Invertase inhibition

Determination of methyl mercury in fish tissue using electrochemical glucose oxidase biosensors based on invertase inhibition... 

This work demonstrates a simple and rapid screening method for determination of methyl mercury in organic solvent using invertase inhibition. We have shown that this method is free from interferences using the solvent extraction combined with the invertase enzyme. 

Competitive inhibition is important in biological control mechanisms for instance, if the product assumes the role of an inhibitor. The enzyme invertase catalyzes the hydrolysis of sucrose into glucose and fructose. As glucose is a competitive inhibitor, it ensures that the reaction does not proceed too far. 

The specificity of levansucrase98 is dependent not only on the d-fructoside but also on the aldoside residue of the substrate. Neither inulin nor methyl D-fructofuranoside was hydrolyzed by levansucrase, and even when these two substrates were hydrolyzed by inulase (prepared from inulin-fermenting Torula yeast) or by yeast invertase respectively, no levan formation occurred with levansucrase. However, neither methyl D-fructofuranoside nor inulin inhibited levan formation from sucrose by levansucrase. No levan was formed from potassium D-glucose... 

This succinyl group avoids the formation of the pyranosidic ring and acts as a spacer to conjugate the mole-cule to resins or biopolymers. The succinylated analog of sucrose 41 (R= HO2CCH2CH2CO-) is not sweet, and it is unable to inhibit invertase. It is however an inhibitor of a-glucosidase from baker s yeast. 

However, pyrimethanil and mepanipyrim do not inhibit proteinase, cellulase or polygalacturnase activity in Botrytis cinerea17 but reduce pectinase and invertase secretion with an associated increase in their intracellular accumulation. This is proposed to be the mechanism of action of the anilinopyrimidines but the biochemical basis of the effect is not known. There is evidence that suggests the involvement of methionine biosynthesis inhibition.18... 

The biosynthesis in yeast of two enzymes that are D-mannoproteins has been studied. A membrane-associated isozyme of invertase (EC 3.2.1.26) has been shown to be a precursor of the external invertase.190 Its molecular weight, as determined by SDS-poly(acrylamide) gel electrophoresis, is 50,000, that is, smaller than that of the external invertase, and it correlates well with the presence of only the inner-core sugars of the final form. It is strictly bound to membranes, possibly those of the endoplasmic reticulum, and it can be completely split191 by endo-/3-N-acetylglucosaminidase H (EC 3.2.1.30). The addition of tunicamycin, which specifically inhibits formation of d-GIcNAc-PP-DoI, inhibits synthesis of external invertase, as well as further formation of the membrane-associated form, which completely disappears after addition of the antibiotic.190 In these aspects, the synthesis of this extracellular enzyme follows the pathway for secreted glycoproteins in animal systems. 

In the mammal, complex polysaccharides which are susceptible to such treatment, are hydrolyzed by successive exposure to the amylase of the saliva, the acid of the stomach, and the disaccharidases (e.g., maltase, invertase, amylase, etc.) by exposure to juices of the small intestine. The last mechanism is very important. Absorption of the resulting monosaccharides occurs primarily in the upper part of the small intestine, from which the sugars are earned to the liver by the portal system. The absorption across die intestinal mucosa occurs by a combination of active transport and diffusion. For glucose, the aclive transport mechanism appears to involve phosphorylation The details are not yet fully understood. Agents which inhibit respiration (e.g., azide, fluoracetic acid, etc.) and phosphorylation (e.g., phlorizin), and those which uncouple oxidation from phosphorylation (e.g., dinitrophenol) interfere with the absorption of glucose. See also Phosphorylation (Oxidative). Once the various monosaccharides pass dirough the mucosa, interconversion of the other... 

Umbelliferose is most probably utilized in vivo through the primary attack of an a-D-galactosidase that would yield sucrose. It has been shown that the oligosaccharide is extremely resistant to hydrolysis by invertase.84 The kinetics of the inhibition showed94 that invertase. does not complex with umbelliferose the inhibition is probably attribu-... 

H. Mohammadi, M. El Razi, A. Amine, A.M.O. Brett and C.M.A. Brett, Determination of mercury (II) by invertase enzyme inhibition coupled with batch injection analysis, Analyst, 27 (2002) 1088-1093. 

Perform the experiments on mercury inhibition in two steps. In the first, a volume of 10 mL of buffer solution containing known concentration of invertase incubate with 10 mL of toluene solvent. 

The degree of inhibition in function of the concentration of the enzyme was studied in presence of 50 ppb of the inhibitor using the biphasic system (aqueous/toluene) as described in the experimental part. The concentration of invertase was varied from 0.05 to 4 gg/mL. 

Fig. 20.4. Calibration curve for inhibition of invertase (0.05 pg/mL) by methyl mercury after 10-min incubation using biphasic system (phosphate buffer/ toluene mixture) and glucose oxidase biosensor. Eapp — +0.60 V vs. Ag/AgCl and reaction time = 5 min.

Invertase (pg/mL) Methyl mercury (ppm) % of inhibition in absence of 1 g of fish % of inhibition in presence of 1 g of fish Recovery... 

To assess the selectivity of our method, the fish samples have been spiked with 2 ppm of mercury (II) and then treated as described before. After toluene incubation with invertase enzyme (0.05 pg/mL) no effect has been observed on enzyme activity. The same amount of mercury (II) has been added in fish samples in presence of 0.4 ppm of methyl mercury. In this case, 50% of inhibition has been noted and corresponds exactly to the value obtained when we studied the calibration cure in absence of mercury (II) (Fig. 20.4). This result is in agreement with what is reported in the literature showing that the methyl mercury is much more soluble in organic solvents than the mercury ions [8]. In addition to this high selectivity of our method, we have shown previously that the enzyme invertase is selective for mercury [3]. In the presence of Zn2+, Cu2+, Cd2+, Pb2+ and Fe3+ no inhibition was detected for these cations. 

The principle of combination of electrochemical glucose oxidase biosensor with the clean-up method for direct extraction and determination of methyl mercury has been successfully demonstrated. The extraction of methyl mercury from the organic solvent has been based on invertase enzyme inhibition. The combination of very low concentration of invertase enzyme and 10 min of incubation time allows the detection of methyl mercury at 5 ppb level. Our method permits the detection of this inhibitor below the legal limit given by the European Union with good recoveries when fish samples were measured. 

Table-TV shows the effect ot fhe LMW fraction on the activity of some of these enzymes in vitro. Maltase, lactase and invertase were competitively inhibited at a concentration of 10 mg/ ml. When the effectsof a range of concentrations (2.5-20 mg/ml) of the LMW fraction were studied, it was revealed that the inhibition was not of the pure competitive type. Table V shows the effect of the HMW fraction. Low concentrations had to be used in the assays, as the intense brown color of this fraction interfered with the spectrophotometric measurements. In spite of this a strong competitive inhibition of lactase and of invertase was found. Maltase was also inhibited, and, to a lesser extent, even trehalase. a-Amylase from saliva was not affected at the concentration tested.

There are scattered reports that phenolic acids inhibit a variety of enzymes, and it is evident that these compounds can block the function of many enzymes if they are sufficiently concentrated at the site of enzymatic functions. Activities of amylase, maltase, invertase, acid phosphatase and protease were suppressed by ferulic acid in tests using maize seeds and seedlings.17,50 Exogenously applied gibberellic acid reversed the effect of ferulic acid on amylase and acid phosphate. 

Foreign enzymes—that is, with no plant equivalent, have been introduced into plants one of the uses of this approach was to address the nature of sucrose transport into the phloem. An invertase derived from the yeast enzyme was targetted to the cell wall of tobacco, potato, tomato, and A. thaliana (Sonnewald et al., 1994). The introduction of invertase decreased yield, presumably through the inhibition of sucrose transport. 

Investigate the inhibition of the enzymatic reaction by p-mercuribenzoate at concentrations of about 10 M Show how an investigation of inhibition or poisoning of the catalyst might be used to establish the number of catalytically active sites on an invertase molecule. 

The polysaccharides associated with yeast invertase do not give a precipitate with Fehling solution, leading to the erroneous conclusion that mannan is absent. However, precipitation is inhibited by proteins, and mannan can be isolated after removal of protein. The proportions of O-methylmannose fragments obtained from the mannan in a methylation study were virtually the same as those obtained from methylated bakers yeast mannan (see Table III, p. 388). 

Many metabolic processes such as glycolysis, Krebs cycle reactions, photosynthesis, protein synthesis, and lipid metabolism are affected by exposure to F. Much of the action of F on these processes can be attributed to F-dependent inhibition of enzymes. Examples of enzymes shown to be inhibited by F include enolase, phosphoglucomutase, phosphatase, hexokinase, PEP carboxylase, pyruvate kinase, succinic dehydrogenase, malic dehydrogenase, pyrophosphatase, phytase, nitrate reductase, mitochondrial ATPase, urease (Miller et al. 1983), lipase (Yu et al. 1987), amylase (Yu et al. 1988), invertase (Yu 1996 Ouchi et al. 1999), and superoxide dismutase (SOD) (Wilde and Yu 1998). 

NADH oxidation, and electron transport (Nriagu 1980). Cadmium is a potent enzyme inhibitor, affecting a variety of plant enzymes such as PEP carboxylase, lipase, invertase (Yu 1997), and others. Extensive reports are available concerning Cd-dependent inhibition of enzymes from animals and humans. Alkaline phosphatase and ATPases of myosin and pulmonary alveolar macrophage cells are examples. 

---