Wolfgang Hösel

Beta-Glucosidases from Cicer arietinum L. Purification and Properties of isoflavone-7-O-glucoside-specific beta-glucosidases.

Beta-Glucosidases specific for isoflavone 7-O-glucosides have been isolated from garbanzo plants, Cicer arietinum L. These aryl-beta-glucohydrolases occur in the different organs of the plant as multiple molecular forms. The major isoenzymes of the roots, the leaves and the hypocotyl were purified to electrophoretic homogeneity. When subjected to isoelectric focussing in polyac rylamide gels the electrophoretically homogeneous glucohydrolases were found to consist of one or two major and several minor enzymically active molecular species. In roots the beta-glucohydrolase isoenzymes constitute a considerable portion of the extractable protein, so that purification to an electrophoretically homogeneous form is easily attainable. All beta-glucosidases analyzed possess molecular weights in the range of 125 000 (ultracentrifugation) to 135 000 (Sephadex G-200) and contain two subunits of molecular weight near 68 000. The pH optimum for enzymic activity is 7--7.5 with a second optimum of 4.5--5. The isoelectric points of the various species range between pH 5.9 AND 7.1. Staining for glycoprotein was positive. Kinetic analysis demonstrated a pronounced specificity of the enzymes for aromatic substrates with glucose as the sugar moiety. alpha-Glucosides as well as disaccharides were not hydrolyzed at all. Isoflavone 7-O-glucosides are the most favoured substrates with a Km of 2 x 10(-5) M, while the Km with aromatic glucosides (i.e. salicin, 4-nitrophenyl glucoside) are 100 times larger. In addition the beta-glucosidases show a pronounced specificity for glucose in the 7-position of the flavonoid nucleus. Using isoflavone aglycones as substrates glucose transferase activity was also demonstrable. The beta-glucohydrolase activity is strongly inhibited by Hg2plus. This inhibition is partially reversible and preferentially influences the Km values of the enzymes compared to V. Agplus, glucono-1,5-lactone, ethyleneglycol monomethyl ether and glycerol are only weakly inhibitory, while glucose, p-chloromercuribenzoate and Cu2plus are without effect.

Characterization of cyanogenic glucosides and β-glucosidases in Triglochin maritima seedlings

Abstract In addition to triglochinin, taxiphyllin has been detected as a cyanogenic glucoside in seedlings of Triglochin maritima. Taxiphyllin at first increases during seedling development and then decreases, whereas tri-glochinin increases to a level higher than that ever reached by taxiphyllin and remains there during further seedling development. Two β-glucosidases have also been characterized in these seedlings. One of these shows a distinct specificity for triglochinin, whereas taxiphyllin appears to be the preferred substrate of the other.

Über den umsatz von flavonolen und isoflavonen in Cicer arietinum

Abstract Pulse labelling experiments have shown that in the leaves and stems of Cicer arietinum L. the flavonols kaempferol, quercetin, and isorhamnetin are subject to turnover. The isoflavones formononetin and biochanin A are also metabolized in the green parts as has earlier been shown to occur in root tissue. Cinnamic acid-(3-14C) proved to be superior to dl -phenylalanine-(1-14C) as a precursor in pulse labelling experiments with flavonoids. The biological half lives of the compounds investigated increase in the order: formononetin, quercetin, biochanin A, kaempferol, and isorhamnetin.

Flavonoide aus Cicer arietinum L.

Zusammenfassung Aus oberirdischen Teilen von Cicer arietinum L. wurden die Flavonole Kampferol, Quercetin und Isorhamnetin sowie das Isoflavon Pratensein isoliert. Als Hauptglykoside des Kampferols wurden Kampferol-3-β-monoglukosid und das bisher unbekannte Kampferol-3-β-glukoapiosid charakterisiert.

Metabolism of Flavonoids

This chapter will deal with various aspects of flavonoid turnover and degradation in higher plants and microorganisms. The microbial dissimilation of flavonoids has been a well understood area of biochemical research for many decades. It has been part of the more general field of microbial transformations of natural aromatic compounds, where it has been shown that all organic molecules are degraded by some form of life to maintain the carbon cycle of nature. Soil and sewage microflora are specifically adapted to perform this task. The results of microbial metabolism will be discussed here to outline the chemical principles governing flavonoid dissimilation and to relate them to the fate of flavonoids in higher plants.

Relationship of coniferin β-glucosidase to lignification in various plant cell suspension cultures

A combination of the phytohormones naphthalene acetic acid and benzylaminopurine (5 μM each) allows lignification in various plant cell cultures. This system has been used to investigate the relationship between the coniferin-hydrolyzingβ-glucosidase activity and lignification. InPetroselinum hortense andTriticum aestipum cell cultures the appearance of this enzymatic activity coincided with lignification. In parsley cell cultures it was moreover shown that this activity appears concomitantly with other lignin biosynthetic enzymes. The unique enzymes of the flavonoid pathway did not appear by this phytohormone treatment. In other cell cultures investigated the correlation between the coniferin-hydrolyzing activity and lignification was not as evident as in the above two cases. This was probably due to the high activity of coniferin glucosidase already present in the normally grown cultures. Coniferinβ-glucosidase was found in all lignified cell cultures.

Characterization of a β-glucosidase from Glycine max which hydrolyses coniferin and syringin

Abstract A β-glucosidase which rapidly hydrolyses the cinnamyl alcohol glucosides coniferin and syringin has been purified from cell cultures, hypocotyls and roots of Glycine max . Isoelectric focusing in a column separated the enzyme from several other β-glucosidases which were inactive against either substrate. Syringin and coniferin were the best substrates tested. Both exhibited identical V max values, whereas the K m of coniferin (0.6 mM) was twice that of syringin (0.3 mM). The widely used synthetic substrates 4-nitrophenyl-β-glucoside and 4-methyl-umbelliferyl-β-glucoside were poorly utilized. Glucono-1,5-lactone was an effective competitive inhibitor with a K i of 0.01 mM. From the observed-substrate specificity, a role in the lignification process of higher plants may be predicted for this β-glucosidase.

Enzymatic transformation of flavonols with a cell-free preparation from Cicer arietinum L.

Abstract 1. 1. Crude enzyme preparations from Cicer arietnum L. were shown to convert the flavonols kaempferol, quercetin, datiscetin and morin in an oxygen-dependent reaction to compounds possessing ultraviolet spectra similar to dihydroflavonols. 2. 2. The enzyme reation only occurred with flavonol aglycones. 3. 3. The enzymatically formed product derived from kaempferol readily regenerated kaempferol when warmed or treated with acid and gave rise to phloroglucinol and p-hydroxybenzoic acid under alkaline conditions. These results indicated that the enzymatically formed product is based upon the flavonoid skeleton. 4. 4. Analysis of the ultraviolet, infrared and NMR spectra of the flavonol conversion products from the four flavonols proved that the new compounds are substituted 2,3-dihydroxyflavanones. 5. 5. Electrophoretic studies indicated that the hydroxyl groups at C-2 and C-3 are cis to each other.

Über den einfluss von licht auf den umsatz von flavonolen und isoflavonen in Cicer arietinum

Abstract Continuous far-red irradiation of garbanzo seedlings ( Cicer arietinum L.) leads to repression of the synthesis of the isoflavones formononetin and biochanin A. The inhibition of isoflavone formation has been shown to occur both in the roots and the aerial parts. In contrast to the two isoflavons, the flavonols kaempferol, quercetin, and isorhamnetin do not significantly accumulate in dark grown seedlings. Tracer experiments, however, have shown that the very small yields of flavonols in etiolated plants are partly due to accelerated turnover. The differences between isoflavones and flavonols regarding their formation and turnover are discussed.

Ueber den abbau von flavonolen durch pflanzliche peroxidasen

Abstract Peroxidases have been shown to catalyse the degradation of flavonols via 2,3-dihydroxyflavanones to benzoic acids. Incubation of (U- 14 C)-kaempferol with pure horseradish peroxidase leads to the same reaction products (2,3,4,5,7,4′-pentahydroxyflavanone, p -hydroxybenzoic acid, 14 CO 2 , several polar, water soluble catabolites as given by enzyme preparations from various plant species. Further reactions of flavonols and their glycosides with peroxidases are discussed. All peroxidase isoenzymes of Sinapis alba and Cicer arietinum , obtained by isoelectric focusing, have been shown to degrade flavonols at the same rate. The peroxidase catalysed degradation of polyphenols is discussed in relation to IAA oxidase.