgi > smooth endoplasmic reticulum (ER) > rough ER, the relative order of dolichol kinase was rough ER > smooth ER > Golgi. In substrate competition studies, the polyisoprenyl pyrophosphate phosphatase activities associated with the Golgi-enriched fractions from rat brain and liver were not affected by a 5-fold excess of UDP or thiamine pyrophosphate, but were inhibited by the isoprenyl pyro-phosphates, farnesyl pyrophosphate and undecaprenyl pyrophosphate. These results demonstrate that membrane vesicles derived from the rough ER and Golgi apparatus from brain and liver contain polyisoprenyl pyrophosphate phosphatase activity capable of converting Dol-P-P or the long-chain Poly-P-P to their respective phosphomonoesters.
|Number of pages||6|
|State||Published - Sep 1991|
Bibliographical noteFunding Information:
The authors thank David Frank and Dr Dean C.Crick for their many helpful suggestions, and express their appreciation to Virginia Hodges for her invaluable secretarial help in preparing the manuscript. This work was supported by National Institutes of Health Grant GM 36065 awarded to C.J.W.
- Membrane fractions
- Polyiso-prenyl pyrophosphate phosphatase
ASJC Scopus subject areas