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Kean, Edward L.; Niu, Naiqian

doi:10.1023/a:1006931230848

Cited by 6 publications

(2 citation statements)

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“…Feedback inhibition of the biosynthesis of GlcNAc-P-Pdolichol 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 (16). Although the reversibility of GPT-1 has previously been demonstrated (17,18), the present report also described the kinetics of inhibition by GlcNAc-P-P-dolichol of its own synthesis.…”

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confidence: 63%

“…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 Hϩ (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 (16). 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).…”

Section: Incubation Conditions and Assay Kinetic Studiesmentioning

confidence: 99%

See 1 more Smart Citation

Regulation of the Biosynthesis ofN-Acetylglucosaminylpyrophosphoryldolichol, Feedback and Product Inhibition

Kean¹,

Wei²,

Anderson³

et al. 1999

Journal of Biological Chemistry

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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 form N-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-Pdolichol 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 M, respectively. Inhibition was also observed under conditions where mannosyl-P-dolichol (Man-P-dol) stimulated the biosynthesis of GlcNAc-P-Pdolichol; the apparent K i values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol were 2.2 and 11 M, 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.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. 1). 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, 1 catalyzed by the enzyme, UDP-GlcNAc:dolichyl-phosphate N-acetylglucosamine 1-phosphate transferase (GPT-1). Factors that modulate the formation of GlcNAc-P-Pdolichol 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 (2), genetic factors (3), and topography of enzymes and substrates (4 -6). Previous studies from this laboratory and others (7-15) have also revealed that another intermediate ...

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mentioning

confidence: 63%

Section: Incubation Conditions and Assay Kinetic Studiesmentioning

confidence: 99%