Abstract
Two independent experiments indicated that the major subcellular site of de novo dolichyl phosphate biosynthesis in rat liver is in the microsomes. First, when purified subcellular fractions were assayed for long chain prenyltransferase activity, the profile obtained indicated exclusively a microsomal localization. Second, when liver slices were incubated 60 min with [14C]acetate and subcellular fractions were subsequently isolated, the microsomal fraction was found to contain greater than two-thirds of the total radioactivity incorporated into dolichyl phosphate. Chemical analysis of dolichyl phosphate in subcellular fractions showed substantial quantities in both the microsomal and mitochondrial-lysosomal fractions. In contrast, dolichol was found primarily in the mitochondrial-lysosomal fraction. The absolute in vitro rate of hepatic dolichyl phosphate synthesis was determined in liver slices and isolated hepatocytes by [14C]acetate labeling using [3H] water incorporation into cholesterol to correct for the dilution of the labeled acetate by the endogenous acetyl-CoA pool. From this value, an in vivo rate of de novo synthesis was estimated as 1.7-2.6 nmol of dolichyl phosphate/liver/day. This rate is more than 30 times the maximum possible rate of accumulation in liver of dietary dolichol (Keller, R. K., Jehle, E., and Adair, W. L., Jr. (1982) J. Biol. Chem. 257, 8985-8989). From the previously determined concentration of dolichyl phosphate in rate liver (Keller, R. K. Tamkun, J. M., and Adair, W. L. (1981) Biochemistry 20, 5831-5836) and the de novo rate of synthesis, it appears that the half-life of hepatic dolichyl phosphate is on the order of days.
Highlights
Two independent experiments indicated that the ma- study by Rip et all ( 6 )reported high concentrations of dolichol jor subcellular site of de novo dolichyl phosphate bio- in rat liver plasma membranes
The absolute in vitro rate of hepatic dolichyl phosphate synthesis was determined ih liver slices and isolated hepatocytes by[‘4C]acetate labeling using [’HI water incorporation into cholesterol to correct for the Dolichyl phosphate biosynthesis diverges from that of cholesterol at the level of farnesyl pyrophosphate
The isoprene chain is lengthened by the addition of 14-18cis-isoprene units to farnesyl pyrophosphate in a reaction catalyzed by a long chainprenyltransferase
Summary
DETERMINATION OF T H E SUBCELLULAR DISTRIBUTION OF DOLICHYL PHOSPHATE AND ITS SITE AND RATE OF DE NOVO BIOSYNTHESIS*. In all previously reported studies on dolichol or dolichyl phosphate biosynthesis, the rates determined were based on the quantity of radioactivity incorporated into product and. Rified subcellular fractions prepared from liver slices which had been incubated with [14CJacetate.The absolute rate of dolichyl phosphate biosynthesis was estimated in rat liver slices and isolated hepatocytes using the methodology of Anderson and Dietschy [25]. The half-life of dolichyl phosphate in rat liver is on the order of days The implications of these hepatocytes, a parenchymal cell fraction was prepared from mid-dark rats exactly as described by Seglen ( 3 3 ) .Approximately 3 X 10' cells (representing -230 mg wet weight of tissue) were incubated in 10-ml Erlenmeyer flasks in a total volume of 2.5 ml of suspension buffer [33] containing 7 mM sodium acetate. First flask contained 15 mCi of ["Hlwater and 5 yCi of ['%]acetate
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