Purification and characterization of a soluble beta-fructofuranosidase from Daucus carota.
Soluble beta-fructofuranosidase with an intracellular location and an isoelectric point of 3.8 (isoenzyme I) was purified and characterized from dry seeds and seedlings of carrot (Daucus carota). The enzyme hydrolyzed sucrose with a Km of 5 mM and a broad pH optimum around 5.0. The purified protein, which was N-glycosylated with high-mannose-containing and high-xylose-containing complex glycans, eluted as a monomeric polypeptide with a molecular mass of 68,000 from a gel-filtration column. On SDS/PAGE, the protein separated in the presence of SDS and 2-mercaptoethanol into three polypeptides with molecular masses of 68, 43 and 25 kDa. The amount of the 68-kDa polypeptide was highest in dry seeds and decreased with increasing age of carrot seedlings. Amino acid sequence analysis and immunological studies showed that the 43-kDa and 25-kDa polypeptides were N-terminal and C-terminal proteolytic fragments of the 68-kDa polypeptide. A comparison of partial amino acid sequences of the soluble beta-fructofuranosidase with the complete sequence of carrot cell-wall beta-fructofuranosidase showed that their N-terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.
Unger
C
C
Friedrich Miescher-Institute, Basel, Switzerland.
Hofsteenge
J
J
Sturm
A
A
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D014357:Trypsin / Q000378:metabolism; D014675:Vegetables / Q000201:enzymology*; D043324:beta-Fructofuranosidase
D007527:Isoenzymes; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D014357:Trypsin