Characterization of beta-Glucosidase Isoenzymes Possibly Involved in Lignification from Chick Pea (Cicer arietinum L.) Cell Suspension Cultures

Hösel, Wolfgang; Surholt, E.; Borgmann, Edith

doi:10.1111/j.1432-1033.1978.tb12190.x

Cited by 58 publications

(27 citation statements)

References 16 publications

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“…Other workers (17) have obtained similar data and have interpreted such results to indicate that the ,Bglucosidase is associated with the cell wall. Still other workers (9,16) have reported that cell wall fractions contain fi-glucosidases active on secondary plant products. When isolated cells of M. alba mesophyll were prepared and examined for the ,B-glucosidase, only a small fraction (7%) of the total activity was observed.…”

Section: Resultsmentioning

confidence: 99%

Subcellular Localization of 2-(β-d-Glucosyloxy)-Cinnamic Acids and the Related β-glucosidase in Leaves of Melilotus alba Desr.

Oba

Conn²,

Canut³

et al. 1981

Plant Physiol.

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The distribution of the glucosides of trans-and cis-2-hydroxy cinnamic acid and of the /8 glucosidase which hydrolyzes the latter glucoside was examined in preparations of epidermal and mesophyll tissue obtained from leaves of sweet clover (Melilotus alba Desr.). The concentrations of glucosides in the two tissues were about equal when compared on the basis of fresh or dry weight. Inasmuch as the epidermal layers account for no more than 10% of the leaf volume, the mesophyll tissue contains 90% or more of the glucosides. Vacuoles isolated from mesophyll protoplasts contained all of the glucosides present initially in the protoplasts. (12,13). One is the glucoside of 2-hydroxy-trans-cinnamic acid (o-coumaric acid) or o-coumaryl glucoside. The other is the glucoside of 2-hydroxy-cis-cinnamic acid (coumarinic acid) or coumarinyl glucoside, also known as 'bound coumarin' (12). A ,B-glucosidase which also occurs in sweet clover leaves hydrolyzes the coumarinyl glucoside (bound coumarin) but is inactive towards the other glucoside (12, 13). When clover leaves are disrupted, the glucosidase liberates coumarinic acid, which spontaneously lactonizes and forms coumarin.Recent studies have shown that dhurrin, another plant glucoside, and the enzymes catalyzing its decomposition are located in different cellular compartments (11). In this paper, we examine the localization of these two glucosides (referred to as the coumarin-glucosides) and the related ,B-glucosidase in leaves of M. alba using epidermal and mesophyll tissues, mesophyll protoplasts, and vacuoles isolated from mesophyll protoplasts. Assay of o-Coumaric Acid and Two Related Glucosides. oCoumaric acid and the two isomeric coumarin-glucosides were determined by the fluorometric method of Haskins and Gorz (5). Tissue samples (lower epidermis or mesophyll with upper epidermis attached) were ground in 95% ethanol in a mortar with a pestle and filtered through a weighed glass filter (Whatman GF/ C). The filtrates from lower epidermis and mesophyll plus upper epidermis were made to 5 and 25 ml, respectively, with 95% ethanol, and their A at 649 nm and 665 nm was measured imnmediately to determine their Chl content (22). Five ml of each alcoholic filtrate was dried in vacuo, and 5.0 ml water were added to dissolve the residue. A 1-ml aliquot of the well-mixed aqueous extract was added to 9 ml 2.5 N NaOH in a Pyrex test tube. After mixing, two 1.0-ml aliquots of the alkaline solution were withdrawn, added to 9 ml water in a test tube, and saved for fluorometric analysis to determine any o-coumaric acid present. The alkaline solution remaining (8.0 ml) was autoclaved for 45 min at 120°C to hydrolyze the coumarin glucosides present in the original sample (12). The autoclaved solution was cooled, and 1-ml samples were diluted to 10 ml with water. These and the earlier nonautoclaved diluted samples were exposed to UV light (peak near 360 nm) for 15 min at a distance of approximately 15 cm from the filter to obtain an equilibrium mixture of the cis-and trans-isomer...

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Section: Resultsmentioning

confidence: 99%

Subcellular Localization of 2-(β-d-Glucosyloxy)-Cinnamic Acids and the Related β-glucosidase in Leaves of Melilotus alba Desr.

Oba

Conn²,

Canut³

et al. 1981

Plant Physiol.

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“…However, we have shown that the so-called "null" genotypes have ␤-glucosidase activity when assayed in solution. The enzyme is not detected on zymograms, because it occurs as large quaternary complexes whose native molecular masses may exceed 1.5 ϫ 10 6 Da. Furthermore, we have shown that the null phenotype is due to a 32-kDa protein called ␤-glucosidase-aggregating factor (BGAF) (GenBank TM AAF71261), which specifically binds and aggregates maize ␤-glucosidase, yielding high molecular weight complexes (12).…”

mentioning

confidence: 99%

“…They are found in all three (Archaea, Eubacteria, and Eukarya) domains of living organisms and play important roles in various biological processes, such as degradation of cellulosic biomass, hydrolysis of glycoconjugates, cyanogenesis, and modification of secondary metabolites (1). In plants, ␤-glucosidase activity is shown to be involved or implicated in several processes, such as defense against pathogens and herbivores (2-3), hydrolysis of phytohormones (4), floral development (5), lignification, and cell wall metabolism (6). In maize, two isozymes of ␤-glucosidase have been isolated and characterized with respect to structure, including three-dimensional structure, and function (7,8).…”

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confidence: 99%

Maize β-Glucosidase-aggregating Factor Is a Polyspecific Jacalin-related Chimeric Lectin, and Its Lectin Domain Is Responsible for β-Glucosidase Aggregation

Kittur¹,

Lalgondar²,

Yu³

et al. 2007

Journal of Biological Chemistry

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In certain maize genotypes, called "null," ␤-glucosidase does not enter gels and therefore cannot be detected on zymograms after electrophoresis. Such genotypes were originally thought to be homozygous for a null allele at the glu1 gene and thus devoid of enzyme. We have shown that a ␤-glucosidase-aggregating factor (BGAF) is responsible for the "null" phenotype. BGAF is a chimeric protein consisting of two distinct domains: the disease response or "dirigent" domain and the jacalin-related lectin (JRL) domain. First, it was not known whether the lectin domain in BGAF is functional. Second, it was not known which of the two BGAF domains is involved in ␤-glucosidase binding and aggregation. To this end, we purified BGAF to homogeneity from a maize null inbred line called H95. The purified protein gave a single band on SDS-PAGE, and the native protein was a homodimer of 32-kDa monomers. Native and recombinant BGAF (produced in Escherichia coli) agglutinated rabbit erythrocytes, and various carbohydrates and glycoproteins inhibited their hemagglutination activity. Sugars did not have any effect on the binding of BGAF to the ␤-glucosidase isozyme 1 (Glu1), and the BGAF-Glu1 complex could still bind lactosyl-agarose, indicating that the sugar-binding site of BGAF is distinct from the ␤-glucosidase-binding site. Neither the dirigent nor the JRL domains alone (produced separately in E. coli) produced aggregates of Glu1 based on results from pull-down assays. However, gel shift and competitive binding assays indicated that the JRL domain binds ␤-glucosidase without causing it to aggregate. These results with those from deletion mutagenesis and replacement of the JRL domain of a BGAF homolog from sorghum, which does not bind Glu1, with that from maize allowed us to conclude that the JRL domain of BGAF is responsible for its lectin and ␤-glucosidase binding and aggregating activities.␤-Glucosidases (␤-D-glucoside glucohydrolase; EC 3.2.1.21) hydrolyze ␤-glycosidic linkage(s) in alkyl and aryl ␤-D-glucosides, glycoproteins, and glycolipids and that between two glucose residues in ␤-linked oligosaccharides. They are found in all three (Archaea, Eubacteria, and Eukarya) domains of liv-

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“…For β-D-glucosidase detection, 4-methylumbelliferyl-β-D-glucuronide was added to a final concentration of 5 mM to the resolving gel mix [39]. After electrophoresis the enzyme protein band was visualized by staining with Coomassie brilliant blue R-250.…”

Section: Purification Of β-D-glucosidasementioning

confidence: 99%

Thermostable ß-D-Glucosidase from Aspergillus flavus: Production, Purification and Characterization

2016

IJCBS

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The present study was focused on production, purification and characterization of thermostable β-Dglucosidase from the culture supernatant of Aspergillus flavus isolated from the wheat flour mill waste. Production of β-D-glucosidase was carried in shake flasks at 30 o C and pH 6.0. The results demonstrated that the β-D-glucosidase activity was high at day 6 of cultivation time, yielded 11.3 U/mL culture broth with a specific activity of 3 Units per milligram of total protein. The β-D-glucosidase purified by ammonium sulphate fractionation, followed by DEAE sepharose fast flow chromatography resulted ~2.07 fold with a final recovery of 75% and specific activity of 6 U/mg protein. The SDS-PAGE and zymogram analysis revealed monomeric protein with a molecular weight of 52.5 kDa. The enzyme was found to be stable up to temperature of 60 o C at pH 5, and retained its activity even after 30 minutes of incubation. The enzyme activity was found resistant (more than 80%) against wide range of surfactants (Tween-20, Tween-80, Triton X-100 and Sodium deoxycholate) at 0.5 and 1% concentration. Among metal ions tested at 5mM and 10 mM concentrations, Mg 2+ , Ca 2+ and Zn 2+ were not affected, whereas Cu 2+ , Hg 2+and Pb 2+ were inhibitory to activity of β-D-glucosidase. Also, the β-D-glucosidase activity (80%) was resistant to product inhibition up to 200 mM glucose and further increase in its concentration decreased the enzyme activity. Kinetic analysis indicated that K m and V max of the enzyme were 0.124 mg/ml and 118 U/ml respectively.

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Characterization of beta-Glucosidase Isoenzymes Possibly Involved in Lignification from Chick Pea (Cicer arietinum L.) Cell Suspension Cultures

Cited by 58 publications

References 16 publications

Subcellular Localization of 2-(β-d-Glucosyloxy)-Cinnamic Acids and the Related β-glucosidase in Leaves of Melilotus alba Desr.

Subcellular Localization of 2-(β-d-Glucosyloxy)-Cinnamic Acids and the Related β-glucosidase in Leaves of Melilotus alba Desr.

Maize β-Glucosidase-aggregating Factor Is a Polyspecific Jacalin-related Chimeric Lectin, and Its Lectin Domain Is Responsible for β-Glucosidase Aggregation

Thermostable ß-D-Glucosidase from Aspergillus flavus: Production, Purification and Characterization

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