Solubilization of a mannose-polymerizing enzyme from Phaseolus aureus.
A soluble enzyme preparation, which catalyses the polymerization of mannose, was obtained by Triton X-100 extraction of a particulate fraction derived from Phaseolus aureus hypocotyls. The product that resulted when GDP-alpha-d-mannose was used as a substrate was a beta-(1-->4)-linked mannan, about three-quarters of which was alkali-insoluble. The mannose-polymerizing enzyme activity was at least as great in the soluble preparation as in the particulate preparation, and the specific activity of the solubilized enzyme was greater by a factor of at least 3.5. Kinetic studies of the soluble enzyme indicate that the apparent K(m) is 55-62mum, and a disproportionate increase in rate is observed at high concentrations. GDP-alpha-d-glucose is a strong competitive inhibitor of the mannose-polymerizing reaction, with an apparent K(i) of 6.2mum. The soluble enzyme is relatively unstable, losing about two-thirds of its original activity in 5h at 0 degrees C or in 24h at -20 degrees C. A solvent (acetone, butanol, diethyl ether)-extracted particulate preparation, which also exhibits the same enzyme activity, is more stable, retaining full activity for at least 5 days at -20 degrees C. There was no polymerizing-enzyme activity in the soluble enzyme preparation when UDP-d-glucose, UDP-d-galactose, UDP-d-xylose, UDP-l-arabinose or UDP-d-glucuronic acid were used as substrates. However, the soluble enzyme preparation would catalyse the polymerization of glucose, with GDP-d-glucose as substrate.
D001089:Arabinose; D002247:Carbon Isotopes; D005690:Galactose; D005947:Glucose; D005965:Glucuronates; D006150:Guanine Nucleotides; D007700:Kinetics; D008358:Mannose / Q000378:metabolism*; D008722:Methods; D010944:Plants / Q000201:enzymology*; D011108:Polymers; D011134:Polysaccharides / Q000096:biosynthesis; D012995:Solubility; D013501:Surface-Active Agents; D014500:Uracil Nucleotides; D014994:Xylose
D002247:Carbon Isotopes; D005965:Glucuronates; D006150:Guanine Nucleotides; D011108:Polymers; D011134:Polysaccharides; D013501:Surface-Active Agents; D014500:Uracil Nucleotides; D014994:Xylose; D001089:Arabinose; D005947:Glucose; D008358:Mannose; D005690:Galactose