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• W2114602369 abstract "The amino acid leucine is efficiently used by the trypanosomatid Leishmania mexicana for sterol biosynthesis. The incubation of [2-13C]leucine withL. mexicana promastigotes in the presence of ketoconazole gave 14α-methylergosta-8,24(24 1The abbreviations used are: HMG3-hydroxy-3-methylglutarylGCgas chromatographyMSmass spectrometryHPLChigh pressure liquid chromatographyTMStetramethylsilylTMSOHtetramethylsilol)-3β-ol as the major sterol, which was shown by mass spectrometry to contain up to six atoms of 13C per molecule. 13C NMR analysis of the 14α-methylergosta-8,24(241)-3β-ol revealed that it was labeled in only six positions: C-2, C-6, C-11, C-12, C-16, and C-23. This established that the leucine skeleton is incorporated intact into the isoprenoid pathway leading to sterol; it is not converted first to acetyl-CoA, as in animals and plants, with utilization of the acetyl-CoA to regenerate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An inhibitor of HMG-CoA synthase (L-659,699) blocked the incorporation of [1-14C]acetate into sterol but had no inhibitory effect on [U-14C]leucine incorporation. The HMG-CoA reductase inhibitor lovastatin inhibited promastigote growth and [U-14C]leucine incorporation into sterol. The addition of unlabeled mevalonic acid (MVA) overcame the lovastatin inhibition of growth and also diluted the incorporation of [1-14C]leucine into sterol. These results are compatible with two routes by which the leucine skeleton may enter intact into the isoprenoid pathway. The catabolism of leucine could generate HMG-CoA that is then directly reduced to MVA for incorporation into sterol. Alternatively, a compound produced as an intermediate in leucine breakdown to HMG-CoA (e.g. dimethylcrotonyl-CoA) could be directly reduced to produce an isoprene alcohol followed by phosphorylation to enter the isoprenoid pathway post-MVA. The amino acid leucine is efficiently used by the trypanosomatid Leishmania mexicana for sterol biosynthesis. The incubation of [2-13C]leucine withL. mexicana promastigotes in the presence of ketoconazole gave 14α-methylergosta-8,24(24 1The abbreviations used are: HMG3-hydroxy-3-methylglutarylGCgas chromatographyMSmass spectrometryHPLChigh pressure liquid chromatographyTMStetramethylsilylTMSOHtetramethylsilol)-3β-ol as the major sterol, which was shown by mass spectrometry to contain up to six atoms of 13C per molecule. 13C NMR analysis of the 14α-methylergosta-8,24(241)-3β-ol revealed that it was labeled in only six positions: C-2, C-6, C-11, C-12, C-16, and C-23. This established that the leucine skeleton is incorporated intact into the isoprenoid pathway leading to sterol; it is not converted first to acetyl-CoA, as in animals and plants, with utilization of the acetyl-CoA to regenerate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An inhibitor of HMG-CoA synthase (L-659,699) blocked the incorporation of [1-14C]acetate into sterol but had no inhibitory effect on [U-14C]leucine incorporation. The HMG-CoA reductase inhibitor lovastatin inhibited promastigote growth and [U-14C]leucine incorporation into sterol. The addition of unlabeled mevalonic acid (MVA) overcame the lovastatin inhibition of growth and also diluted the incorporation of [1-14C]leucine into sterol. These results are compatible with two routes by which the leucine skeleton may enter intact into the isoprenoid pathway. The catabolism of leucine could generate HMG-CoA that is then directly reduced to MVA for incorporation into sterol. Alternatively, a compound produced as an intermediate in leucine breakdown to HMG-CoA (e.g. dimethylcrotonyl-CoA) could be directly reduced to produce an isoprene alcohol followed by phosphorylation to enter the isoprenoid pathway post-MVA. 3-hydroxy-3-methylglutaryl gas chromatography mass spectrometry high pressure liquid chromatography tetramethylsilyl tetramethylsilol Parasitic trypanosomatid protozoa of the genusLeishmania cause diseases in tropical and subtropical regions of the world. The treatment of leishmaniasis still relies upon the drugs introduced many years ago (1Croft S.L. Urbina J.A. Brun R. Hide G. Mottram J.C. Coombs G.H. Holmes P.H. Trypanosomiasis and Leishmaniasis Biology and Control. CAB International, Wallingford, UK1997: 245-258Google Scholar), which have toxic side effects, and there is now a great need for more effective new chemotherapeutic drugs (1Croft S.L. Urbina J.A. Brun R. Hide G. Mottram J.C. Coombs G.H. Holmes P.H. Trypanosomiasis and Leishmaniasis Biology and Control. CAB International, Wallingford, UK1997: 245-258Google Scholar, 2Van den Bossche, H. (1993) Microbiol. Eur.Nov./Dec. 20–28.Google Scholar). This has prompted the search for new metabolic targets for drugs and has resulted in the recognition of sterol synthesis inhibitors as a potential candidate (2Van den Bossche, H. (1993) Microbiol. Eur.Nov./Dec. 20–28.Google Scholar, 3Chance M.L. Goad L.J. Hide G. Mottram J.C. Coombs G.H. Holmes P.H. Trypanosomiasis and Leishmaniasis Biology and Control. CAB International, Wallingford, UK1997: 163-176Google Scholar). The importance of an active sterol biosynthetic pathway in trypanosomatids for growth and viability has been demonstrated using antifungal agents that are inhibitors of sterol biosynthesis. Thus, the imadazole- and triazole-based drugs (e.g. ketoconazole and itraconazole), which inhibit the 14α-methylsterol 14-demethylase, and the allylamines (e.g.terbinafine), which inhibit squalene epoxidase (2Van den Bossche, H. (1993) Microbiol. Eur.Nov./Dec. 20–28.Google Scholar, 3Chance M.L. Goad L.J. Hide G. Mottram J.C. Coombs G.H. Holmes P.H. Trypanosomiasis and Leishmaniasis Biology and Control. CAB International, Wallingford, UK1997: 163-176Google Scholar, 4Haughan P.A. Goad L.J. Coombs G.H. North M.J. Biochemical Protozoology. Taylor and Francis, London1991: 312-328Google Scholar, 5Berman J.D. Holz Jr., G.G. Beach D.H. Mol. Biochem. Parasitol. 1984; 12: 1-13Crossref PubMed Scopus (59) Google Scholar, 6Goad L.J. Holz Jr., G.G. Beach D.H. Mol. Biochem. Parasitol. 1985; 15: 257-279Crossref PubMed Scopus (59) Google Scholar, 7Urbina J.A. Payares G. Molina J. Sanoja C. Liendo A. Lazardi K. Piras M.M. Piras R. Perez N. Wincker P. Ryley J.F. Science. 1996; 273: 969-971Crossref PubMed Scopus (176) Google Scholar, 8Beach D.H. Goad L.J. Holz G.G. Biochem. Biophys. Res. Commun. 1986; 136: 851-856Crossref PubMed Scopus (62) Google Scholar, 9Goad L.J. Berens R.L. Marr J.J. Beach D.H. Holz G.G. Mol. Biochem. Parasitol. 1989; 32: 179-190Crossref PubMed Scopus (67) Google Scholar, 10Urbina J.A. Lazardi K. Marchan E. Visbal G. Aguirre T. Piras M.M. Piras R. Maldonado R.A. Payares G. DeSouza W. Antimicrob. Agents Chemother. 1991; 37: 580-591Crossref Scopus (119) Google Scholar, 11Goad L.J. Holz G.G. Beach D.H. Biochem. Pharmacol. 1985; 34: 3785-3788Crossref PubMed Scopus (17) Google Scholar), have been shown to block sterol synthesis in a number of Leishmania andTrypanosoma species with retardation of growth and death of the parasite. In our studies on sterol biosynthesis in Leishmania species, we have recently demonstrated (12Ginger M.L. Chance M.L. Goad L.J. Biochem. J. 1999; 342: 397-405Crossref PubMed Scopus (31) Google Scholar, 13Ginger M.L. Prescott M.C. Reynolds D.G. Chance M.L. Goad L.J. Eur. J. Biochem. 2000; 267: 2555-2566Crossref PubMed Scopus (34) Google Scholar) that leucine is the major source of the carbon used for de novo sterol biosynthesis. By contrast, acetate or substrates from which acetyl-CoA is generated by metabolism (e.g. glucose, palmitic acid, alanine, serine, and isoleucine) are very poorly incorporated into sterol, although they are used efficiently for the synthesis of the fatty acid moieties of triacylglycerol and phospholipid. The utilization of leucine for sterol biosynthesis has been shown previously in animal tissues (14Bloch K. J. Biol. Chem. 1944; 155: 255-263Abstract Full Text PDF Google Scholar, 15Miettinen T.A. Penttila I.M. Ann. Med. Exp. Biol. Fenn. 1971; 49: 20-28PubMed Google Scholar, 16Rosenthal J. Angel A. Farkas J. J. Physiol. ( Lond. ). 1974; 226: 411-418Google Scholar, 17Stillway L.W. Weigland D.A. Reifler J.F. Buse M.G. Lipids. 1977; 12: 1012-1018Crossref PubMed Scopus (12) Google Scholar, 18Hida A. Uchijima Y. Seyama Y. J. Biochem. ( Tokyo ). 1998; 124: 648-653Crossref PubMed Scopus (10) Google Scholar), plants (19Anastasis, P., Freer, I., Overton, K. H., Picken, D., Rycroft, D. S., and Singh, T. B. (1985) J. Chem. Soc. Chem. Commun., 148–149.Google Scholar, 20Anastasis, P., Freer, I., Overton, K. H., Picken, D., Rycroft, D. S., and Singh, T. B. (1987) J. Chem. Soc. Perkin Trans. I, 2427–2436.Google Scholar, 21Suga T. Tange K. Iccho K. Hirate T. Phytochemistry. 1980; 19: 67-70Crossref Scopus (14) Google Scholar), and fungi (22Chichester C.O. Yokoyama H. Nakayama T.O.M. Lacton A. Mackinney G. J. Biol. Chem. 1959; 234: 598-602Abstract Full Text PDF PubMed Google Scholar, 23Domenech C.E. Giordono W. Avalos J. Cerda-Olmedo E. Eur. J. Biochem. 1996; 239: 720-725Crossref PubMed Scopus (41) Google Scholar). In animals and plants, a major route for leucine catabolism has been demonstrated to be located in the mitochondrion (24Weinstock S.B. Kopito R.R. Endemann G. Tomera J.F. Marinier E. Murray D.M. Brunengrabers H. J. Biol. Chem. 1984; 259: 8939-8944Abstract Full Text PDF PubMed Google Scholar, 25Anderson M.D. Che P. Song J. Nicolau B.J. Wurtele E.V. Plant Physiol. 1998; 118: 1127-1138Crossref PubMed Scopus (77) Google Scholar). The pathway proceeds through the production of α-ketoisocaproate, isovaleryl-CoA, 3-methylcrotonyl-CoA, and 3-methylglutaconyl-CoA (Scheme FS1) to give 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA),1 which is then cleaved by a lyase to produce acetyl CoA and acetoacetate. The acetyl-CoA generated in this way can be either fed into the citric acid cycle or alternatively transported out of the mitochondrion into the cytosol, where the acetyl-CoA may be utilized for the biosynthesis of a range of compounds including fatty acids and isoprenoids such as sterols. The entry of the acetyl-CoA into the isoprenoid pathway requires the regeneration of HMG-CoA, which is then reduced to mevalonic acid (Scheme FS1). Conclusive evidence that leucine enters isoprenoids in plants by this indirect route and involving production of acetyl-CoA has been provided by incubation of 13C-labeled leucine with a callus culture ofAndrographis paniculata (19Anastasis, P., Freer, I., Overton, K. H., Picken, D., Rycroft, D. S., and Singh, T. B. (1985) J. Chem. Soc. Chem. Commun., 148–149.Google Scholar, 20Anastasis, P., Freer, I., Overton, K. H., Picken, D., Rycroft, D. S., and Singh, T. B. (1987) J. Chem. Soc. Perkin Trans. I, 2427–2436.Google Scholar). 13C NMR analysis of the 13C-enriched sesquiterpenoid and phytosterols (19Anastasis, P., Freer, I., Overton, K. H., Picken, D., Rycroft, D. S., and Singh, T. B. (1985) J. Chem. Soc. Chem. Commun., 148–149.Google Scholar, 20Anastasis, P., Freer, I., Overton, K. H., Picken, D., Rycroft, D. S., and Singh, T. B. (1987) J. Chem. Soc. Perkin Trans. I, 2427–2436.Google Scholar) produced by the callus showed unequivocally that the leucine was metabolized to acetyl-CoA and acetoacetate prior to incorporation into the isoprenoid pathway. We have demonstrated previously (12Ginger M.L. Chance M.L. Goad L.J. Biochem. J. 1999; 342: 397-405Crossref PubMed Scopus (31) Google Scholar, 13Ginger M.L. Prescott M.C. Reynolds D.G. Chance M.L. Goad L.J. Eur. J. Biochem. 2000; 267: 2555-2566Crossref PubMed Scopus (34) Google Scholar) that [U-14C]leucine incubated withLeishmania mexicana and other trypanosomatid species was very efficiently incorporated into sterol and to some limited extent into fatty acids. However, by contrast, [1-14C]acetate was readily incorporated into fatty acids but poorly utilized for sterol production. These observations are incompatible with a route inLeishmania that requires leucine degradation to proceed to acetyl CoA before reutilization for isoprenoid production (19Anastasis, P., Freer, I., Overton, K. H., Picken, D., Rycroft, D. S., and Singh, T. B. (1985) J. Chem. Soc. Chem. Commun., 148–149.Google Scholar, 20Anastasis, P., Freer, I., Overton, K. H., Picken, D., Rycroft, D. S., and Singh, T. B. (1987) J. Chem. Soc. Perkin Trans. I, 2427–2436.Google Scholar). We have therefore undertaken the studies described here to investigate the metabolic route whereby L. mexicana uses leucine for sterol biosynthesis, since this may produce evidence for a target for new antileishmanial drug development. [1-14C]Acetate, sodium salt (57 mCi/mmol), and [U-14C]leucine (299–314 mCi/mmol) were obtained from Amersham Pharmacia Biotech. [2-13C]Leucine (99% enrichment) was obtained from Promochem (Welwyn Garden City, UK). [2-13C]Acetate, [1-13C]glucose, ketoconazole, and mevalonic acid were obtained from Sigma-Aldrich. Compound L-659,699 was a gift from Merck. The strain of Leishmania used in this study was L. mexicana (MNYC/62/BZ/M379). Promastigotes were cultured at 26 °C in HO-minimum essential medium (26Berens R.L. Brun R. Krassner S.M. J. Parasitol. 1976; 62: 360-365Crossref PubMed Scopus (155) Google Scholar), supplemented with 10% (v/v) heat-inactivated fetal calf serum.14C- and 13C-labeled substrates were added to the media to give the final concentrations indicated under “Results.” Cell density of cultures was determined by counting using a Neubauer hemocytometer. Cultures were normally established with an initial cell density of 106 cells/ml; at the termination of the culture period, the cells were harvested by centrifugation as described previously (12Ginger M.L. Chance M.L. Goad L.J. Biochem. J. 1999; 342: 397-405Crossref PubMed Scopus (31) Google Scholar, 27Haughan P.A. Chance M.L. Goad L.J. Biochem. J. 1995; 308: 31-38Crossref PubMed Scopus (42) Google Scholar). Lovastatin was administered to cultures from a stock solution (2 mg/ml) in Me2SO; compound L-659,699 was dissolved (2 mg/ml) in sterile H2O saturated with NaHCO3 for administration to the cultures. Amastigotes were obtained after the infection of macrophages isolated from the peritoneal cavities of female CD1 mice with stationary phase promastigotes (72 h of growth) (28Bates P.A. Parasitol. Today. 1993; 9: 143-146Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 29Haughan P.A. Chance M.L. Goad L.J. Biochem. Pharmacol. 1992; 44: 2199-2206Crossref PubMed Scopus (33) Google Scholar). Promastigotes were allowed to infect macrophages for 24 h at 32 °C before the medium overlying the macrophages was decanted. The cells were rinsed with Locke's solution (containing the following per liter: 9 g of NaCl, 0.42 g of KCl, 0.4 g of CaCl2·H2O, 0.2 g of NaHCO3, and 1 g of glucose) to remove any remaining extracellular promastigotes. RPMI supplemented 15% (v/v) with heat-inactivated fetal calf serum and containing [2-13C]leucine in place of unlabeled free leucine was then added to the infected macrophages, and the cultures were left at 32 °C for 48 h. At this time, amastigote forms were prepared by the method described by Haughanet al. (29Haughan P.A. Chance M.L. Goad L.J. Biochem. Pharmacol. 1992; 44: 2199-2206Crossref PubMed Scopus (33) Google Scholar). Parasite lipids were isolated after extraction with chloroform/methanol (2:1) as described previously (12Ginger M.L. Chance M.L. Goad L.J. Biochem. J. 1999; 342: 397-405Crossref PubMed Scopus (31) Google Scholar, 27Haughan P.A. Chance M.L. Goad L.J. Biochem. J. 1995; 308: 31-38Crossref PubMed Scopus (42) Google Scholar). Radioactive lipid extracts were analyzed by analytical TLC and radioscanning using silica gel TLC plates and chloroform/ethanol (98:2) as the developing solvent. Sterols were isolated and analyzed by GC or GC-MS as the TMS ether derivatives following previously described protocols (12Ginger M.L. Chance M.L. Goad L.J. Biochem. J. 1999; 342: 397-405Crossref PubMed Scopus (31) Google Scholar, 27Haughan P.A. Chance M.L. Goad L.J. Biochem. J. 1995; 308: 31-38Crossref PubMed Scopus (42) Google Scholar). Steryl acetates were prepared by treatment of the free sterol with pyridine/acetic anhydride (1:1) followed by usual work up of the steryl acetate. The steryl acetates were separated by preparative TLC on silica gel impregnated with 10% AgNO3and developed with freshly distilled chloroform. Sterols were quantified by capillary GC analysis using 5α-cholestane as a standard. The NMR spectra were measured on deuteriochloroform solutions using a Varian INOVA 600 spectrometer operating at 599.9 MHz for protons and 150.9 MHz for 13C nuclei. 1H NMR spectra were recorded using the following parameters: 7000 Hz spectrum width, 3-s preacquisition delay, 90° pulse, 5-s acquisition time, 10,000 transients, 64K data points No weighting function was used before Fourier transformation. Broad band proton-decoupled 13C NMR spectra were obtained using the following parameters: 35,000-Hz spectrum width, 22° pulse, 0.936-s acquisition time, 70,000 transients, 64K data points, line broadening of 1 Hz before Fourier transformation. Promastigotes were cultured for 72 h in the presence of 0.1 mg/ml ketoconazole (administered from a stock solution of 1 mg/ml in Me2SO) in HO-minimum essential medium. The sterols were isolated as previously described (12Ginger M.L. Chance M.L. Goad L.J. Biochem. J. 1999; 342: 397-405Crossref PubMed Scopus (31) Google Scholar, 26Berens R.L. Brun R. Krassner S.M. J. Parasitol. 1976; 62: 360-365Crossref PubMed Scopus (155) Google Scholar), and the 14α-methylergosta-8,24(241)-dien-3β-ol was characterized by GC-MS and 1H NMR analyses. For the experiments studying [2-13C]leucine incorporation into the sterol, the medium contained [2-13C]leucine (78 mg/liter) in place of the unlabeled free leucine normally present. In total, 50 cultures (50 ml in each culture) were grown, and the cells were harvested in batches and extracted with 3 × 100 ml of chloroform/methanol (2:1) using procedures similar to the those used for the smaller scale extraction (12Ginger M.L. Chance M.L. Goad L.J. Biochem. J. 1999; 342: 397-405Crossref PubMed Scopus (31) Google Scholar, 27Haughan P.A. Chance M.L. Goad L.J. Biochem. J. 1995; 308: 31-38Crossref PubMed Scopus (42) Google Scholar). The 13C-labeled N14α-methylergosta-8,24(241)-dien-3β-ol was then purified from the lipid by reversed-phase HPLC using an Econosphere C18 column (250 × 4.6 mm; inner diameter, 5 mm; supplied by Alltech) to separate it from cholesterol and other minor sterols. Compounds were eluted isocratically using acetonitrile/water (9:1), and sterols were detected by UV absorbance at 215 nm. The 14α-methylergosta-8,24(241)-dien-3β-ol was eluted at 26–30 min, and cholesterol was eluted at 32–36 min. The solvent volume of the eluate was carefully reduced by rotary evaporation, and the sterol was extracted into petroleum ether before taking to dryness and storage at −20 °C. The purity of the isolated13C-labeled 14α-methylergosta-8,24(241)-dien-3β-ol (∼3 mg) was checked by GC-MS and then analyzed by 13C NMR spectroscopy We have utilized [2-13C]leucine to investigate if the leucine carbon skeleton is incorporated by L. mexicana directly into the sterols by reduction of HMG-CoA to MVA. The major sterols of L. mexicana are cholesterol obtained from the medium, and the biosynthesized ergosta-5,7,24(241)-trien-3β-ol (4Haughan P.A. Goad L.J. Coombs G.H. North M.J. Biochemical Protozoology. Taylor and Francis, London1991: 312-328Google Scholar, 12Ginger M.L. Chance M.L. Goad L.J. Biochem. J. 1999; 342: 397-405Crossref PubMed Scopus (31) Google Scholar,13Ginger M.L. Prescott M.C. Reynolds D.G. Chance M.L. Goad L.J. Eur. J. Biochem. 2000; 267: 2555-2566Crossref PubMed Scopus (34) Google Scholar) with smaller variable amounts of ergosta-5,7,22-trien-3β-ol, stigmasta-5,7,24(241)-trien-3β-ol, and precursors such as ergosta-7,24(241)-dien-3β-ol. A sterol mixture of this nature presents certain problems for the type of 13C NMR study envisaged. First, the complexity of the sterol mixture demands careful purification of one of the biosynthesized ergosta types of sterol so that signal assignments to 13C-enriched carbons can be made with accuracy and without ambiguity. Second, the major sterols of L. mexicana are Δ5,7-compounds that are notoriously unstable in small amounts due to oxidation, and this may cause difficulties in the purification, storage, and NMR analysis of these sterols. This problem has been considered in detail by Schroepfer et al. (30Wilson W.K. Sumpter R.M. Warren J.J. Rogers P.S. Ruan B.F. Schroepfer G.J. J. Lipid Res. 1996; 37: 1529-1555Abstract Full Text PDF PubMed Google Scholar), specifically in relation to the NMR analysis of Δ5,7-sterols. Finally, the success of the study depends upon the extent of enrichment of the biosynthesized sterol with13C. A large pool of preexisting sterol from the inoculum will dilute the 13C-enriched sterol species and could make13C-enriched carbons difficult to detect. Accordingly, we looked for a new approach to the problem and decided to use an inhibitor of sterol biosynthesis. An inhibitor was required that would cause the accumulation of a large amount of a relatively stable sterol intermediate that would normally occur in only trace amounts in the parasite, so dilution of the newly synthesized [13C]sterol by preexisting material would not be significant. Sterol biosynthesis inhibitors suited to this purpose are the imadazole and triazole types of antifungal drugs. These compounds block the action of the cytochrome P450-dependent 14α-methylsterol 14-demethylase with the result that the normal sterols are depleted and one or more 14α-methylsterols accumulate, often in large amounts (31Van den Bossche H. Berg D. Plempl M. Sterol Biosynthesis Inhibitors: Pharmaceutical and Agrochemical Aspects. Ellis Harwood Ltd., Chichester, UK1988: 79-119Google Scholar). It has been demonstrated previously that antifungal imidazoles and triazoles also inhibit the 14α-demethylation step in sterol biosynthesis in severalLeishmania species with the resulting appearance of 4α,14α-dimethylergosta-8,24(241)-dien-3β-ol and 14α-methylergosta-8,24(241)-dien-3β-ol in appreciable amounts (4Haughan P.A. Goad L.J. Coombs G.H. North M.J. Biochemical Protozoology. Taylor and Francis, London1991: 312-328Google Scholar, 6Goad L.J. Holz Jr., G.G. Beach D.H. Mol. Biochem. Parasitol. 1985; 15: 257-279Crossref PubMed Scopus (59) Google Scholar). These 14α-methylsterols are considerably more stable during isolation, storage, and NMR analysis than are Δ5,7-sterols. Consequently, we decided that using the 14-demethylase inhibitor ketoconazole offered the best opportunity for the isolation of a pure 13C-enriched sterol undiluted by preexisting endogenous sterol, which was required for the13C NMR analysis to determine the route of incorporation of leucine into the isoprenoid pathway. Preliminary experiments were first undertaken to determine the optimum conditions for incubation of the L. mexicana promastigotes with ketoconazole to accumulate a 14α-methylsterol in sufficient amount for isolation and 13C NMR analysis. The incubation of promastigotes of L. mexicana with ketoconazole (0.1 and 1.0 μg/ml) for 72 h followed by isolation and GC-MS examination of the sterols showed that, as anticipated, the ergosta-5,7,24(241)-trien-3β-ol found in the control was replaced by 14α-methylergosta-8,24(241)-dien-3β-ol in the ketoconazole-treated cultures (TableI). Cholesterol taken up from the medium was present in both the control and treated cells. The 14α-methylergosta-8,24(241)-dien-3β-ol was identified by the mass spectrum of the TMS ether and by the 1H NMR spectrum (6Goad L.J. Holz Jr., G.G. Beach D.H. Mol. Biochem. Parasitol. 1985; 15: 257-279Crossref PubMed Scopus (59) Google Scholar, 32Goad L.J. Akihisa T. Analysis of Sterols. Blackie Academic and Professional, Chapman Hall, London1997Crossref Google Scholar). MS m/z (rel. intensity): 484 [M]+ (43Blum J.J. J. Protozool. 1991; 38: 527-531Crossref PubMed Scopus (11) Google Scholar), 469 [M-methyl]+ (100), 385 [M-methyl-part side chain]+ (13Ginger M.L. Prescott M.C. Reynolds D.G. Chance M.L. Goad L.J. Eur. J. Biochem. 2000; 267: 2555-2566Crossref PubMed Scopus (34) Google Scholar), 379 [M-methyl-TMSOH]+ (79), 303 (36Heise N. Opperdoes F.R. Z. Naturforsch. C. 2000; 55: 477-4763Crossref Scopus (21) Google Scholar), 295 [M-TMSOH-methyl-part side chain]+ (29Haughan P.A. Chance M.L. Goad L.J. Biochem. Pharmacol. 1992; 44: 2199-2206Crossref PubMed Scopus (33) Google Scholar), 281 (17Stillway L.W. Weigland D.A. Reifler J.F. Buse M.G. Lipids. 1977; 12: 1012-1018Crossref PubMed Scopus (12) Google Scholar), 227 [M-TMSOH-side chain and ring D]+ (28Bates P.A. Parasitol. Today. 1993; 9: 143-146Abstract Full Text PDF PubMed Scopus (84) Google Scholar), 213 [M-methyl-TMSOH-side chain and ring D]+ (41Blum J.J. J. Bioenerg. Biomembr. 1994; 26: 147-155Crossref PubMed Scopus (36) Google Scholar).1H NMR (chloroform-d): δ 0.70 s(H3-18), 0.94 s (H3-19), 0.92d (H3-21), 0.88 s(H3-32), 1.01 d and 1.02 d(H3-26 and H3-27), 4.65 br s and 4.70 br s (H2-241).Table IThe effects of ketoconazole on the growth and sterol composition of promastigotes of L. mexicanaKetoconazole concentration0 (control)0.1 μg/ml1.0 μg/mlCells × 106/ml at end of 72-h growth16.515.712.3Total sterol (ng/106 cells)1309060Sterol composition (%)Cholesterol7121514α-Methylergosta-8,22,24(241)-trien-3β-ol4tr14α-Methylergosta-8,24(241)-dien-3β-ol67794α, 14α-Dimethylcholesta-8,24-dien-3β-ol116Ergosta-5,7,24(241)-trien-3β-ol81Unidentified C28-sterol6Stigmasta-5,7,24(241)-trien-3β-ol6Lanosterol (4,4,14α-trimethylcholesta-8,24-dien-3β-ol)6trPromastigotes of L. mexicana (1.0 × 106cells/ml) were cultured for 72 h alone (control) or in the presence of 0.1 or 1.0 μg/ml ketoconazole. The cell density was determined at the end of the growth period, and the cells were harvested, the lipid was extracted, and the sterols were isolated and analyzed by GC-MS as described under “Materials and Methods”; tr indicates trace amount (<0.5%). Open table in a new tab Promastigotes of L. mexicana (1.0 × 106cells/ml) were cultured for 72 h alone (control) or in the presence of 0.1 or 1.0 μg/ml ketoconazole. The cell density was determined at the end of the growth period, and the cells were harvested, the lipid was extracted, and the sterols were isolated and analyzed by GC-MS as described under “Materials and Methods”; tr indicates trace amount (<0.5%). Ketoconazole at 0.1 μg/ml retarded growth by only about 5% compared with the control, and the total sterol content of the treated cells was ∼70% of the control value. At the higher ketoconazole concentration (1.0 μg/ml), the growth was around 75% of the control, and the total sterol was about 50% of the control. In a further experiment, theL. mexicana promastigotes were incubated with ketoconazole (0.1 μg/ml) and [U-14C]leucine (2.6 μCi) for 72 h to ensure that the accumulating 14α-methylsterol was being biosynthesized from leucine derived from the medium rather than from some internal source of unlabeled precursor(s). The lipids were extracted and found to contain 4.2% of the radioactivity added to the culture medium, while analytical TLC with radioscanning showed that the 14α-methylsterol was the major labeled material. Recovery of the labeled sterol from the TLC plate, acetylation, and rechromatography by TLC on silver nitrate-impregnated silica gel showed that the radioactivity accompanied a material with the sameR f as 14α-methylergosta-8,24(241)-dien-3β-yl acetate. The above experiments showed that our approach to obtain a pure sterol for the 13C NMR analysis was feasible. Therefore, multiple cultures (50 × 50 ml) of L. mexicanapromastigotes were grown in HO-minimum essential medium (plus 10% (v/v) heat-inactivated fetal calf serum) in which the free (i.e. nonprotein) leucine was replaced with [2-13C]leucine, and 0.1 μg/ml ketoconazole was added. The cells were cultured for 72 h and harvested, and the lipid was extracted. The total sterol was isolated from the lipid, and the 14α-methylergosta-8,24(241)-dien-3β-ol was then separated from the cholesterol and other minor sterols by HPLC (see “Materials and Methods”). The cholesterol was shown by GC-MS analysis to contain only the natural abundance of 13C, and there was no detectable labeling from the [2-13C]leucine. This observation established unequivocally that the cholesterol inL. mexicana must be taken up from the medium and that it is not the product of de novo synthesis in the parasite. The purity of the isolated 14α-methylergosta-8,24(241)-dien-3β-ol (∼3 mg) was 95% as judged by GC analysis. The mass spectrum of the TMS ether showed clusters of ions for the molecular and fragment ions arising from several labeled species of the sterol containing from one to six13C atoms (Fig. 1). Ions due to unlabeled sterol were very minor, showing that there was excellent incorporation of [2-13C]leucine into the sterol in accord with our previous studies with [U-14C]leucine incorporation, which had revealed that at least 80% of the sterol carbon originated from leucine (12Ginger M.L. Chance M.L. Goad L.J. Biochem. J. 1999; 342: 397-405Crossref PubMed Scopus (31) Google Scholar, 13Ginger M.L. Prescott M.C. Reynolds D.G. Chance M.L. Goad L.J. Eur. J. Biochem. 2000; 267: 2555-2566Crossref PubMed Scopus (34) Google Scholar). The molecular ion region comprised a cluster of ions at m/z 484 (unlabeled sterol), 485, 486, 487, 488, 489, and 490, with the last two predominating (Table II). The fragment ion clusters atm/z 469–475 and 379–385, which arise by loss of a methyl and methyl and TMSOH from the [M]+ ion, respectively, showed a similar distribution of molecular species containing 1–6 13C-enriched positions (Fig. 1). The ions at m/z 304–307 showed fragments containing up" @default.
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• W2114602369 title "The Biosynthetic Incorporation of the Intact Leucine Skeleton into Sterol by the Trypanosomatid Leishmania mexicana" @default.
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