The effect of kinetin on the indoleacetic acid level and indoleacetic acid oxidase activity in roots of young plants

Kinetin treatment increased the extractable IAA content in roots of young plants of Phaseolus vulgaris L., Zea mays L. and Avena sativa L. The highest increase was obtained with roots of beans and the lowest with oat roots. Maize was intermediate between these two species. Kinetin treatment decreased the activity of IAA-oxidase but the correlation between the decrease of the activity of this enzyme and the increase in the level of IAA was not good. The decrease of the oxidase activity was greatest in oat roots where kinetin had very little effect on the IAA level, and was rather small in bean roots, where kinetin treatment significantly increased the IAA level.     

N-carbobenzyloxy-D-aspartic acid as a competitive inhibitor of indole-3-acetyl-L-aspartic acid hydrolase of Enterobacter agglomerans

The gene for a specific IAA-asp hydrolase from Enterobacteragglomerans, iaaspH, is a potentially useful tool for modificationofIAA homeostasis in higher plants that use the IAA-asp oxidation pathway forauxin catabolism. In order to optimize the utility of this gene for plantmodification and to increase the success of obtainingiaaspH transformed plants from culture, we haveinvestigated aspects of IAA-asp hydrolase catalysis. The catalyticcharacteristics of the IAA-asp hydrolase from Enterobacteragglomerans was studied using ten compounds that are structuralanalogues of IAA-asp. These compounds were tested as potential IAA-asphydrolasesubstrates as well as for inhibition of IAA-asp hydrolysis. Among them,N-carbobenzyloxy-D-aspartic acid (N-CBZ-D-asp) and N-CBZ-L-asp were found to bethe strongest inhibitors with more than 80% inhibition of IAA-asp hydrolysis.Aspartyl-L-aspartic acid and a asp-ser-asp-pro-arg peptide also showed stronginhibitory activities, reducing rates of IAA-L-asp hydrolysis, when added atequal molar amounts relative to the substrate, by 60% and 65%, respectively.N-CBZ-D-asp was chosen for further kinetic studies and for studies of itstoxicity in relation to seed germination because it was a strong inhibitor,exhibited a very low rate of hydrolysis by the IAA-asp hydrolase and wascommercially available. N-CBZ-D-asp was shown to be a competitive inhibitor forthe Enterobacter agglomerans IAA-asp hydrolase with aK _i value of 1.22 mM. Studies oftomato seed germination showed that N-CBZ-D-asp did not affect the rate of seedgermination at up to 1 mM, but the growth rate of seedlings wassignificantly reduced when the concentration in the medium was 0.5mM and higher. These results indicate that, at suitableconcentrations, N-CBZ-D-asp should be a useful tool for control of low levelexpression of the iaaspH in transgenic plants duringcritical stages of plant regeneration from culture.     

Cytokinin control in subcellular localization of indoleacetic acid oxidase and peroxidase

IAA oxidase and peroxidase were found in all subcellular fractions of tobacco callus cells. The bound and cytoplasmic fractions differed greatly in IAA oxidase/peroxidase ratio and in isoperoxidase composition. The IAA oxidase/peroxidase ratio was particularly high in the plasma membrane-rich fraction. Kinetin had profound effects on IAA oxidase and peroxidase. The appearance of fast migrating isoperoxidases in response to 0·2 μM kinetin was found only in cytoplasmic, plasma membrane and ribosome-rich fractions; a high concentration of kinetin inhibited their formation. High kinetin concentrations also lowered the specific activity of IAA oxidase and peroxidase in all subcellular fractions, but the effect was much greater on peroxidase than on IAA oxidase, thus resulting in a drastic increase in IAA oxidase/peroxidase ratio. Evidently the activities of IAA oxidase and peroxidase were not equivalent and should be considered separately.     

Cherry fruit development: The use of a microassay for studying indoleacetic acid oxidase

An improved procedure is described for extraction and assay of indoleacetic acid oxidase from seeds of sour cherry (Prunus cerasus L.). The extraction procedure was optimized for pH, buffer, polyvinylpolypyrrolidone (PVP) and tissue: buffer ratio. Greatest extraction efficiency was obtained at pH 4.0, 0.2 M acetate buffer, tissue: PVP ratio of 1:2.5 and tissue: buffer ratio of 50 ml per g of seed. The enzyme was assayed at 30°C using indoleacetic acid-1-¹⁴C as substrate and radioassaying the ¹⁴CO₂ evolved. Mn²⁺ and 2,4-dichlorophenol enhanced enzyme activity but were not obligatory. A minimum substrate concentration of 60 M was needed for quantitative evaluation. This assay was sensitive and reproducible, enzyme activity being demonstrated in as little as 0.8 mg of seed tissue with a coefficient of variation of 1 to 9%.     

Fumaric acid is a competitive inhibitor of wheat germ lipoxygenase

Fumaric acid is shown to be a competitive inhibitor of wheat germ lipoxygenase.     

Acarbose is a competitive inhibitor of mammalian lysosomal acid alpha-D-glucosidases

Intraperitoneal injections (approximately 400 mg/kg of body weight) of acarbose, an inhibitor of acid (1----4)-alpha-D-glucosidase, perturb the metabolism of glycogen in the liver, resulting in excess storage of lysosomal glycogen. The metabolism of skeletal muscle glycogen was unaffected, suggesting that acarbose either does not enter the tissue or that the muscle alpha-D-glucosidase is not inhibited. The hydrolysis of maltose and glycogen by the acid alpha-D-glucosidases from rat liver, rat skeletal muscle, and human placenta was inhibited competitively by acarbose. Thus, the lack of effect of acarbose upon the metabolism of muscle glycogen is due to its inability to enter the tissue.     

Promotion of indoleacetic Acid oxidase isoenzymes in tobacco callus cultures by indoleacetic Acid

Indoleacetic acid oxidase in tobacco callus cultures (Nicotiana tabacum L., cv. White Gold) was composed of at least two groups of isoenzymes, which were distinctly different in electrophoretic mobilities and in responses to growth substances. Indoleacetic acid had dual effects; at low concentrations it promoted the development of two fast-migrating indoleacetic acid oxidase isoenzymes, but at high concentrations it increased the level of other indoleacetic acid oxidase isoenzymes with low and moderate electrophoretic mobilities. However, indoleacetic acid was not unique in such effects; 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid were effective at concentrations lower than that of indoleacetic acid.     

N2O as a substrate and as a competitive inhibitor of nitrogenase

We have investigated the inhibitory effect of N2O on NH3 formation by purified component proteins from Klebsiella pneumoniae and have confirmed that the inhibition is competitive with respect to N2 and that N2O is reduced to N2, which in turn is further reduced to NH3. In addition, we have shown that N2O is unable to support HD formation from D2 and H2O. N2-supported HD formation from D2 and H2O was found to be inhibited by N2O. In contrast to N2, N2O was found to suppress nitrogenase-mediated H2 evolution completely at infinitely high pN2O. H2 was found to inhibit N2O-supported NH3 production but not N2O-supported N2 production. The steady-state kinetics of N2O reduction showed a good fit to Michaelis-Menten kinetics with a Km for N2O of 5 mM at 30 degrees C, corresponding to 24 kPa of N2O. A model is proposed that fits the observed results.     

Lithospermic acid as a novel xanthine oxidase inhibitor has anti-inflammatory and hypouricemic effects in rats

Lithospermic acid (LSA) was originally isolated from the roots of Salvia mitiorrhiza, a common herb of oriental medicine. Previous studies demonstrated that LSA has antioxidant effects. In this study, we investigated the in vitro xanthine oxidase (XO) inhibitory activity, and in vivo hypouricemic and anti-inflammatory effects of rats. XO activity was detected by measuring the formation of uric acid or superoxide radicals in the xanthine/xanthine oxidase system. The results showed that LSA inhibited the formation of uric acid and superoxide radicals significantly with an IC50 5.2 and 1.08 microg/ml, respectively, and exhibited competitive inhibition. It was also found that LSA scavenged superoxide radicals directly in the system beta-NADH/PMS and inhibited the production of superoxide in human neutrophils stimulated by PMA and fMLP. LSA was also found to have hypouricemic activity on oxonate-pretreated rats in vivo and have anti-inflammatory effects in a model of gouty arthritis. These results suggested that LSA is a competitive inhibitor of XO, able to directly scavenge superoxide and inhibit superoxide production in vitro, and presents hypouricemic and anti-inflammatory actions in vivo.     

Beta-selenaproline as competitive inhibitor of proline activation

Beta-Selenaproline, a proline analog having the beta-methylene group substituted by a selenium atom, has been tested in ATP-PPi exchange reaction catalyzed by either Escherichia coli or rat liver aminoacyl-tRNA synthetases. It has been shown that with both enzymatic systems beta-selenaproline does not give rise to ATP-PPi exchange, but specifically inhibits proline activation. The inhibition is of fully competitive type and the Ki values, lower than the Km values for proline, show that beta-selenaproline binds to the synthetases with high affinity. The inability to form the complex with AMP, taking into account also the behavior of gamma-selenaproline and other proline analogs, has been ascribed to the presence of the selenium atom in the beta-position.     

Electrophoretic studies of the relationship of peroxidases,polyphenol oxidase,and indoleacetic acid oxidase to cold tolerance of alfalfa

Two cultivars of alfalfa (Medicago sativa L.), cold-tolerant Vernal and cold-sensitive Sonora, were grown under summer, winter, and dehardening environments to investigate the relationship of soluble proteins and enzyme activity and solubility characteristics to cold tolerance.Evaluations of cold-tolerance levels developed in crown and root samples were compared with results of soluble protein analyses and were in agreement with previously reported observations. Soluble protein content was associated with increases in cold tolerance and related to the environment from which samples were obtained; however, the degree of protein differences within samples of the same cultivar as well as between the two cultivars seemed to be influenced by the type of extractant used.Polyacrylamide disc gel electrophoresis of the extracted soluble proteins was performed on the basis of equal dry weights and equal quantities of protein. Amido black straining of gels indicated mainly quantitative changes and slight qualitative differences in component bands influenced by environment and extractant.Gels assayed for peroxidase, polyphenol oxidase, and indoleacetic acid oxidase enzymes exhibited mainly quantitative differences in constitutive isoenzyme components of both cultivars which were associated with environmental changes. Enzyme activities generally increased in winter, as cold tolerance and soluble protein content increased, and decreased during dehardening. The few qualitative differences in isoenzyme bands that were detected, appeared to be influenced by cultivar, environment, extractant, or substrate specificity differences.Variation in isoenzyme components between cultivars was maximum in summer samples, and minimum in winter samples, suggesting that overall reaction rates or activities of individual isoenzymes, preceding or during hardening, could be a limiting factor in cold-tolerance development.     

Solvent as a competitive inhibitor for Candida antarctica lipase B

In enzyme-catalyzed reactions, the choice of solvent often has a marked effect on the reaction outcome. In this paper, it is shown that solvent effects could be explained by the ability of the solvent to act as a competitive inhibitor to the substrate. Experimentally, the effect of six solvents, 2-pentanone, 3-pentanone, 2-methyl-2-pentanol, 3-methyl-3-pentanol, 2-methylpentane and 3-methylpentane, was studied in a solid/gas reactor. As a model reaction, the CALB-catalyzed transacylation between methyl propanoate and 1-propanol, was studied. It was shown that both ketones inhibited the enzyme activity whereas the tertiary alcohols and the hydrocarbons did not. Alcohol inhibition constants, K(i)(I) were changed to "K(i)", determined in presence of 2-pentanone, 3-pentanone, and 3-methyl-3-pentanol, confirmed the marked inhibitory character of the ketones and an absence of inhibition of 3-methyl-3-pentanol. The molecular modeling study was performed on three solvents, 2-pentanone, 2-methyl-2-pentanol and 2-methyl pentane. It showed a clear inhibitory effect for the ketone and the tertiary alcohol, but no effect for the hydrocarbon. No change in enzyme conformation was seen during the simulations. The study led to the conclusion that the effect of added organic component on lipase catalyzed transacylation could be explained by the competitive inhibitory character of solvents towards the first binding substrate methyl propanoate.     

Water as a competitive inhibitor of lipase-catalysed esterification in organic media

Summary Kinetic parameters were determined for esterification of dodecanol and decanoic acid in hexane catalysed by lipases from Rhizomucor miehei and Candida rugosa, after pre-equilibration to different values of thermodynamic water activity (a_w). V_m increases with increasing a_w, but so do the K_m values for both substrates. The effect on K_m for the alcohol probably represents competition between the first product and the second substrate, as expected for Ping-Pong kinetics. The rise in K_m for the acid probably reflects the displacement of water molecules during substrate binding.