Mitsuhiko Satô

Purification and Properties of Flavin- and Molybdenum-Containing Aldehyde Oxidase from Coleoptiles of Maize

Aldehyde oxidase (AO; EC 1.2.3.1) that could oxidize indole-3-acetaldehyde into indole-3-acetic acid was purified approximately 2000-fold from coleoptiles of 3-d-old maize (Zea mays L.) seedlings. The apparent molecular mass of the native enzyme was about 300 kD as estimated by gel-filtration column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was composed of 150-kD subunits. It contained flavin adenine dinucleotide, iron, and molybdenum as prosthetic groups and had absorption peaks in the visible region (300–600 nm). To our knowledge, this is the first demonstration of the presence of flavin adenine dinucleotide and metals in plant AO. Other aromatic aldehydes such as indole-3-aldehyde and benzaldehyde also served as good substrates, but N-methylnicotinamide, a good substrate for animal AO, was not oxidized. 2-Mercaptoethanol, p-chloromercu-ribenzoate, and iodoacetate partially inhibited the activity, but well-known inhibitors of animal AO, such as menadione and estradiol, caused no reduction in activity. These results indicate that, although maize AO is similar to animal enzymes in molecular mass and cofactor components, it differs in substrate specificity and susceptibility to inhibitors. Immunoblotting analysis with mouse polyclonal antibodies raised against the purified maize AO showed that the enzyme was relatively rich in the apical region of maize coleoptiles. The possible role of this enzyme is discussed in relation to phytohormone biosynthesis in plants.

Metabolism of phenolic substances by the chloroplasts-II. : Conversion by the isolated chloroplasts of p-coumaric acid to caffeic acid☆

Abstract The isolated chloroplasts of Saxifraga stolonifera are able to oxidize p-coumaric acid to caffeic Acid. This activity is also found in the chloroplasts of several other plants, and its possible role in the biogenesis of polyphenols is discussed.

The latency of spinach chloroplast phenolase

Abstract The latent phenolase in spinach chloroplast membranes could be activated by treatment with various detergents. Examination by thin-layer gel filtration showed the presence of two active proteins (one with lower MW called protein A and the other, protein B). The protein B was converted to A by dilution or on standing, and the latter conversely to the former by concentration. On freezing, an extract of the acetone powder of the chloroplasts, phenolase activity was strikingly reduced, and this is ascribed to an association of the protein A and a low MW (diffusible) substance giving rise to an inactive enzyme-inhibitor complex. The activity declined from autumn to winter, and it appears that the second type of latency due to the formation of the above complex is also involved.

Involvement of peroxidase in differential sensitivity to γ-radiation in seedlings of two Nicotiana species

Abstract Mechanisms underlying differential radiosensitivity were investigated using seeds and seedlings from two Nicotiana species, Nicotiana debneyi and Nicotiana tabacum with 60 Co γ -radiation up to 500 Gy. A contrasting response to radiation was found between the seeds and seedlings in these species. Seeds were more radiosensitive in N. debneyi than in N. tabacum with respect to germination rate and subsequent seedling development. Conversely, irradiation of seedlings damaged N. tabacum more than N. debneyi , as revealed by inhibition of further seedling growth, chlorophyll degradation, and abnormalities in morphology. Subsequent experiments on seedlings revealed that the amount of the radical, OH ⋅ , generated in irradiated tissues did not appear to differ between the species or with increasing doses of γ -radiation and that endogenous antioxidants which might eliminate radicals non-enzymatically were not altered by irradiation in either species irrespective of radiation doses. Although radiation induced increases in the activities of some antioxidant enzymes (peroxidase, catalase and superoxide dismutase) and changed isozyme patterns, none of these features could explain the difference in response to radiation in these plants. However, activity of peroxidase in non-irradiated tissues was 5-fold higher in N. debneyi than in N. tabacum . It was thus suggested that the difference in response to radiation between these two species was ascribed to the difference in the potential activity of this enzyme to remove toxic hydrogen peroxide which was produced in irradiated tissues.

The conversion of phenolase of p-coumaric acid to caffeic acid with special reference to the role of ascorbic acid

Abstract Caffeic acid was isolated as a product of p -coumaric acid oxidation catalysed by phenolase. Time course of p -coumaric acid conversion was studied in the absence and presence of ascorbic acid, and caffeic acid accumulation was found to be correlated with p -coumaric acid disappearance in the ascorbic acid-containing system. In the above reaction, p -coumaric acid undergoes a coupled oxidation with ascorbic acid as follows: p -coumaric acid+ascorbic acid+O 2 → caffeic acid. The effect on the initial reaction stage (lag phase) of concentrations of enzyme, monohydric phenol and ascorbic acid was studied and favourable conditions for obtaining caffeic acid in quantity are discussed.

Metabolism of phenolic substances by the chloroplasts—III. : Phenolase as an enzyme concerning the formation of esculetin☆

Abstract The chloroplasts of Saxifraga stolonifera are able to convert cis -caffeic acid to esculetin. This reaction is enzymic and the enzyme responsible is phenolase ( ortho -diphenol: O 2 oxidoreductase, EC. 1.10.3.1). An ortho -quinone produced from cis -caffeic acid by the enzyme may convert spontaneously to esculetin. The results present a new explanation for the first step in the formation of the lactone ring of esculetin.

Inhibition by oxalates of spinach chloroplast phenolase in unfrozen and frozen states

Abstract Spinach chloroplast phenolase was inhibited by oxalic acid and its salts. Complete inhibitions were induced instantly in the acidic region (e.g. by 1 and 5 mM oxalate at pH 5 and 5.5, respectively), and in the neutral region pre-incubation of the enzyme with oxalates could also lead to complete loss of activity. The inhibition mode was non-competitive for phenol substrate with K i of 0.9 mM pH 6.8. Reduction of enzyme activity in a crude extract of chloroplasts induced by freezing at neutral pH was due to the presence of ammonium oxalate. With 0.5 mM oxalate, the inhibition attained 75% under frozen conditions, whilst no inhibition could be detected in the enzyme which had not been frozen. Free oxalic acid and K + and Na + salts also caused freezing inhibition. Glyoxylic and oxamic acids acted as inhibitors with less efficiency. With a pure mushroom tyrosinase (phenolase), essentially the identical results were obtained using the same conditions.

BIOSYNTHESIS OF DIHYDROXYCOUMARINS IN DAPHNE ODORA AND CICHORIUM INTYBUS

Abstract The biosynthesis of daphnin and daphnetin-8-glucoside in Daphne odora and of cichoriin in Cichorium intybus was studied using labelled cinnamic acids as precursors, and it is suggested that these compounds are produced mainly via p-coumaric acid, but not via caffeic acid.

A transglucosylase in Daphne odora converting daphnin to daphnetin 8-glucoside

Abstract The hydrolysis of daphnin (daphnetin 7-glucoside) and the transglucosylation from this glucoside to daphnetin forming daphnetin 8-glucoside have been studied using an enzyme preparation from the flowers of Daphne odora . Both the reactions seem to be controlled by the same enzyme having a high substrate specificity. Some other properties of this enzyme are described.

Reactivation by copper of phenolase pre-inactivated by oxalate

Abstract The activity of spinach chloroplast phenolase which had been repressed by ammonium oxalate was restored by adding copper. Oxalate appears to bind to the enzyme at a single site, the binding paralleling the inhibition produced at neutral pH. The inhibition of oxalate is due to its binding with copper at the active centre to form an inactive complex, the oxalate moiety of which is releasable when more copper is added. Similar reactivation by copper was obtained with pure mushroom phenolase.