Studies on myrosinases. I. Purification and characterization of a myrosinase from white mustard seed (Sinapis alba, L.) (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Enzymology, 1973
Myrosinases (EC 3.2.3.I) are a group of isoenzymes present in Cruciferaes which catalyze the hydrolysis of glucosinolates. The myrosinases from rapeseed (Brassica napus L.) have been purified and the main component isolated and characterized. It is a glycoprotein with a molecular weight of 135 ooo which consists of two peptide chains with a molecular weight of 65 ooo and contains about 14% carbohydrate. It exists in 3 forms with different carbohydrate compositions and the isoelectric points are 4.96 , 4.99 and 5.06, respectively. This work describes the isolation and physicochemical characterization and makes a comparison between the myrosinases from rapeseed and white mustard (Sin@is alba L.).
Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties
Phytochemistry, 2009
Myrosinases (EC 3.2.1.147) are b-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes (TGG1-TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS-PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activationinhibition responses towards ascorbic acid with maximal activity around 0.7-1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-b-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed.
Food Biochemistry 5 (1) 39-61.
Myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) activity in dehulled seeds of Brassica napus cultivar Tower, B. campestris cultivar Candle and B. juncea (oriental mustard) was localized by cytochemical and biochemical procedures. It was found to be associated with the plasmalemma (plasma membrane) in the majority of embryonic cells which is contrary to the general belief that myrosinase is found only in a few specialized idioblasts called 'myrosin cells', The presence of lead and sulfur in electron-opaque deposits resulting from in situ myrosinase activity was confirmed by energy dispersive X-ray analyses. Association of myrosinase activity with the plasmalemma was also verified by fractionation and biochemical procedures. Although there were indications of some myrosinase activity in aleurone grains (protein bodies), the evidence was inconclusive.
2001
also present in seedling cotyledons. However, from this tissue, Extraction of Sinapis alba seeds under native conditions soluthey could be extracted with non-denaturing buffers. In addi-bilized 3 myrosinase isoforms, pool I, II and III, which could tion, cotyledons contained a 65-kDa MB myrosinase not be separated by ion exchange chromatography. Sequencing of found in seeds. In contrast, seedling cotyledons contained only numerous peptides of the I and III isoforms showed that they minute amounts of pool I and no pool III MA myrosinases, belonged to the Myrosinase A (MA) family of myrosinases emphasizing the tissue-specific expression of the corresponding and that they were encoded by different genes. Western blot analysis of S. alba seed proteins, extracted with a sodium gene families. Sequence analysis of myrosinase cDNAs generated cDNA by reversed transcription-polymerase chain reac-dodecyl sulphate-containing buffer, using an anti-myrosinase tion using degenerate primers with mRNA isolated from seeds, monoclonal antibody, showed the presence of two additional cotyledons and leaves confirmed the result that the MA myrosinase isoforms with approximate molecular sizes of 62 and 59 kDa. These myrosinases, which only could be solubi-isoforms were expressed only in seed tissue, while MB myrolized from seeds by inclusion of denaturing agents in the sinases were found in all tissues investigated. Furthermore, extraction buffer, were by sequence analysis identified as MB seed and leaf contained unique MB myrosinase transcripts, myrosinases. These isoenzymes or very similar forms were suggesting organ-specific expression of individual MB genes.
Characterisation of myrosinase in polish varieties of rapeseed
2006
It has been shown that the myrosinase activity in rapeseed and in white mustard seeds is similar and does not depend on the variety of rapeseed. The two molecular forms of rapeseed myrosinase with different isoelectric points, K, values towards sinigrin, response to ascorbic acid and stability were isolated. The enzyme was strongly stimulated by ascorbic acid and had the same hydrolytic potential against rapeseed glucosinolates as the myrosinase from white mustard. However, the rapeseed enzyme was much less stable during storage. The fast inactivation of myrosinase in both, flaked and intact seeds was achieved after incubation at 9&100"C and 90-100 % relative humidity. In the flaked seeds, however, a substantial amount of the endogenous glucosinolates had already been decomposed before complete inactivation of the enzyme. Thus, it is suggesting that the enzyme should be inactivated in the intact seeds before processing.
Plant Molecular Biology Reporter, 2009
Myrosinase (EC 3.2.1.147) catalyzes cleavage of glucosinolates, which consist of a thioglucoside moiety linked to amino acid-derived side chains. Myrosinase activity and expression profiles were investigated together with glucosinolate contents in Capparis ovata (caper) in order to characterize the glucosinolate-myrosinase system. The desulfoglucosinolates-glucocapparin, glucoiberin, progoitrin, epiprogoitrin, sinigrin, gluconapin, glucosinalbin, and glucobrassicin-were extracted and quantified from leaves, seeds, flowers, flower buds, and young shoots. The major desulfoglucosinolate was glucocapparin, which accumulated to values of 39.35±0.09 and 25.56±0.11 μmol g −1 dry weight in seed and leaf extracts, respectively. Myrosinase has high activity in caper seeds, leaves, flowers, and flower bud tissues having the highest total activities in seed extracts (79.23±0.18 U). However, specific activities were the highest in flower bud extracts (200.44±0.09 U mg −1 protein). The myrosinase protein migrated as a single band with a molecular weight of 65 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and on Western blots probed with the myrosinase-specific 3D7 antibodies. Native gel electrophoresis revealed two putative myrosinase isoenzymes in seeds, leaves, and flower tissues. The caper homolog of the Arabidopsis thaliana TGG1 gene was differentially expressed in seeds, leaves, flowers, and flower buds with the highest expression levels in leaves and flower bud tissues.
Industrial Crops and Products, 2013
Myrosinase (EC 3.2.3.1) found in Brassicaceae plants, is the enzyme responsible for hydrolysis of glu-cosinolates. As a result a variety of biologically active metabolites are liberated, whose importance in crop protection and especially in cancer chemoprevention is rapidly gaining recognition. The growing practical application of glucosinolate degradation products requires that sensitive and reliable methods of myrosinase activity determination in different types of plant samples are established. With the use of commercial myrosinase prep, we systematically optimised conditions of measurement of this enzyme activity by spectrophotometric and pH-stat methods. The parameters evaluated included: sample preparation, choice of substrate, its concentration, reaction temperature and detection wavelength. Two substrates with different spectral properties were chosen: sinigrin (SIN) and glucotropaeolin (GTL). For both substrates, the best reliability was achieved at reaction temperature of 37 • C and substrate concentration of 0.2 mM and 5mM for spectrophotometric and pH-stat methods, respectively. GTL exhibiting higher absorption at the recommended detection wavelength of 230 nm ensured greater sensitivity of spectrophotometric determination of myrosinase activity in the case of transparent plant samples. GTL seemed to increase also the sensitivity of pH-stat method, however, in this case homogenisation of plant samples turned out to be most important. The optimised conditions were then verified for a range of plant samples. Based on these results, the optimised protocols of myrosinase activity determination for both methods are proposed.
Plant physiology, 2002
The enzyme myrosinase (EC 3.2.3.1) degrades the secondary compounds glucosinolates upon wounding and serves as a defense to generalist pests in Capparales. Certain myrosinases are present in complexes together with other proteins such as myrosinase-binding proteins (MBP) in extracts of oilseed rape (Brassica napus) seeds. Immunhistochemical analysis of wild-type seeds showed that MBPs were present in most cells but not in the myrosin cells, indicating that the complex formation observed in extracts is initiated upon tissue disruption. To study the role of MBP in complex formation and defense, oilseed rape antisense plants lacking the seed MBPs were produced. Western blotting and immunohistochemical staining confirmed depletion of MBP in the transgenic seeds. The exclusive expression of myrosinase in idioblasts (myrosin cells) of the seed was not affected by the down-regulation of MBP. Using size-exclusion chromatography, we have shown that myrosinases with subunit molecular masses o...
Journal of agricultural and food chemistry, 2014
Myrosinase (β-thioglucosidase glucohydrolase, EC 3.2.1.147) from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50-55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis-Menten model, considering substrate inhibition, gave Vmax equal to 0.246 μmol min(-1), Km equal to 0.086 mM, and K(I) equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase.