Eddie C. Smith

Scopoletin: A substrate for an isoperoxidase from Nicotiana tabacum tissue culture W-38☆

Abstract Scopoletin was found to be a substrate for a single anodic isoperoxidase isolated from tobacco callus tissue W-38. Isolation of this peroxidase was accomplished using DEAE-cellulose chromatography. This isoperoxidase catalysed the destruction of scopoletin in the presence of H2O2 only. An enzyme assay for the scopoletin reaction was developed. The pH optimum of the enzyme was 5·5 and the apparent Kms for scopoletin and H2O2 were 0·6 and 0·9 rnM respectively.

Ferulic acid: a substrate for two isoperoxidases from Nicotiana tabacum tissue cultures

Abstract An anodic isoperoxidase (A 2 ) from tobacco tissue culture W-38 and a cathodic isoperoxidase (C 4 ) from tobacco tissue suspension culture WR-132 have been separated and characterized. Both isoperoxidases catalysed oxidation of ferulic acid in the presence of H 2 O 2 . When the reaction mixture was subjected to TLC, ferulic acid was found to have been converted to an unknown compound which, after treatment with ammonia, fluoresces green in UV light. Both the isoperoxidases A 2 and C 4 appear to follow simple Michaelis-Menten kinetics with respect to guaiacol as the substrate. The K m s for guaiacol are 4 and 4·5 mM for isoperoxidases C 4 and A 2 , respectively. The pH optimum for both enzymes is about 6·0. The effect of various phenolic and related compounds on the activity of each isoperoxidase is reported and discussed.

The effect of some phenolic compounds on the activity of 6-phosphogluconate dehydrogenase from tobacco tissue cultures

Abstract Anodic polyacrylamide gel electrophoresis of extracts of cultures of tobacco tissue Nicotiana tabacum W-38 revealed the presence of two 6-phosphogluconate dehydrogenases (6PGD). The slow and the fast anodic migrating zones were designated I and II, respectively. After purification, enzymes from both zones exhibited no major differences in their affinity towards 6-phosphogluconate (6PG) or NADP + , and were found to have approximately the same pH optima and MWs (69 000–72 000). The coumarins scopoletin and esculetin showed some inhibitory effect on each isozyme at 0.4 mM. Below 0.3 mM, however, esculetin stimulated the activity of zone I when lower amounts of 6PG ( S 0.25 ) were used. The glucosylated compounds, scopolin and esculin, were much more inhibitory towards the 6PGDs than their respective aglycones. Ferulic, p -coumaric and caffeic acids seemed to have an inhibitory effect dependent on 6PG concentration. A larger inhibition was observed in each case at the lower 6PG levels used. Zone I activity appeared to be inhibited to a greater degree than zone II activity by 0.4 mM p -coumaric acid with low 6PG. Of the phenolic compounds tested, chlorogenic acid was most effective, completely inhibiting the enzyme activity at 0.4 mM. Of the non-phenolic compounds investigated, glucose 1,6-diphosphate inhibited both isoenzymes of 6PGD at lower 6PG concentrations. On the other hand, 2,3-diphosphoglycerate activated both isoenzymes up to 200% of their original activity.

Isolation and characterization of two isoperoxidases from tobacco tissue cultures

Abstract Two isoperoxidases (A f and C n ) from the medium of tobacco tissue suspension culture WR-132 grown in darkness have been purified to apparent homogeneity and partially characterized. C n and A f have MWs of ca 30 000 and 54 000, respectively. A f has ca 5.1% carbohydrate, but none could be detected in C n . Both isoperoxidases appear to follow simple Michaelis-Menten kinetics with respect to guaiacol as the substrate. The K m s for guaiacol are 4 and 13.3 mM for A f and C n , respectively, while both isoperoxidases have a pH optimum at 6.5. C n , is dissimilar to other isoperoxidases from tobacco tissue cultures, but A f is very similar to isoperoxidase A 3 from W-38 tobacco tissue culture.

Isoperoxidases from tobacco tissue cultures

Abstract Two anodic isoperoxidases (A1 and A2) from tobacco tissue culture W-38 and two cathodic isoperoxidases (C3 and C4) from tobacco suspension culture WR-132 have been separated and characterized. Molecular weights for each of the isoperoxidases have been determined by two different methods. Only C4 contained a carbohydrate component. The substrate specificity and the pH optima for the four enzymes with each of five substrates were determined.

Isoenzymes of glucose-6-phosphate dehydrogenase from tobacco cells

Abstract Two anodic isoenzymes of glucose-6-phosphate dehydrogenase (G6PDH) were isolated from tobacco suspension culture WR-132, utilizing fractional ammonium sulfate precipitation and DEAE-cellulose chromatography. The pH optimum was 9.0 for isoenzyme G6PDH I and 8.0–8.3 for G6PDH IV. Isoenzyme G6PDH I exhibited Michaelis-Menten kinetics for both substrates, G6P and NADP+, with Kms of 0.22 mM and 0.06 mM, respectively. G6PDH IV exhibited Michaelis-Menten kinetics for G6P with a Km of 0.31 mM. The NADP+ double reciprocal plot showed an abrupt transition between two linear sections. This transition corresponds to an abrupt increase in the apparent Km and Vmax values with increasing NADP+, denoting negative cooperativity. The two Kms for high and low NADP+ concentrations were 0.06 mM and 0.015 mM, respectively. MWs of the isoenzymes as determined by SDS disc gel electrophoresis were 85 000–91 000 for G6PDH I and 54 000–59 000 for G6PDH IV. Gel filtration chromatography on Sephadex G-150 showed MWs of 91 000 for G6PDH I and 115 000 for G6PDH IV. A probable dimeric structure for IV is suggested, with two NADP+ binding sites.

Indoleacetic acid inhibition of a phenylalanine ammonia-lyase preparation from suspension cultures of WR-132 tobacco

Abstract The effect of indoleacetic acid, 2,4-dichlorophenoxyacetic acid, tryptophan, scopoletin, scopolin, and related compounds on the activity of a phenylalanine ammonia-lyase preparation from tobacco tissue WR-132 grown in suspension culture has been investigated. Under the experimental conditions used, IAA and its probable plant precursor trytophan, were found to inhibit the enzyme strongly. Scopoletin showed slightly lesser inhibition. The synthetic auxin, 2,4-D, produced little effect on the enzyme except at the higher concentration used. Scopolin, kinetin, gibberellic acid, and β-indole-3-propionic acid showed no effect. The concentration of β-mercaptoethanol used in each assay was found to affect the values obtained for the percentage inhibition of phenylalanine ammonia-lyase by IAA. The possible physiological significance of these inhibitions is discussed.

Effect of phenolic compounds on glucose-6-phosphate dehydrogenase isoenzymes

Abstract Effector studies with two isoenzymes (I and IV) of glucose-6-phosphate dehydrogenase (G6PDH) from tobacco suspension culture WR-132 revealed that chlorogenic acid, at 0.4 mM, inhibited both isoenzymes almost 100%, with the inhibition decreasing as the concentration of the acid was reduced. At 0.3 and 0.4 mM, the coumarin glucosides scopolin and esculin were inhibitory, whereas their aglucones scopoletin and esculetin were less inhibitory, and at low concentrations of glucose-6-phosphate (G6P), the latter two were actually stimulatory for G6PDH I. Of the possible effectors studied, only scopoletin and esculetin exhibited a significant activation of G6PDH I under these conditions. However, with G6PDH IV these two effectors do not show the same marked activation at the low G6P concentrations. The phenolic acids, caffeic and ferulic, were less inhibitory than the coumarins tested. The activation of G6PDH I by scopoletin, a compound which accumulates in tobacco under certain stress conditions, gives a possible clue as to the resulting enhanced activity of the hexose monophosphate pathway that has been reported for some plants subjected to stress conditions.

Evidence for a collagen-like protein within spicules of coelenterates

Abstract 1. Calciferous spicules were obtained fromBriareum asbestinum, a gorgonian or “soft” coral. 2. The spicules were washed extensively and treated with potassium hydroxide to remove the exterior sheath and any proteins on the outside. 3. After solubilization of the calcium carbonate with acid, an insoluble material remained. 4. Peptides with molecular weights between 1600 and 5000 were isolated from this core material. 5. These peptides had amino acid compositions characteristic of the collage like proteins.

Inhibition of glucose-6-phosphate dehydrogenase from tobacco tissue culture by scopoletin and scopolin

Abstract The pH optimum for glucose-6-phosphate dehydrogenase from tobacco tissue culture was determined to be between 8 and 9. The plot of the reaction velocity vs. glucose-6-phosphate concentration showed a non-sigmoidal, hyperbolic saturation curve. A similar curve was obtained for the NADP + saturation function. Compounds that may be related to glucose-6-phosphate metabolism were tested for their effects on the enzyme. It was found that the phenolic compounds, scopoletin, scopolin, esculin and ferulic acid, inhibited the enzyme. Similar inhibitions by scopolin and scopoletin were observed for three other NADPH producing dehydrogenases from the same plant cells; namely, 6-phosphogluconate dehydrogenase, NADP + specific isocitrate dehydrogenase and NADP + specific malate dehydrogenase.