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The uptake of C95[l-3H]dolichol in the form of liposomes into rat brain after the intracerebral and intraperitoneal injection was inves­tigated. Efficient, time-dependent uptake of dolichol into the brain was observed exclusively after the intraventricular injection. Within 24 h after the injection about 10% of the applied dolichol was found in the brain and 1.5% in liver. The distribution of dolichol in various parts of rat brain decreased in the order: cerebellum > midbrain > grey matter and brain stem > white matter. Sevtn days after the injection total radioactivity in the brain decreased and concomitantly a sig­nificant increase was observed in blood circulating and liver of the rat. The highest activity was found in grey matter and it remained a few times higher in comparison with that in whitematter. About 80% of the dolichol taken up by the brain membrane was recovered in the follow­ing subcellular fractions: crude nuclear fraction > microsomes > mitochondria > synaptosomes > myelin.
The contents of the heterogenous group of polyisoprenoids was found about two orders of magnitude lower in seeds than the amount of polyprenols and/or their carboxylic esters accumulated during vegetation season in leaves. In contrast to leaves, no seeds were found containing more than 0.5 mg of these lipids per gram of dry tissue. Almost 50% had less than 0.01 mg/g — the amount which is the limit of detection by the procedure used in this work. In gymnosperms (10 representatives of Cupressaceae, Pinaceae and Taxaceae) the polyprenol spectra in seeds and in needles were similar. In angiosperms (25 representatives of 13 botanical families) the polyisoprenoid mixture in seeds resembled the minor, additional subfamily found in leaves.
A protein catalyzing dolichol transfer between membranes has been purified from bovine liver up to 600-fold by acid precipitation, ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic interaction chromatography. The protein displays a relative molecular mass of 15000 on SDS-gel electrophoresis. Kinetics as well as the influence of a series of effectors were studied. The transfer activity is inhibited by sphingomyelin, sulfhydryl groups and cationic amphiphilic amines with a bulky heterocyclic aromatic function. High salt concentration decreases the transfer efficiency. Transfer of dolichol between vesicles and mitochondria is not affected by the presence of moderate amounts of cholesterol in the donor vesicles. The overall characteristics of dolichol transfer activity are discussed in comparison to these of other lipid transfer proteins.
In vitro cultivated plant cells and tissues were found to synthesize polyisoprenoids. Taxus baccata suspension cell cultures accumulated polyisoprenoids of the same pattern as the parental tissue; methyl jasmonate or chitosan treatment almost doubled their content. All the root cultures studied accumulated dolichols as predominant polyisoprenoids. Roots of Ocimum sanctum grown in vitro accumulated approx. 2.5-fold higher amount of dolichols than the roots of soil-grown plants. Dolichols dominated over polyprenols in all Triticum sp. tissues studied.
Three different mass spectrometric method suitable for the analysis of polyprenyl and dolichyl phosphates and their glycosylated forms are described. Fast atom bombardment mass spectrometry (FAB MS) of glycosyl monophosphopolyprcnols produces negative ions characteristic of the intact molecule. Tandem mass spectrometry of (M-H)~ anions allows the determination of masses of both glycosyl and lipid moieties. Thus, for example, FAB-MS/MS of a mixture of native glycosyl monophosphopolyprenols isolated from ethambutol-treated Mycobacterium smegmatis enabled us to detect two novel pentosyl monophosphopolyprenols. Two other methods are proposed for the analysis of prenyl phosphates, as these compounds do not produce fragments in FAB-MS/MS at low collisional energy. By Desorption Electron Impact ionization (DEI) an intense (M-H3PO4)* ion as well as fragments corresponding to the successive loss of isoprene residues (68 Da) can be observed. Alternatively, Desorption Chemical Ionization yields ions corresponding to the loss of 66, 78 and 98 Da (i.e. of a part or the entire phosphate moiety) of a prenyl phosphate molecule. Tandem mass spectrometry of the (M-H-98)" ion gives a series of intense fragments differing by 68 mass units over the whole mass range.
Dolichols isolated from leaves of the fern Matteucia struthiopteris were present as a mixture of prenologues composed of 14 up to 20 isoprene units with Dol-16 dominating. They comprised approximately 0.004% of the fresh weight of fresh plant tissue and were accompanied by traces of polyprenols (Pren-14 up to Pren-17, Pren-16 dominating). Their structure was confirmed by electropray ionization mass spectrometry (ESI-MS). This is the first time that dolichols have been reported as dominating polyisoprenoid alcohols in plant photosynthetic tissue.
The results obtained on dolichol metabolism, in two itt vivo model systems, the developing rat liver and the regenerating rat liver, which provide different timing and interplay of proliferation and differentiation processes, have been reported. The regenerating liver presents a marked increase of both synthesis and content of dolichol, a decreased cholesterol/dolichol ratio, unchanged synthesis and content of dolichyl phosphate, or dolichol-kinase and dolichyl phosphate-phosphatase activities; no significantly modified distribution of dolichol homologs, with respect to the control. Total content of dolichols is growing during perinatal development. At fetal stages only short chain dolichols are detectable, while the content of dolichyl phosphate is very low and the activity of dolichyl phosphate-phosphatase is high. The study of the role of liver in dolichol supply to the body in the partially hepatectomized rat shows an increased content of dolichol in the blood; blood dolichol is essentially provided by the release from liver and dolichol traffic in the blood is mediated by multiple carriers.
GDPMan is a key substrate in glycoprotein formation. This is especially true for lower eukaryotes where, in addition to the involvement in N-glycan biosynthesis and GPI-anchor formation, GDPMan takes part in the process which is unique for yeast and fungi i.e. O-mannosylation. Several lines of evidence have been presented that the level of GDPMan affects the process occurring in the Golgi compartment i.e. the elongation of outer mannose chain of glycoproteins in Saccharomyces cerevisiae. Results from our laboratory indicate that the availability of GDPMan affects also the early steps of glycoprotein formation ascribed to the endoplasmic reticulum, i.e. assembly of the dolichol-linked oligosaccharide as well as mannosyl-phosphodolichol (MPD) formation. The biochemical basis of carbohydrate deficient glycoprotein syndrome, a severe neurological disorder related to the GDPMan deficiency, is also discussed.
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