R.Kennedy Keller

Rapid synthesis of isoprenoid diphosphates and their isolation in one step using either thin layer or flash chromatography.

A rapid procedure for the preparation of short-chain (C s-C.) isoprenoid diphosphates is described . It is based on the method of Cornforth and Popjak [Methods Enzymol., 15 (1969) 359-390] which utilizes bis-triethylammonium phosphate in trichioroacetonitriie as the phosphorylating reagent . The reaction takes place in 15 min, and product isolation, previously requiring several steps, is done in a single step using either preparative thin-layer chromatography or flash chromatography on silica . From a single TLC plate, up to 50 μ mol of pure farnesyl diphosphate (i .e., ca. 20 mg) can be isolated, while up to 1200 μ mol can be isolated using a standard flash chromatography column.

Dolichol and Dolichyl Phosphate Levels in Brain Tissue from English Setters with Ceroid Lipofuscinosis

Abstract: Human ceroid lipofuscinosis (CL) is an inherited disease marked by cerebromacular degeneration and early death. We have utilized the canine model to investigate the possible role of dolichol and dolichyl phosphate in the developmental pathology of CL. We found that while brain levels of dolichol increase with age in both affected and unaffected dogs, the amount in the diseased animal was similar to that in controls. Brain levels of dolichyl phosphate ranged from 20 to 35 μg/g in control dogs at all ages examined, but increased substantially during development in the affected dogs, a value of 113 ± 24 μg/g (mean ± SD) being obtained in the end‐stage animals. In addition to the results obtained in the canine model, dolichyl phosphate levels in human brain tissues from a 5‐year‐old with late infantile CL and from a 19‐year‐old with juvenile CL were found to be 153 and 382 μg/g, respectively, compared with a control that assayed 26 μg/g. The preliminary findings with human tissues provide further evidence for an association of elevated brain dolichyl phosphate levels with CL. Whether the increase is primary or secondary remains to be determined.

Squalene synthase inhibition alters metabolism of nonsterols in rat liver

We have used the potent squalene synthase inhibitor squalestatin I to investigate the regulation of isoprenoid metabolism in rat liver Fresh-frozen liver pieces from normal rats and rats infused with squalestatin I at 16 micrograms h-1 for 16 h were assayed for farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) by HPLC after dephosphorylation. Levels of FPP and GGPP were 5.4 +/- 1.6 nmol g-1 and 1.6 +/- 0.7 nmol g-1 (n = 13) wet wt., respectively, in control livers and 110 + 41 nmol g-1 and 3.0 +/- 2.2 nmol g-1 (n = 13) in livers from squalestatin I infused rats. In order to determine the relative level of isopentenyl pyrophosphate, liver slices from normal and squalestatin I infused rats were labeled to steady-state with [3H]acetate. Analysis of isoprenoid pyrophosphate intermediates by radio-HPLC after dephosphorylation indicated that squalestatin I brought about a 20-fold increase in the relative level of FPP (confirming direct analysis) and a 5-fold increase in the relative level of IPP. No change in either of these compounds was observed in livers from cholesterol-fed rats. To determine if squalestatin I altered the synthesis of nonsterol products, rats were subjected to long term subcutaneous infusion. After 14 days of infusion of 15 micrograms h-1, the median chain length of hepatic dolichol and dolichyl phosphate increased from C95 to C115 and the levels of these lipids increased approximately 3-fold. In addition, dolichyl phosphate mannose synthase activity in microsomes from squalestatin I treated rats was increased relative to controls when assayed in the absence of dolichyl phosphate. Squalestatin I affected ubiquinone metabolism to a lesser extent: chain lengths shifted from a Q10/Q9 ratio of 0.118 +/- 0.021 in the normal rat to 0.185 +/- 0.016 in the squalestatin I treated animals, and levels rose by approximately 90%. These results suggest that the isoprenoid pyrophosphate intermediates are shared by the cholesterol, dolichol and ubiquinone pathways and further show that the dolichol and ubiquinone pathways are not saturated. Apparently, under normal conditions, the levels of these intermediates are maintained relatively constant by coordinate enzyme regulation, thereby ensuring a constant rate of synthesis of nonsterols.

In vivo biosynthesis of cholesterol in the rat retina

Previous reports have suggested that the rate of de novo cholesterol synthesis in the adult vertebrate retina is extremely slow. We investigated cholesterol biosynthesis in the adult rat retina in vivo, following intravitreal injection of [3H]acetate. HPLC analysis of retinal non‐saponifiable lipid extracts revealed co‐elution of radioactivity with endogenous cholesterol mass within 4.5 h post‐injection. Incorporation of [3H]acetate into cholesterol was markedly reduced by co‐injection of known inhibitors of the cholesterol pathway. In contrast to previous results with retinas from other species, no radiolabel or mass corresponded to squalene, except in lipid extracts from retinas treated with NB‐598, a squalene epoxidase inhibitor. These results demonstrate, for the first time, the capacity of the adult vertebrate retina to rapidly synthesize cholesterol de novo.

Microdetermination of dolichol in tissues

A method is described for the rapid purification and analysis of tissue dolichol on a nanomole scale. The assay is based on a radioisotope dilution technique in which [3H]dolichyl palmitate is used as tracer and acetylation with [14C]acetic anhydride serves as the quantitating reaction. The acetylation conditions were characterized with respect to time, temperature and concentration of reagents. When this method was applied to pig liver, a value was obtained which fell within the range of previously published results based on a gravimetric assay. In chicken, dolichol levels were found to be high in oviduct and low or absent in red blood cells and plasma.

Quantitation of dolichyl phosphate and dolichol in major organs of the rat as a function of age

Previous studies in mice and humans have shown age-related increases in the levels of dolichol in all organs investigated. In the present study, the levels of dolichyl phosphate, the physiologically active form of dolichol, as well as dolichol and cholesterol were determined in five major organs of the rat from 4 to 14 wk of age. As observed for mice and humans, the levels of dolichol increased in all tissues examined, especially testis where an eightfold increase was found. Cholesterol levels remained relatively constant in all tissues examined except brain, where a threefold increase was observed. Hepatic dolichyl phosphate levels decreased slightly during growth while nonhepatic tissues showed moderate (1.2–1.7-fold) increases. It is proposed that steady-state levels of hepatic dolichyl phosphate are maintained in the face of constant de novo synthesis by a combination of two pathways: export, either via the circulation or the previously demonstrated fecal route (Connelly and Keller [1984]bioscience Reports 4, 771–776) and conversion to dolichol with subsequent accumulation.

Extraction of dolichyl phosphate and its quantitation by straight phase high-performance liquid chromatography

A procedure for the rapid quantitation of dolichyl phosphate by high-performance liquid chromatography on silica is described. The compound elutes as a single peak at 6 ml in excellent yield. The method employs isocratic elution and requires no column treatment between runs; the limit of sensitivity is in the nanogram range. Dolichyl-11-phosphate, which elutes at 7 ml, can be used as an internal standard, thereby eliminating the requirement for preparation of [3H]dolichyl phosphate. The procedure was used in development of a facile assay for free dolichyl phosphate in rat liver. For the assay of total dolichyl phosphate (free and chemically bound), it was found that when rat liver is first saponified and then extracted with ethyl ether, the amount of dolichyl phosphate present in the ether extract is significantly greater than the sum of the amounts found in extracts derived by treating the tissue first with chloroform/methanol (2/1) and then with chloroform/ethanol/water (10/10/3). Using these new procedures, the level of total dolichyl phosphate in rat liver was found to be 14.7 +/- 3.5 micrograms g-1 wet wt (n = 28). Levels in six other organs are also reported.

Subcellular localization and substrate specificity of dolichol kinase from rat liver

When purified subcellular fractions were prepared from rat liver and assayed for dolichol kinase activity using pig liver dolichol as a substrate, the microsomes were found to contain the highest specific activity and greater than 75% of the total activity. With regard to substrate specificity, the microsomal enzyme showed a marked preference for saturation of the alpha-isoprene: dolichol-16 and -19 were 2.5-fold more active than the corresponding polyprenols. For a given class of prenol, the 16 and 19 isoprenologs exhibited similar activity, whereas the 11 isoprenolog appeared less active. The enzyme was twice as active against the naturally occurring polyprenol-16 (alpha-cis-isoprene) compared to synthetic alpha-trans-polyprenol-16. Taken together, the data indicate that the alpha-isoprene specificity follows the order: saturated greater than cis greater than trans. In addition, all-trans-2,3-dihydrosolanesol was not a substrate, suggesting that at least one cis isoprene residue is required.

Tunicamycin does not block ovalbumin secretion in the oviduct.

Abstract In order to examine the role of carbohydrate in the secretion of ovalbumin, oviduct minces were incubated in the presence of tunicamycin, an inhibitor of dolichol-mediated glycosylation. Ovalbumin secretion was monitored immunologically and found to be identical, within experimental error, in the absence and presence of tunicamycin. These results, coupled with the recent finding of Palmiter et al . [Proc. Natl. Acad. Sci. (1978) 75 , 94–98] indicate that neither a transient hydrophobic pre-piece nor carbohydrate is required for ovalbumin secretion.

Dolichol metabolism in the rat

Abstract Dolichyl phosphate (Dol-P) is a long chain polyisoprenoid which transfers saccharide residues in N -linked glycoprotein synthesis. It is synthesized by a quantitatively small branch of the cholesterol pathway at a rate which, at least in liver, appears to be independent of the rate of sterol synthesis. This article reviews recent work on the fate of dolichol compounds and the regulation of Dol-P synthesis.