ABSTRACT
Invertase was purified from rose (Fructus cynosbati) hips by ammonium sulfate fractionation and hydroxyapatite column chromatography. The enzyme was obtained with a yield of 4.25% and about 10.48-fold purification and had a specific activity of 8.59 U/mg protein. The molecular mass of invertase was estimated to be 66.51 kDa by PAGE and 34 kDa by SDS-PAGE, indicating that the native enzyme was a homodimer. The enzyme was a glycoprotein and contained 5.86% carbohydrate. The Km for sucrose was 14.55 mM and the optimum pH and temperature of the enzyme were 4.5 and 40°C, respectively. Sucrose was the most preferred substrate of the enzyme. The enzyme also hydrolyzed D(+) raffinose, D(+) trehalose and inulin (activity 39.88, 8.12 and 4.94%, respectively of that of sucrose), while D(+) lactose, cellobiose and D(+) maltose showed no effect on the enzyme. The substrate specificity was consistent with that for a β-fructofuranoside, which is the most popular type in the higher plants. The enzyme was completely inhibited by HgCl2, MnCl2, MnSO4, FeCl3, Pb(NO3)2, ammonium heptamolybdate, iodoacetamide and pyridoxine hydrochloride. It was also inhibited by Ba(NO3)2 (86.32%), NH4Cl (84.91%), MgCl2 (74.45%), urea (71.63%), I2 (69.64%), LiCl (64.99%), BaCl2 (50.30%), Mg(NO3)2 (49.90%), CrCl3 (31.90%) and CuSO4 (21.45%) and but was activated by Tris (73.99%) and methionine (12.47%).
Subject(s)
Carbohydrate Metabolism , Chemical Fractionation/methods , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Fruit/enzymology , Hydrogen-Ion Concentration , Molecular Weight , Rosa/enzymology , Substrate Specificity , Temperature , beta-Fructofuranosidase/antagonists & inhibitors , beta-Fructofuranosidase/chemistry , beta-Fructofuranosidase/isolation & purification , beta-Fructofuranosidase/metabolismABSTRACT
The ascorbate-glutathione (ASC-GSH) cycle has an important role in defensive processes against oxidative damage generated by drought stress. In this study, the changes that take place in apoplastic and symplastic ASC-GSH cycle enzymes of the leaf and petiole were investigated under drought stress causing leaf rolling in Ctenanthe setosa (Rose.) Eichler (Marantaceae). Apoplastic and symplastic extractions of leaf and petiole were performed at different visual leaf rolling scores from 1 to 4 (1 is unrolled, 4 is tightly rolled and the others are intermediate forms). Glutathione reductase (GR), a key enzyme in the GSH regeneration cycle, and ascorbate (ASC) were present in apoplastic spaces of the leaf and petiole, whereas dehydroascorbate reductase (DHAR), which uses glutathione as reductant, monodehydroascorbate reductase (MDHAR), which uses NAD(P)H as reductant, and glutathione were absent. GR, DHAR and MDHAR activities increased in the symplastic and apoplastic areas of the leaf. Apoplastic and symplastic ASC and dehydroascorbate (DHA), the oxidized form of ascorbate, rose at all scores except score 4 of symplastic ASC in the leaf. On the other hand, while reduced glutathione (GSH) content was enhanced, oxidized glutathione (GSSG) content decreased in the leaf during rolling. As for the petiole, GR activity increased in the apoplastic area but decreased in the symplastic area. DHAR and MDHAR activities increased throughout all scores, but decreased to the score 1 level at score 4. The ASC content of the apoplast increased during leaf rolling. Conversely, symplastic ASC content increased at score 2, however decreased at the later scores. While the apoplastic DHA content declined, symplastic DHA rose at score 2, but later was down to the level of score 1. While GSH content enhanced during leaf rolling, GSSG content did not change except at score 2. As well, there were good correlations between leaf rolling and ASC-GSH cycle enzyme activities ...