Purification and characterization of neutral and alkaline invertase from carrot.
Neutral and alkaline invertase were identified in cells of a suspension culture of carrot (Daucus carota L.) and purified to electrophoretic homogeneity. Neutral invertase is an octamer with a molecular mass of 456 kD and subunits of 57 kD, whereas alkaline invertase is a tetramer with a molecular mass of 504 kD and subunits of 126 kD. Both enzymes had sharp pH profiles, with maximal activities at pH 6.8 for neutral invertase and pH 8.0 for alkaline invertase, and both hydrolyzed sucrose with typical hyperbolic kinetics and similar Km values of about 20 mM at pH 7.5. Neutral invertase also hydrolyzed raffinose and stachyose and, therefore, is a beta-fructofuranosidase. In contrast, alkaline invertase was highly specific for sucrose. Fructose acted as a competitive inhibitor of both enzymes, with Ki values of about 15 mM. Glucose was a noncompetitive inhibitor of both neutral and alkaline invertase, with a Ki of about 30 mM. Neither enzyme was inhibited by HgCl2. Alkaline invertase was markedly inhibited by CaCl2, MgCl2, and MnCl2, and neutral invertase was not. In contrast to alkaline invertase, neutral invertase was inhibited by the nucleotides ATP, CTP, GTP, and UTP.
Lee
H S
HS
Friedrich Miescher-Institut, Basel, Switzerland.
Sturm
A
A
D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D018552:Daucus carota / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D043324:beta-Fructofuranosidase
D004791:Enzyme Inhibitors; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase