Glucosamine metabolism in Drosophila salivary glands. Separation of metabolites and some characteristics of three enzymes involved.
The conversion of fructose 6-phosphate to mucopolysaccharide precursors was studied in extracts of Drosophila virilis salivary glands. 1. Methods for chromatography of sugar phosphates were adapted and modified to allow routine separation and quantitation of radioactivity of the metabolites from milligram amounts of tissue. Anion exchange chromatography was performed on Dowex 1-X8 employing steps of increasing ammonium formate. Final isolation of each compound was achieved by various thin-layer chromatographic systems. 2. Data obtained by isotope incorporation into glucosamine 6-phosphate compare well with results of the Morgan-Elson colorimetric assay for amino-sugars. 3. Glucosaminephosphate isomerase (glutamine-forming) (EC 5.3.1.19) in gland extracts has a Km of 0.35 mM for fructose 6-phosphate, and of 0.25 mM for glutamine. The enzyme is inhibited at glutamine concentrations exceeding 1 mM and by UDP-N-acetylglucosamine (50% at 0.6 mM). Feedback inhibition by UDP-N-acetylglucosamine is enhanced by AMP and by glucose 6-phosphate. 4. Glucosaminephosphate isomerase (EC 5.3.1.10) has a twenty fold lower affinity towards fructose 6-phosphate (Km = 6.0 mM) compared to the glutamine-forming isomerase. Km (NH+4) is 7.4 mM. In the presence of 20 mM glucose 6-phosphate, the pH optimum is shifted from 6.6 to 7.4, and V increased by a factor of 2.5. Furthermore, the affinity is approximately doubled for both substrates. 5. Glucosamine acetyltransferase (EC 2.3.1.3) has a Km of 2 mM for glucoseamine 6-phosphate. Its activity is not rate-limiting in salivary glands. Since N-acetylglucosamine 6-phosphate and 1-phosphate were found near equilibrium concentrations, acetylglucosamine phosphomutase (EC 2.7.5.2) must also be present in the extracts.
D000123:Acetyltransferases / Q000378:metabolism*; D019747:Aldose-Ketose Isomerases; D000818:Animals; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004330:Drosophila; D005636:Fructosephosphates; D005944:Glucosamine / Q000031:analogs & derivatives / Q000378:metabolism*; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000378:metabolism*; D007700:Kinetics; D012469:Salivary Glands / Q000201:enzymology*
D005636:Fructosephosphates; D000123:Acetyltransferases; C020580:glucosamine acetyltransferase; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); C020582:glucosamine-6-phosphate isomerase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; D005944:Glucosamine