Purification and Characterization of Neutral and Alkaline Invertase from Carrot

Abstract

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 K, values of about 20 mM at pH 7.5.Neutral invertase also hydrolyzed raffinose and stachyose and, therefore, is a 6-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 HgCI,. Alkaline invertase was markedly inhibited by CaCI,, MgCI,, and MnCI,, and neutral invertase was not. In contrast to alkaline invertase, neutral invertase was inhibited by the nucleotides ATP, CTP, GTP, and UTP.

Point Mutation of a Plastidic Invertase Inhibits Development of the Photosynthetic Apparatus and Enhances Nitrate Assimilation in Sugar-treated Arabidopsis Seedlings

“…At present, just why the sicy-192 mutant is recessive cannot be explained, although there is the following possibility. It has been reported that A/N invertase acts as homo-octamer or homo-tetramer in higher plants (41). Only when the holoenzyme consists of mutated subunits, will INV-E:C294Y become more stable.…”

Cloning of cDNA for a cell wall-bound acid invertase from tomato (Lycopersicon esculentum) and expression of soluble and cell wall-bound invertases in plants and wounded leaves of L. esculentum and L. peruvianum.

“…Alkaline and neutral invertases are cytosolic and have neutral to weakly alkaline pH optima (Chen and Black, 1992;Van den Ende and Van Laere, 1995;Ross et al, 1996;Lee and Sturm, 1996). Alkaline and neutral invertases appear to be involved in the breakdown of sucrose to meet the metabolic demand for cytosolic hexoses (Ricardo, 1974;Masuda et al, 1988;Stommel and Simon, 1990; Lee and Sturm, 1996). cDNAs and genomic DNAs for soluble and cell wallbound acid invertases have been isolated from various plants (Sturm and Chrispeels, 1990;Arai et al, 1992;Klann et al, 1992;Elliott et al, 1993;Unger et al, 1994;Roitsch et al, 1995;Weber et al, 1995).…”

“…Both soluble and cell wall-bound invertases are involved in responses to wounding and pathogens (Matsushita and Uritani, 1974;Sturm and Chrispeels, 1990;Benhamou et al, 1991;Ohyama et al, 1995;Herbers et al, 1996;Zhang et al, 1996). Alkaline and neutral invertases are cytosolic and have neutral to weakly alkaline pH optima (Chen and Black, 1992;Van den Ende and Van Laere, 1995;Ross et al, 1996;Lee and Sturm, 1996). Alkaline and neutral invertases appear to be involved in the breakdown of sucrose to meet the metabolic demand for cytosolic hexoses (Ricardo, 1974;Masuda et al, 1988;Stommel and Simon, 1990; Lee and Sturm, 1996).…”

Purification and characterization of an alkaline invertase from shoots of etiolated rice seedlings

“…It is well known that alkaline invertase participates in the regulation of hexose and sucrose levels in the cytoplasm. For example, an increase in the concentration of glucose and fructose due to a decreased demand for hexoses would decrease sucrose metabolism directly through the inhibition of invertases (Lee & Sturm, 1996). We found that fructose and glucose inhibited rice alkaline invertase.…”

“…Ca# + and Mg# + had no effect on activity, whereas heavy metal ions, especially Hg# + , Cu# + and Ag + , decreased activity. Several reagents known to inhibit plant invertases (Karuppiah et al, 1989 ;Lin & Sung, 1993 ;Charng et al, 1994 ;Sung & Huang, 1994 ;Lee & Sturm, 1996) were tested (Table 2). At 5 mM, pyridoxine, pyridoxal, aniline and iodoacetamide decreased the activity to 40 %, 58 %, 30 % and 65 %, respectively.…”