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V S Butt

Publications and source records attributed to V S Butt.

18 recordsLinked to original sources
PubMed1995

The origin of the oxidative burst in plants.

A large number of publications recently have drawn strong analogies between the production of active oxygen species in plant cells and the "oxidative burst" of the phagocyte, even to the point of constructing elaborate models involving receptor mediated G-protein activation of a plasmalemma NADPH oxidase in plant cells. However there are potentially other active oxygen species generating systems at the plant cell surface. The present work examines these alternatives with particular emphasis on the rapid production of active oxygen species, in common with a number of other systems, by suspension-cultured cells of French bean on exposure to an elicitor preparation from the fungal pathogen Colletotrichum lindemuthianum. The cells show a rapid increase in oxygen uptake which is followed shortly afterwards by the appearance of a burst of these active oxygen species, as measured by a luminescence assay, which is probably all accounted for by hydrogen peroxide. An essential factor in this production of H2O2 appears to be transient alkalinization of the apoplast where the pH rises to 7.0-7.2. Dissipation of this pH change with a number of treatments, including ionophores and strong buffers, substantially inhibits the oxidative burst. Little evidence was found for enhanced activation of a membrane-bound NADPH oxidase. However the production of H2O2 under alkaline conditions can be modelled in vitro with a number of peroxidases, one of which, an M(r) 46,000 wall-bound cationic peroxidase, is able to sustain H2O2 production at neutral pH unlike the other peroxidases which only show low levels of this reaction under such conditions and have pH optima at values greater than 8.0. On the basis of such comparative pH profiles between the cells and the purified peroxidase and further inhibition studies a direct production of H2O2 from the wall peroxidase in French bean cells is proposed. These experiments may mimic some of the responses to plant pathogens, particularly the hypersensitive response, which is an important feature of resistance. A cell wall peroxidase-origin for the oxidative burst is clearly different from a model consisting of receptor activation of a plasmalemma-localised NADPH oxidase generating superoxide. An alternative simple and rapid mechanism thus exists for the generation of H2O2 which does not require such multiple proteinaceous components.

Fabaceae↗
PubMed1979

Synthesis and removal of phenylalanine ammonia-lyase activity in illuminated discs of potato tuber parenchyme.

(1) The synthesis and removal of phenylalanine ammonia-lyase (EC 4.3.1.5) in illuminated discs of potato (Solanum tuberosum cv King Edward) tuber tissue has been investigated by density labelling with deuterium (2H) from deuterium oxide (2H2O) followed by centrifugation to equilibrium in a CsC1 density gradient. (2) Temporal changes in enzyme level have been described in terms of the equation (dE/dt) = ks-kdE where (dE/dt) is the rate of change of enzyme level per unit of tissue (E) with respect to time (t), ks is the rate constant for synthesis of the enzyme and kd is the rate constant for the removal of active enzyme. (3) The optimal concentration of 2H2O was determined by analysis of the relationship between 2H2O concentration, development of enzyme activity and the magnitude of the increase in buoyant density of phenylalanine ammonia-lyase. A concentration of 2H2O of about 40% (v/v) was found to be optimal, allowing achievement of maximal or near maximal increases in the buoyant density of the enzyme without inhibition of the development of enzyme activity, thereby circumventing the major drawback of 2H2O as a source of density label. (4) The overlapping distribution profiles of enzyme activity after density gradient centrifugation were resolved by an iterative method of best fit which allows estimation of the proportions of pre-existing, unlabelled enzyme and newly synthesised, labelled enzyme at the end of the labelling period. This technique has been developed to obtain the rate constants for enzyme synthesis and for removal of active enzyme throughout the period of rapid change in enzyme level. (5) It is demonstrated that the initial rapid increase in phenylalanine ammonia-lyase activity in illuminated discs reflects an increase in the rate constant for enzyme synthesis in the absence of activation of pre-existing enzyme and in the absence of removal of active enzyme. The abrupt transition to a phase of decline in enzyme activity is caused by (a) a reduction in the rate constant for enzyme synthesis and (b) a dramatic increase in the rate constant for removal of active enzyme. The subsequent stabilisation of the enzyme is caused by decay of both rate constants to relatively low levels. (6) The results are consistent with hypothesis that rapid modulation of enzyme levels during tissue differentiation is achieved by simultaneous changesin the rate constants for both enzyme synthesis and for removal of active enzyme.

Ammonia-Lyases↗
PubMed1975

Purification and properties of S-adenosyl-L-methionine: caffeic acid O-methyltransferase from leaves of spinach beet (Beta vulgaris L).

1. An enzyme catalysing the methylation of caffeic acid to ferulic acid, using S-adenosyl-L-methionine as methyl donor, has been extracted from leaves of spinach beet and purified 75-fold to obtain a stable preparation. 2. The enzyme showed optimum activity at pH 6.5, and did not require the addition of Mg2+ for maximum activity. 3. It was most active with caffeic acid, but showed some activity with catechol, protocatechuic acid and 3,4-dihydroxybenzaldehyde. The Km for caffeic acid was 68 muM. 4. 4. The Km for S-adenosyl-L-methionine was 12.5 muM. S-Adenosyl-L-homocystein (Ki = 4.4 muM) was a competitive inhibitor of S-adenosyl-L-methionine. 5. The synthesis of S-adenosyl-L-homocysteine from adenosine and L-homocysteine and its consequent effect on caffeic acid methylation were demonstrated with a partially-purified preparation from spinach-beet leaves, which possessed both S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) and adenosine nucleosidase (EC 3.2.2.7) activities. This preparation was also able to catalyse the rapid breakdown of S-adenosyl-L-homocysteine to adenosine and adenine; the possible significance of this reaction in relieving the inhibition of caffeic acid methylation by S-adenosyl-L-homocystein is discussed.

Adenosine↗
PubMed1974

Oxidative decarboxylation of glycollate and glyoxylate by leaf peroxisomes.

1. The conditions under which peroxisomal preparations from leaves of spinach beet and spinach catalyse the release of (14)CO(2) from [1-(14)C]glycollate and [1-(14)C]glyoxylate were investigated. 2. At pH8, (14)CO(2) production from [1-(14)C]glyoxylate was accompanied by equivalent quantities of formate. The accumulation of oxalate and the effects of various reagents, especially catalase inhibitors, show that glyoxylate is non-enzymically oxidized by H(2)O(2), which is generated by the oxidation of glyoxylate to oxalate by the action of glycollate oxidase. 3. (14)CO(2) is shown to be generated from [1-(14)C]glycollate at pH8 by a similar reaction, but the H(2)O(2) is generated mainly by the oxidation of glycollate to glyoxylate. 4. The physiological significance of these reactions is discussed, with special reference to their role in photorespiration.

Animals↗
PubMed1972

The expression of catechol oxidase activity during the hydroxylation of p-coumaric acid by spinach-beet phenolase.

1. The conditions under which oxygen consumption in excess of that required for the hydroxylation of p-coumaric acid to caffeic acid, catalysed by spinach-beet phenolase, can be suppressed, have been examined. 2. With dimethyltetrahydropteridine as electron donor, oxygen uptake was exactly equivalent to the caffeic acid produced, provided that p-coumaric acid was in excess, but with excess of reductant, oxygen uptake caused by the further oxidation of caffeic acid was also observed. 3. With equal concentrations of ascorbate and p-coumaric acid, equivalent oxygen uptake and caffeic acid production was found only in the first stages of the reaction, whereas with NADH substituted for ascorbate, oxygen uptake was in excess throughout. 4. When ascorbate was used, the period of the reaction over which this equivalence was found was decreased at high reaction rates and not observed at all with aged enzyme preparations; equivalence was restored by adding bovine serum albumin to these aged preparations. 5. Equivalence between oxygen consumption and caffeic acid production was observed with NADH, if small quantities of dimethyltetrahydropteridine were also added. 6. It is concluded that hydroxylation proceeds without the concomitant production of caffeic acid only if the enzyme is stabilized for hydroxylation by p-coumaric acid and the reductant, and is protected from attack by o-quinones.

Ascorbic Acid↗
PubMed1970

The action of o-dihydric phenols in the hydroxylation of p-coumaric acid by a phenolase from leaves of spinach beet (Beta vulgaris L.).

1. Under defined conditions, the hydroxylation of p-coumaric acid catalysed by a phenolase from leaves of spinach beet (Beta vulgaris L.) was observed to develop its maximum rate only after a lag period. 2. By decreasing the reaction rate with lower enzyme concentrations or by increasing it with higher concentrations of reductants, the length of the lag period was inversely related to the maximum rate subsequently developed. 3. Low concentrations of caffeic acid or other o-dihydric phenols abolished this lag period. With caffeic acid, the rate of hydroxylation was independent of the reductant employed. 4. Hydroxylation was inhibited by diethyldithiocarbamate, but with low inhibitor concentrations hydroxylation recovered after a lag period. This lag could again be abolished by the addition of high concentrations of caffeic acid or other o-dihydric phenols. 5. Catechol oxidase activity showed no lag period, and did not recover from diethyldithiocarbamate inhibition. 6. The purified enzyme contained 0.17-0.33% copper; preparations with the highest specific activity were found to have the highest copper content. 7. The results are interpreted to suggest that the oxidation of o-dihydric phenols converts the enzymic copper into a species catalytically active in hydroxylation. This may represent the primary function for the catechol oxidase activity of the phenolase complex. The electron donors are concerned mainly, but not entirely, in the reduction of o-quinones produced in this reaction.

Caffeine↗
PubMed1969

The hydroxylation of p-coumaric acid by an enzyme from leaves of spinach beet (Beta vulgaris L.).

1. An enzyme from the leaves of spinach beet (Beta vulgaris L.) that catalyses the hydroxylation of p-coumaric acid to caffeic acid in the presence of ascorbate has been purified about 1000-fold on a protein basis. 2. It is activated by high concentrations of ammonium sulphate and sodium chloride. 3. The preparation shows both hydroxylase and catechol oxidase activities, in a constant ratio throughout the purification procedure; they are similarly activated by salts. 4. Ascorbate acts as a reductant in quantities equivalent to the caffeic acid produced by hydroxylation. 5. Ascorbate can be replaced by tetrahydrofolic acid, NADH, NADPH or 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, but not by caffeic acid. Among these, the pteridine is the most effective, but the reaction is not inhibited by aminopterin. In experiments with saturating concentrations of NADH and the pteridine, these reductants compete in the reaction and are equivalent on a molar basis. 6. No cofactor has been separated from the enzyme by prolonged dialysis. 7. The relation of the enzyme to other hydroxylases and phenolases is discussed.

Ascorbic Acid↗