A fast and gentle method for the isolation of myrosinase complexes from Brassicaceous seeds.
Myrosinase is a beta-thioglucosidase glucohydrolase that catalyses the hydrolysis of the thioglucoside bond in glucosinolates, allelochemicals present in Brassicaceous plants. These isoenzymes have been found to form complexes with other proteins; however, traditional isolation procedures involving ammonium sulphate precipitation and/or ion exchange chromatography do not allow for the isolation of these complexes. The present paper reports a fast and gentle procedure for the isolation of myrosinases in the complex form. Partial purification by Con A affinity chromatography followed by Sephadex G-200 gel filtration allowed for the isolation of myrosinase complexes from seeds of Brassica carinata, B. oleracea var. capitata, B. napus and Sinapis alba. Myrosinases in the Brassicas formed complexes of different molecular weight (500-600 kDa, 270-350 kDa and 140-200 kDa) whereas in seeds of S. alba it was only possible to isolate and detect 140-200 kDa complexes. In all species the complexes were formed by isoenzymes with isoelectric points between 4.8 and 5.6 and in some cases up to 6.8. SDS-PAGE confirmed that the myrosinase isoenzymes were composed by several protein subunits of molecular weights ranging between 10 and 110 kDa. The relative amount and enzymatic activity of the myrosinase complexes varied amongst the species studied. The isolation of myrosinase complexes in their native form is of great importance for the study of the hydrolysis of glucosinolates under autolysis conditions.
Bellostas
Natalia
N
Department of Natural Sciences, Biochemistry and Natural Product Chemistry, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Denmark.
Petersen
Iben Lykke
IL
Sørensen
Jens Christian
JC
Sørensen
Hilmer
H
D019607:Brassicaceae / Q000201:enzymology*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D007525:Isoelectric Focusing; D008970:Molecular Weight; D011485:Protein Binding; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D013997:Time Factors
D021122:Protein Subunits; D006026:Glycoside Hydrolases; C022739:thioglucosidase