Effect of Castanospermine and Related Polyhydroxyalkaloids on Purified Myrosinase from Lepidium sativum Seedlings

Abstract

Myrosinase (ft-thioglucoside glucohydrolase, EC 3.2.3.1) was purified to apparent homogeneity from light-grown cress (Lepidium sativum L.) seedlings. This enzyme, which catalyzes hydrolysis of the glucosinolate sinigrin (K,, 115 micromolar) at an optimum pH of 5.5 in sodium citrate buffer, had a native molecular weight of 130 ± 5 kilodaltons and an isoelectnc point of 4.7 to 4.9. SDS-PAGE revealed two polypeptides with molecular weights of 62 and 65 kilodaltons. values of 5 and 6 micromolar, respectively. In contrast, the related polyhydroxyalkaloids swainsonine and deoxynojirimycin were without effect upon these hydrolyses.

An unusual case of ‘uncompetitive activation’ by ascorbic acid: purification and kinetic properties of a myrosinase from Raphanus sativus seedlings

“…The difference in the apparent molecular masses of the two subunits may reflect differential glycosylation of two identical or similar polypeptides, because the larger peptide stained more weakly with Coomassie Brilliant Blue, which is known to stain glycoproteins less strongly than simple proteins. Myrosinases that have been purified from other plant sources, such as S. alba [11,19] and cress seedlings [13], have native and subunit molecular masses similar to those reported here for the daikon enzyme.…”

“…Myrosinase has been purified and characterized from several sources, including white mustard (Sinapis alba) [11,12], cress (Lepidium sati um) [13], yellow mustard (Brassica juncea) [14], rape seed (Brassica napus) [15] and wasabi (Wasabia japonica) [16]. It has also been reported to occur in the vegetative tissue of Raphanus sati us (daikon or Japanese radish) [17,18].…”

Enzymic Properties of Purified Myrosinase from Lepidium sativum Seedlings

“…After washing extensively with buffer A to remove unbound pro teins, bound proteins were eluted using a linear gradient (20 ml) of 40 m M to 225 m M NaCl in buf fer A. Fractions (1 ml) were collected and assayed for myrosinase activity. Active fractions were pooled, desalted by chromatography on a Sephadex G-25 column (44 x 2.5 cm) equilibrated with buffer A, and subjected to isoelectric focusing using a Bio-Rad Rotofor Preparative IEF Cell and Pharmalyte ampholyte carriers (pH 4 .5 -5 .4 , 2% (v/v) final concentration) as described previously [7], Focused fractions (approx. 2 ml) were assayed for myrosinase and a-mannosidase activities.…”

“…Cress myrosinase was extracted in imidazole buffer and purified to homogeneity as previously described [7] excepting that DEAE-cellulose chro m atography was replaced by FPLC as follows. The unbound proteins obtained from Reactive Red 120-Agarose chromatography were pooled and applied to a Pharmacia Mono Q H R 5/5 anion exchange column pre-equilibrated with 20 m M imidazole-HCl, pH 6.2 (buffer A).…”

“…In 1988, we reported that cell-free extracts of cress seedlings catalyzed the sulfation of desulfobenzylglucosinolate and desulfoallylglucosinolate after endogenous m yro sinase activity had been removed by Concanavalin A-Sepharose 4B chrom atography [6]. M ore re cently, we described the purification of cress m yro sinase to apparent homogeneity and its potent competitive inhibition by the indolizidine alkaloid castanospermine [7].…”