Regulation of the Biosynthesis ofN-Acetylglucosaminylpyrophosphoryldolichol, Feedback and Product Inhibition
1999; Elsevier BV; Volume: 274; Issue: 48 Linguagem: Inglês
10.1074/jbc.274.48.34072
ISSN1083-351X
AutoresEdward L. Kean, Zenglu Wei, Vernon Anderson, Nanjing Zhang, Lawrence M. Sayre,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoThe assembly of the core oligosaccharide region of asparagine-linked glycoproteins proceeds by means of the dolichol pathway. The first step of this pathway, the reaction of dolichol phosphate with UDP-GlcNAc to formN-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-P-P-dolichol), is under investigation as a possible site of metabolic regulation. This report describes feedback inhibition of this reaction by the second intermediate of the pathway,N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-GlcNAc-P-P-dolichol), and product inhibition by GlcNAc-P-P-dolichol itself. These influences were revealed when the reactions were carried out in the presence of showdomycin, a nucleoside antibiotic, present at concentrations that block the de novo formation of GlcNAc-GlcNAc-P-P-dolichol but not that of GlcNAc-P-P-dolichol. The apparent K i values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol under basal conditions were 4.4 and 2.8 μm, respectively. Inhibition was also observed under conditions where mannosyl-P-dolichol (Man-P-dol) stimulated the biosynthesis of GlcNAc-P-P-dolichol; the apparent K i values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol were 2.2 and 11 μm, respectively. Kinetic analysis of the types of inhibition indicated competitive inhibition by GlcNAc-P-P-dolichol toward the substrate UDP-GlcNAc and non-competitive inhibition toward dolichol phosphate. Inhibition by GlcNAc-GlcNAc-P-P-dolichol was uncompetitive toward UDP-GlcNAc and competitive toward dolichol phosphate. A model is presented for the kinetic mechanism of the synthesis of GlcNAc-P-P-dolichol. GlcNAc-P-P-dolichol also exerts a stimulatory effect on the biosynthesis of Man-P-dol, i.e. a reciprocal relationship to that previously observed between these two intermediates of the dolichol pathway. This network of inhibitory and stimulatory influences may be aspects of metabolic control of the pathway and thus of glycoprotein biosynthesis in general. The assembly of the core oligosaccharide region of asparagine-linked glycoproteins proceeds by means of the dolichol pathway. The first step of this pathway, the reaction of dolichol phosphate with UDP-GlcNAc to formN-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-P-P-dolichol), is under investigation as a possible site of metabolic regulation. This report describes feedback inhibition of this reaction by the second intermediate of the pathway,N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-GlcNAc-P-P-dolichol), and product inhibition by GlcNAc-P-P-dolichol itself. These influences were revealed when the reactions were carried out in the presence of showdomycin, a nucleoside antibiotic, present at concentrations that block the de novo formation of GlcNAc-GlcNAc-P-P-dolichol but not that of GlcNAc-P-P-dolichol. The apparent K i values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol under basal conditions were 4.4 and 2.8 μm, respectively. Inhibition was also observed under conditions where mannosyl-P-dolichol (Man-P-dol) stimulated the biosynthesis of GlcNAc-P-P-dolichol; the apparent K i values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol were 2.2 and 11 μm, respectively. Kinetic analysis of the types of inhibition indicated competitive inhibition by GlcNAc-P-P-dolichol toward the substrate UDP-GlcNAc and non-competitive inhibition toward dolichol phosphate. Inhibition by GlcNAc-GlcNAc-P-P-dolichol was uncompetitive toward UDP-GlcNAc and competitive toward dolichol phosphate. A model is presented for the kinetic mechanism of the synthesis of GlcNAc-P-P-dolichol. GlcNAc-P-P-dolichol also exerts a stimulatory effect on the biosynthesis of Man-P-dol, i.e. a reciprocal relationship to that previously observed between these two intermediates of the dolichol pathway. This network of inhibitory and stimulatory influences may be aspects of metabolic control of the pathway and thus of glycoprotein biosynthesis in general. N-acetylglucosaminylpyrophosphoryldolichol N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol mannosylphosphoryldolichol 2-{[tris(hydroxymethyl)methyl] amino}ethanesulfonic acid UDP-GlcNAc:dolichyl-phosphate N-acetylglucosamine 1-phosphate transferase UDP-GlcNAc:GlcNAc-P-P-dolichol, N-acetylglucosamine transferase chloro- form/methanol It has been well established that the dolichol pathway is the means whereby the core region of asparagine-linked glycoproteins is assembled (see review, Ref. 1Hemming F.W. Montreuil J. Vliegenthart J.F.K. Schachter H. Glycoproteins. Elsevier Science Publishers B. V., Amsterdam1995: 127-143Google Scholar). Our understanding of the mechanisms that regulate this complex series of reactions, however, is still limited. To this end we have directed our attention to the initial reaction of the pathway, the reaction between dolichol phosphate and UDP-GlcNAc producing GlcNAc-P-P-dolichol,1catalyzed by the enzyme, UDP-GlcNAc:dolichyl-phosphateN-acetylglucosamine 1-phosphate transferase (GPT-1). Factors that modulate the formation of GlcNAc-P-P-dolichol could have an effect on the rate of synthesis of the other intermediates of the dolichol pathway and thus influence nascent glycoprotein biosynthesis in general. Several factors have previously been described that could have a regulatory influence on this reaction as follows: hormonal effects (2Hayes G.R. Lucas J.J. J. Biol. Chem. 1983; 258: 15095-15100Abstract Full Text PDF PubMed Google Scholar), genetic factors (3Lennon K. Pretel R. Kesselheim J. te Hessen S. Kukuruzinska M.A. Glycobiology. 1995; 5: 633-642Crossref PubMed Scopus (32) Google Scholar), and topography of enzymes and substrates (4Abeijon C. Hirschberg C.B. J. Biol. Chem. 1990; 265: 14691-14695Abstract Full Text PDF PubMed Google Scholar, 5Kean E.L. J. Biol. Chem. 1991; 266: 942-946Abstract Full Text PDF PubMed Google Scholar, 6Dan N.D. Middleton R.B. Lehrman M.A. J. Biol. Chem. 1996; 271: 30717-30724Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Previous studies from this laboratory and others (7Kean E.L. J. Biol. Chem. 1982; 257: 7952-7954Abstract Full Text PDF PubMed Google Scholar, 8Kean E.L. Biochim. Biophys. Acta. 1983; 750: 268-273Crossref PubMed Scopus (14) Google Scholar, 9Kean E.L. Biochim. Biophys. Acta. 1983; 752: 488-490Crossref PubMed Scopus (5) Google Scholar, 10Kean E.L. J. Biol. Chem. 1985; 260: 12561-12571Abstract Full Text PDF PubMed Google Scholar, 11Kean E.L. DeBrakeleer D.J. Arch. Biochem. Biophys. 1986; 250: 146-152Crossref PubMed Scopus (12) Google Scholar, 12Kean E.L. Rush J.S. Waechter C.J. Biochemistry. 1994; 33: 10508-10512Crossref PubMed Scopus (24) Google Scholar, 13Kaushal G.P. Elbein A.D. J. Biol. Chem. 1985; 260: 16303-16309Abstract Full Text PDF PubMed Google Scholar, 14Shailubhai K. Dong-Yu B. Saxena E.S. Vijay I.K. J. Biol. Chem. 1988; 263: 15964-15972Abstract Full Text PDF PubMed Google Scholar, 15Carson D.D. Farrar J.D. Laidlaw J. Wright D.A. J. Biol. Chem. 1990; 265: 2947-2955Abstract Full Text PDF PubMed Google Scholar) have also revealed that another intermediate of the pathway, mannosyl-P-dolichol (Man-P-dol), acts as an allosteric activator of GPT-1, resulting in the stimulation of GlcNAc-P-P-Dol synthesis. A reciprocal relationship has now been revealed whereby Man-P-Dol formation is stimulated by GlcNAc-P-P-dolichol. The present study has revealed other potential aspects of regulation of the initial reaction of the dolichol pathway. Feedback inhibition of the biosynthesis of GlcNAc-P-P-dolichol was demonstrated by the second intermediate of the pathway, GlcNAc-GlcNAc-P-P-dolichol. The formation of the latter compound is catalyzed by a separate GlcNAc-transferase, UDP-GlcNAc:GlcNAc-P-P-dolichol, N-acetylglucosamine transferase (GT-2), the kinetics of which have recently been described (16Kean E.L. Niu N. Glycoconj. J. 1998; 15: 11-17Crossref PubMed Scopus (4) Google Scholar). Although the reversibility of GPT-1 has previously been demonstrated (17Heifetz A. Keenan R.W. Elbein A.D. Biochemistry. 1979; 18: 2186-2192Crossref PubMed Scopus (307) Google Scholar, 18Harford J.B. Waechter C.J. Arch. Biochem. Biophys. 1979; 197: 424-435Crossref PubMed Scopus (15) Google Scholar), the present report also described the kinetics of inhibition by GlcNAc-P-P-dolichol of its own synthesis. 2Preliminary reports of some of these studies have been made (Kean, E. L., Niu, N., and Imperiali, B. (1996)Glycobiol. 6, 740; Kean, E. L. (1997)Glycoconj. J. 14, (suppl) S35). We have examined the effect of these inhibitory influences on the biosynthesis of GlcNAc-P-P-dolichol at the basal level and under stimulatory conditions in the presence of Man-P-dol. On the basis of these relationships a model is suggested as a mechanism of action of UDP-GlcNAc:dolichol phosphate, GlcNAc-1-phosphate transferase. Microsomes were prepared from the retinas of 15–16-day-old embryonic chicks as described previously (10Kean E.L. J. Biol. Chem. 1985; 260: 12561-12571Abstract Full Text PDF PubMed Google Scholar). Purified, recombinant yeast mannosyl-P-dolichol synthase was obtained from Dr. John Schutzbach. Dolichol phosphate was purchased from Sigma. UDP-[3H]GlcNAc and GDP-[14C]mannose were purchased from NEN Life Science Products. UDP-[3H]GlcNAc, UDP-[14C]GlcNAc, and GDP[3H]mannose were purchased from American Radioactive Chemicals, Inc.d-(+)-Showdomycin was obtained from Dr. Sung Ho Kang, Department of Chemistry, Korea Advanced Institute of Science and Technology, Taejon, Korea. N-Benzyl-2′-deoxyshowdomycin was obtained from Dr. R. S. Hosmane, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore. Large scale preparations were performed by incubating microsomes from the retina of the embryonic chick with UDP-[14C]GlcNAc and dolichol phosphate, as described previously (10Kean E.L. J. Biol. Chem. 1985; 260: 12561-12571Abstract Full Text PDF PubMed Google Scholar). The incubations were performed in the presence also of Man-P-Dol and the antibiotic, showdomycin. As described previously, the former stimulates the production of GlcNAc-P-P-dolichol (7Kean E.L. J. Biol. Chem. 1982; 257: 7952-7954Abstract Full Text PDF PubMed Google Scholar, 8Kean E.L. Biochim. Biophys. Acta. 1983; 750: 268-273Crossref PubMed Scopus (14) Google Scholar, 9Kean E.L. Biochim. Biophys. Acta. 1983; 752: 488-490Crossref PubMed Scopus (5) Google Scholar, 10Kean E.L. J. Biol. Chem. 1985; 260: 12561-12571Abstract Full Text PDF PubMed Google Scholar, 11Kean E.L. DeBrakeleer D.J. Arch. Biochem. Biophys. 1986; 250: 146-152Crossref PubMed Scopus (12) Google Scholar, 12Kean E.L. Rush J.S. Waechter C.J. Biochemistry. 1994; 33: 10508-10512Crossref PubMed Scopus (24) Google Scholar), and the latter both inhibits the formation of GlcNAc-GlcNAc-P-P-dolichol and also brings about an increase in the production of the mono-GlcNAc derivative (19Kean E.L. Wei Z. Glycoconj. J. 1998; 15: 405-414Crossref PubMed Scopus (6) Google Scholar). The product was isolated by solvent partitioning according to the procedure of Folch et al. (20Folch J. Lees M. Sloane-Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) and purified by chromatography on DEAE-cellulose, as described previously (10Kean E.L. J. Biol. Chem. 1985; 260: 12561-12571Abstract Full Text PDF PubMed Google Scholar). Its concentration was determined by Dionex chromatography, as described below. By using dolichol kindly provided by Dr. Tadeusz Chojnacki of the Institute of Biochemistry and Biophysics, Warsaw, Poland, GlcNAc-P-P-dolichol was initially synthesized by the method of Imperiali and Zimmerman (21Imperiali B. Zimmerman J.W. Tetrahedron Lett. 1990; 31: 6485-6488Crossref Scopus (34) Google Scholar), which requires azeotropic drying (toluene or pyridine) at the stage of the oxalyl chloride-mediated coupling of dolichol phosphate to the per-O-acetyl-GlcNAc-P pyridinium salt. Acceptable yields of the latter intermediate could be obtained only by careful azeotropic drying of all commercial ingredients (including tetraethylammonium chloride and dibenzylphosphate) and use of freshly distilled solvents. In the published method (21Imperiali B. Zimmerman J.W. Tetrahedron Lett. 1990; 31: 6485-6488Crossref Scopus (34) Google Scholar), oxalyl chloride activation of dolichol phosphate generates the highly activated dolichylphosphoryl dichloride, which actually is an intermediate in the POCl3-mediated preparation of dolichol phosphate but which could not successfully be coupled directly to the protected GlcNAc-P, and was instead hydrolyzed to dolichol phosphate, which was then purified and reactivated with oxalyl chloride. During this study, it was found that the requisite dolichylphosphoryl dichloride could in fact be generated from dolichol in a directly usable form using excess POCl3 in hexane, followed by a brief extraction with water to remove HOPOCl2, and then evaporation of hexane and remaining POCl3 under high vacuum. Following coupling, purification of the protected pyrophosphate prior to NaOMe-mediated deacetylation was achieved by silica gel 60 column chromatography using CHCl3/MeOH/H2O (65:25:4, by volume) as the eluant. These modifications, which avoid the need to isolate and purify dolichol phosphate, resulted in the best overall yield of GlcNAc-P-P-dolichol from dolichol. The enzymatically and chemically synthesized GlcNAc-P-P-dolichol functioned in a similar manner in these studies. Large scale preparations of [14C]Man-P-Dol were made by incubating dolichol phosphate, GDP[14C]mannose (1.7 dpm/pmol), buffer, and metal ions as described previously (12Kean E.L. Rush J.S. Waechter C.J. Biochemistry. 1994; 33: 10508-10512Crossref PubMed Scopus (24) Google Scholar) with extracts fromMicrococcus luteus provided by Dr. Charles J. Waechter and Dr. Jeffrey S. Rush of the Department of Biochemistry, University of Kentucky College of Medicine, Lexington, KY, or a purified, recombinant yeast dolichyl-P-synthase from yeast provided by Dr. John S. Schutzbach, Department of Biochemistry, University of Alabama, Birmingham, AL. Purification was carried out by chromatography on DEAE-cellulose acetate as described previously (22Kean E.L. J. Supramol. Struct. 1977; 7: 381-395Crossref PubMed Scopus (11) Google Scholar, 23Kean E.L. J. Biol. Chem. 1977; 252: 5622-5629Abstract Full Text PDF PubMed Google Scholar), followed by preparative thin layer chromatography at 4 °C on 0.5-mm thick plates of Silica Gel 60 (Merck) as described previously (10Kean E.L. J. Biol. Chem. 1985; 260: 12561-12571Abstract Full Text PDF PubMed Google Scholar). As indicated by guide strips, the Man-P-dolichol region was scraped from the plates and recovered by leaching the gel in the cold with C/M (chloroform/methanol)/water (10:10:3) followed by C/M (2:1). After solvent partitioning by the procedure of Folch et al. (20Folch J. Lees M. Sloane-Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), the concentration of [14C]Man-P-Dol was calculated from the specific activity of GDP[14C]mannose used in its preparation. Incubations were carried out for 10–20 min at 37 °C in the presence of dolichol phosphate (16–20 μm), Triton X-100 (0.15%), UDP-[3H]GlcNAc (52 μm; 169 dpm/pmol), MgCl2 (27 mm), showdomycin, orN-benzyl-2′-deoxyshowdomycin, as indicated, and enzyme (0.2–0.25 mg of protein) in a total volume of 0.15 ml (basal conditions). Reactions were also carried out in the presence of Man-P-Dol (2.1–6.4 μm) (stimulatory conditions). The incubations were performed in the absence or presence of exogenously added GlcNAc-P-P-dolichol or GlcNAc-GlcNAc-P-P-dolichol (as indicated in the tables and figures). The dolichol derivatives were evaporated to dryness with nitrogen, vortexed vigorously with 0.015 ml of 1.5% Triton X-100, after which the other components of the reaction mixture were added. The incubations, carried out at 37 °C, were started by the addition of the enzyme preparation and stopped by the addition of C/M (2:1). After solvent partitioning by the Folch procedure (20Folch J. Lees M. Sloane-Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) the radioactivity in the washed lower phase was determined by scintillation spectrometry, as described previously (7Kean E.L. J. Biol. Chem. 1982; 257: 7952-7954Abstract Full Text PDF PubMed Google Scholar, 10Kean E.L. J. Biol. Chem. 1985; 260: 12561-12571Abstract Full Text PDF PubMed Google Scholar). Embryonic chick retina microsomes were incubated at 37 °C for 15 min in a medium containing GDP-[3H]mannose (1.9 μm, 200 dpm/pmol), dolichol phosphate (16 μm), MnCl2 (20 mm), Triton X-100 (0.2%), Tes buffer (0.2 m, pH 7.45), in the presence or absence of GlcNAc-P-P-dolichol (as indicated), and enzyme in a total volume of 0.15 ml, as described previously (24Kean E.L. Exp. Eye Res. 1977; 25: 405-417Crossref PubMed Scopus (13) Google Scholar). The products were analyzed by scintillation spectrometry after Folch washing, as described above. Dilutions of purified, full-length, recombinant yeast Man-P-Dol synthase (25Schutzbach J.S. Zimmerman J.W. Forsee W.T. J. Biol. Chem. 1993; 268: 24190-24196Abstract Full Text PDF PubMed Google Scholar) were made in a buffer 3J. S. Schutzbach, personal communication. containing 10% glycerol, 0.015 m Tris-HCl, pH 7.5, 1% Nonidet P-40, 2 mm dithiothreitol, and 2 mg/ml bovine serum albumin (the latter acted to stabilize the enzyme upon dilution). Incubations were carried out at 37 °C for 10 min essentially as described by Schutzbach et al. (25Schutzbach J.S. Zimmerman J.W. Forsee W.T. J. Biol. Chem. 1993; 268: 24190-24196Abstract Full Text PDF PubMed Google Scholar) in a medium containing 0.5% Nonidet P-40 (w/v), 25 mm Tris-HCl, pH 7.5, 5 mmMnCl2, 2.5 mm MgCl2, 0.25 mm EDTA, 5 mm dithiothreitol, 32 μm dolichol phosphate, GDP-[3H]Man (18 μm, 12–22 dpm/pmol) in the presence or absence of GlcNAc-P-P-dolichol (as indicated) and enzyme in a total volume of 0.15 ml. The products were analyzed by scintillation spectrometry after Folch washing, as above. After incubation followed by solvent partitioning as above, the material in the washed organic phase was evaporated to dryness and subjected to mild acid hydrolysis in 1 ml of 0.1 n HCl in 80% tetrahydrofuran for 100 min at 50 °C as described previously (26Lucas J.J. Waechter C.J. Lennarz W.J. J. Biol. Chem. 1975; 250: 1992-2002Abstract Full Text PDF PubMed Google Scholar). After evaporation to dryness and redissolving in water, the material was applied to a column containing 0.5 ml each of AG-2-X8 (200–400 mesh) acetate and AG-50-X8 H+ (200–400 mesh), the column eluted with 20 ml of water, the eluate evaporated to dryness, and the residual material redissolved in water as described previously (16Kean E.L. Niu N. Glycoconj. J. 1998; 15: 11-17Crossref PubMed Scopus (4) Google Scholar). To an aliquot was added 5 nmol of fucose to serve as an early eluting reference marker and 10 nmol each of GlcNAc and GlcNAc-GlcNAc to serve as internal standards for analysis by high pH anion exchange chromatography (Dionex Corp., Sunnyvale, CA). The mixture was injected onto a CarboPAc 1 column (4 × 250 mm) with a Carbo PAc guard column (3 × 25 mm) and eluted isocratically with a mixture of 25% of 100 mm NaOH plus 75% 1 mm NaOH at a flow rate of 1 ml/min. The elution of the standards was followed by pulsed amperometric detection (high pH anion exchange chromatography-pulsed amperometric detection), and the products of the reactions by measuring their radioactivity by scintillation spectrometry of 0.5-ml fractions collected from the pulsed amperometric detection cell, as described previously (27Prasad A.V.K. Plantner J.J. Kean E.L. Exp. Eye Res. 1992; 54: 913-920Crossref PubMed Scopus (10) Google Scholar). The relative contributions of GPT-1 and GT-2 was investigated by analyzing the distribution of the tritium label in the GlcNAc residues of their respective products, GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol, as described previously (28Kean E.L. Biochem. Cell Biol. 1992; 70: 413-421Crossref PubMed Scopus (13) Google Scholar, 29Kean E.L. Glycoconj. J. 1996; 13: 675-680Crossref PubMed Scopus (7) Google Scholar). In short, this involves the following procedures carried out sequentially. After incubation, the GlcNAc lipids extracted into the chloroform-rich layer after solvent partitioning are subjected to mild acid hydrolysis. GlcNAc and GlcNAc-GlcNAc thus formed are separated by paper chromatography, recovered from the chromatogram, and reduced with NaBH4. After mixed bed ion exchange chromatography, strong acid hydrolysis N-deacetylates the products and cleaves chitobiose. The products are then subjected to high voltage paper electrophoresis in 1% sodium borate buffer, the electrophoretogram cut into 1-cm zones, and the radioactivity determined by scintillation spectrometry. The mobilities of standard [3H]GlcNH2 and [3H]GlcNH2OH were determined in the same manner. By these procedures glucosaminitol would have been derived from GlcNAc-P-P-dolichol after hydrolysis and glucosamine from the non-reducing end of GlcNAc-GlcNAc-P-P-dolichol after hydrolysis and glucosaminitol from the reducing end. The concentration of GlcNAc-P-P-dolichol was determined after mild acid hydrolysis, as above, by quantitative Dionex chromatography of the liberated GlcNAc and by the Morgan-Elson reaction as described by Reissig et al. (30Reissig J.L. Strominger J.L. LeLoir L.F. J. Biol. Chem. 1955; 217: 959-966Abstract Full Text PDF PubMed Google Scholar). The concentration of GlcNAc-GlcNAc-P-P-dolichol, provided by Dr. B. Imperiali, Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, was determined by Dionex chromatography ofN,N′-diacetylchitobiose liberated by mild acid hydrolysis. The concentration of dolichol phosphate and the GlcNAc-P-P-dolichol used in the early phases of this work (provided by Dr. Imperiali) was determined by analysis for total phosphate as described previously (10Kean E.L. J. Biol. Chem. 1985; 260: 12561-12571Abstract Full Text PDF PubMed Google Scholar). Total microsomal phospholipids were determined in a similar manner after solvent partitioning (20Folch J. Lees M. Sloane-Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) of retina microsomes. Thin layer chromatography was performed using 20 × 20-cm glass plates precoated with a 0.25- or 0.50-mm layer of Silica Gel 60 without fluorescent indicator. The following solvent systems were used: 1) chloroform/methanol/acetic acid/water (25:15:4:2, by volume); 2) chloroform/methanol/ water (65:25:4, by volume). The location of radioactive material was accomplished by measuring the radioactivity by scintillation spectrometry of 1 × 3-cm zones scraped from the chromatogram. The migration of non-radioactive material was detected by the anisaldehyde spray reagent or by exposure to iodine vapor, as described previously (24Kean E.L. Exp. Eye Res. 1977; 25: 405-417Crossref PubMed Scopus (13) Google Scholar). Incubations carried out using dolichol phosphate and UDP-[3H]GlcNAc as substrates would result in the formation of [3H]GlcNAc-P-P-dolichol and [3H]GlcNAc-[3H]GlcNAc-P-P-dolichol. Thus, in assays carried out by solvent partitioning, any effect added compounds might have specifically on the new synthesis of [3H]GlcNAc-P-P-dolichol would be masked by the accompanying formation of the labeled chitobiosyl product. This difficulty was resolved by carrying out the incubations in the presence of the nucleoside antibiotic, showdomycin, which inhibits the formation of the chitobiosyl compound, enhancing the formation of GlcNAc-P-P-dolichol (19Kean E.L. Wei Z. Glycoconj. J. 1998; 15: 405-414Crossref PubMed Scopus (6) Google Scholar). The N-benzyl-2′deoxy derivative was shown to have the same effect (19Kean E.L. Wei Z. Glycoconj. J. 1998; 15: 405-414Crossref PubMed Scopus (6) Google Scholar). Thus, using concentrations of the showdomycins that extensively inhibited the formation of GlcNAc-GlcNAc-P-P-dolichol, the effect of exogenously added non-radioactive GlcNAc-P-P-Dol on the de novo synthesis of the labeled compound could be readily determined. Apparent K i and V maxvalues were calculated from a non-linear least squares analysis of the data using GraFit (31Leatherbarrow R.J. GraFit Version 3.0. Erithacus Software, Ltd., Staines, UK1992Google Scholar) fit to an expression for general inhibition derived from Equation 1 for mixed inhibition with a constant substrate concentration. v(inhibited)=V(uninhibited)(1+[I]/Kiapparent)Equation 1 Analyses of the types of inhibition were carried out in the following manner. The steady state kinetic data were fitted using the unweighted non-linear least squares method implemented in the computer program GraFit (31Leatherbarrow R.J. GraFit Version 3.0. Erithacus Software, Ltd., Staines, UK1992Google Scholar). Inhibition constants were determined by a global fit of the data from an entire experiment. The best fit of the data is one that minimizes the sum of the square of the observed and calculated velocities for an entire data set (32Cleland W.W. Methods Enzymol. 1979; 63: 103-138Crossref PubMed Scopus (2021) Google Scholar). The standard deviations of the data points were within 3% of the highest velocity. The data were initially fitted to Equation 2 for non-competitive or mixed inhibition. By using the criterion that if the standard error of the slope or intercept inhibition constants were over 50% of the fitted value, that parameter was excluded from the final fit. If so, the data were then fit to Equation 3 or Equation 4 for competitive and uncompetitive inhibition, respectively. The more restrictive mechanism was accepted if the χ-squared value was not increased by omitting the poorly defined inhibition constant. v=Vmax·SKm(1+I/Kis)+S(1+I/Kii)Equation 2 v=Vmax·SKm(1+I/Kis)+SEquation 3 v=Vmax·SKm+S(1+I/Kii)Equation 4 Incubations were carried out under initial rate conditions for the yeast and retina microsomal enzymes. Apparent K aand V max values were calculated from Lineweaver-Burk double-reciprocal plots of the data after analysis by computer using the Kcat program (BioMetalics, Princeton, NJ). Incubations were carried out under optimal conditions of GlcNAc lipid synthesis as described under “Experimental Procedures” in the presence or absence of exogenously added GlcNAc-P-P-dolichol. The incubation mixtures additionally contained concentrations of showdomycin that inhibited 93–99% of the formation of GlcNAc-GlcNAc-P-P-dolichol. As seen in Fig.1 A, with increasing concentrations of GlcNAc-P-P-dolichol there was increasing inhibition (over 70%) of GlcNAc lipid synthesis. This effect on GlcNAc-P-P-dolichol biosynthesis occurred at the basal level and when the reaction was performed in the presence of Man-P-Dol shown previously to greatly stimulate its formation (10Kean E.L. J. Biol. Chem. 1985; 260: 12561-12571Abstract Full Text PDF PubMed Google Scholar). In these studies, either under basal or stimulatory conditions, exogenously added GlcNAc-P-P-dolichol was present over a range from 2- to 86-fold over the [3H]GlcNAc-P-P-dolichol formed in the absence of the inhibitor. (The curves in Fig. 1 were drawn in accord with an analysis of the data by Equation 1 as described under “Experimental Procedures.”) The nature of the products formed under these conditions after analysis by Dionex chromatography is seen in Fig.2. Fig. 2 A shows a typical example of the distribution of the mono-GlcNAc and chitobiosyl products formed under basal conditions in the absence of showdomycin. As seen in Fig. 2 B, under basal conditions in the presence of showdomycin, there was now extensive inhibition of the formation of the chitobiosyl product (solid line) and an increase in the formation of GlcNAc-P-P-dolichol (solid line) as described previously (19Kean E.L. Wei Z. Glycoconj. J. 1998; 15: 405-414Crossref PubMed Scopus (6) Google Scholar). When the reaction was carried out in the presence also of exogenously added GlcNAc-P-P-dolichol (18 μm), there was now an 86% decrease (diamond symbols) in the de novo formation of [3H] GlcNAc-P-P-dolichol. Exogenously added GlcNAc-P-P-dolichol was present in 360-fold molar excess over the [3H]GlcNAc-P-P-dolichol formed in its absence. Similar results were obtained when the reactions were performed under stimulatory conditions, i.e. in the presence of Man-P-dolichol (Fig. 2 C). The formation of GlcNAc-GlcNAc-P-P-dolichol under these conditions was reduced 97% in the presence of showdomycin (filled circles). When the reaction was carried out in the presence also of exogenously added GlcNAc-P-P-dolichol (18 μm), the formation of [3H]GlcNAc-P-P-dolichol was decreased 88% (diamonds). The added GlcNAc-P-P-dolichol was present in 56-fold molar excess over that formed in its absence. The reaction between GlcNAc-P-P-dolichol and UDP-GlcNAc catalyzed by GT-2 results in the formation of the chitobiosyl derivative. As seen in Fig. 1 B, when GlcNAc lipid synthesis was examined in the presence of exogenously added GlcNAc-GlcNAc-P-P-dolichol, inhibition of about 90% of that formed in the absence of the inhibitor was attained. As with the mono-GlcNAc derivative (Fig. 1 A), inhibition was also induced and to a similar extent when the reaction was stimulated by the addition of Man-P-Dol as seen also in Fig. 1 B. Shown in Fig. 2 D is an identification of the products from experiments of this type using Dionex chromatography. The presence of showdomycin resulted in the essentially complete (96%) inhibition of the formation of the chitobiosyl derivative. When the incubation was performed in the presence also of exogenously added GlcNAc-GlcNAc-P-P-dolichol (diamond symbols), the formation of GlcNAc-P-P-dolichol was inhibited 94% of that formed in its
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