Purification and characterization of a soluble β‐fructofuranosidase from Daucus carota

Abstract

Soluble 8-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 carola). The enzyme hydrolyzed sucrose with a K , 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 68000 from a gelfiltration 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 p-fructofuranosidase with the complete sequence of carrot cell-wall P-fructofuranosidase showed that their N-terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.

Invertases. Primary Structures, Functions, and Roles in Plant Development and Sucrose Partitioning

“…A 70-kD monomeric form of vacuolar invertase of mung bean hypocotyls was found to be split into a 30-kD N-terminal and a 38-kD C-terminal fragment (Arai et al, 1991). Likewise, the 68-kD monomer of isoform I of vacuolar invertase from carrot was fragmented into N-and C-terminal polypeptides of 43 and 25 kD, respectively (Unger et al, 1992(Unger et al, , 1994. Under native conditions, these fragments appear to be tightly associated and, in a complex, possess enzyme activity.…”

“…Acid invertases have been purified from several plant species (for summary, see Unger et al, 1992). The enzymes have a K m for Suc in the low-millimolar range.…”

Antisense Repression of Vacuolar and Cell Wall Invertase in Transgenic Carrot Alters Early Plant Development and Sucrose Partitioning

“…Subsequently, immunological detection of cell wall invertase or isoform I of vacuolar invertase was performed by using an electrochemiluminescence-based detection system, as described by the manufacturer (Amersham). The antibodies against cell wall invertase (Laurière et al, 1988) and isoform I of vacuolar invertase (Unger et al, 1992) were diluted 1:3000 and 1:2000, respectively.…”

“…Homogeneously growing callus cultures were obtained after several rounds of subculture. Soluble and cell wall proteins of geneticinresistant calli were extracted and analyzed on protein gel blots with antibodies raised against vacuolar invertase (Unger et al, 1992; Figure 1A) or cell wall invertase (Laurière et al, 1988;Figure 1B).…”

Sucrolytic Enzyme Activities in Cotyledons of the Faba Bean (Developmental Changes and Purification of Alkaline Invertase)

“…Alkaline invertase is thought to be a cytosolic enzyme (Ricardo and ap Rees, 1970), although no direct evidence is available as yet. By comparison with acid invertases, which have been purified and cloned from severa1 plant sources (Pressey, 1966;Nakagawa et al, 1971;Cheng et al, 1990;Sturm and Chrispeels, 1990;Unger et al, 1992;Milling et al, 1993;Hedley et al, 1994), alkaline invertase has not been well characterized. The purification of alkaline invertase to homogeneity has been reported for soybean hypocotyls (Chen and Black, 1992) and more recently for chicory roots (Van den Ende and Van Laere, 1995).…”