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Purification and characterisation of a non-plant myrosinase from the cabbage aphid Brevicoryne brassicae (L.)

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Abstract

Plant myrosinases and glucosinolates constitute a defence system in cruciferous plants towards pests and diseases. We have purified for the first time a non-plant myrosinase from the cabbage aphid Brevicoryne brassicae (L.) to homogeneity. The protein was N-terminally blocked and protease (trypsin and lys c) degradation gave peptides of which five were sequenced. The protein is a dimer with subunits of mass 54 kDa±500 Da. Western blot analysis with an anti-aphid myrosinase antibody showed a strong cross reaction with a protein extract from the Brassica specialist, B. brassicae. The anti-aphid myrosinase antibody does not cross react with plant myrosinase neither does an anti-plant myrosinase antibody cross react with aphid myrosinase.

Introduction

Glucosinolates and their degradation products are responsible for the characteristic taste and odour of crops such as horseradish, cabbage, mustard and broccoli (isothiocyanates are responsible for the ‘bite’ and pungency) and therefore in these crops the glucosinolate content is valued.

The enzyme responsible for the hydrolysis of glucosinolates is known as myrosinase (E.C. number 3.2.3.2, also known as: β-thioglucosidase, β-thioglucoside glucohydrolase). The enzyme mediated hydrolysis of glucosinolates leads to a labile aglycone, which rapidly undergoes spontaneous rearrangement, eliminating sulphur, to yield a variety of toxic metabolites such as isothiocyanates, thiocyanates, cyanoepithioalkanes and nitriles. The reaction products depend on pH and other factors such as the presence of ferrous ions, epithiospecifier protein and the nature of the glucosinolate side chain. Plant myrosinase genes have been well characterised but there have been no reports so far on genes of non-plant myrosinases (Bones and Rossiter, 1996).

There are a number of reports of myrosinase-like activity from a number of sources including fungi (Ohtsuru and Hata, 1973), intestinal bacteria (Tani et al., 1974), mammalian tissue (Goodman et al., 1959) and cruciferous aphids (MacGibbon and Allison, 1968). In 1968, McGibbon and Allison observed that goitrin (5-vinyl-2-oxazolidinethione, the hydrolysis product of 2-hydroxy-3-butenyl glucosinolate) was liberated from crushed B. brassicae which had been feeding on Brassica napus. Five other species of aphid were examined and displayed no myrosinase activity. The species of aphid examined were: Macrosiphum avanae (F.), Myzus persicae (Sulz.), Rhopalosiphum padi (L.), Aphid craccivora C. L. Koch., Macrosiphum rosae (L.). M. persicae and M. rosae are polyphagous aphids whose diet can include crucifers. The other aphids do not feed on glucosinolate containing plants. Further work by this group showed that the ‘glucosinolase’ (myrosinase) also occurs in the turnip aphid, Lipaphis erysimi and activity (in both aphids) was restricted to the head and thorax regions, although no specific internal organ could be associated with the activity. L. erysimi showed consistently lower levels of myrosinase activity than B. brassicae (MacGibbon and Beuzenberg, 1978). We have set out to purify and characterise the myrosinase with a view to examining both the biological function of the myrosinase–glucosinolate system in the aphid as well as the enzyme mechanism.

Section snippets

Purification of aphid myrosinase

Freeze-dried aphids (8.7 g) were ground in extraction buffer (20 mM Tris, 0.15 M NaCl, 0.02% azide, leupeptin (10 μg/ml) and 0.1 mM PMSF, pH 7.5). The extract was centrifuged at 12,000g for 30 min to remove solid matter and the supernatant fractionated with ammonium sulphate. The active fraction (40–60%) was run on a Sephacryl (S-200) gel filtration column in Tris buffer (20 mM Tris, 0.15 M NaCl, pH 7.5, 0.02% sodium azide) and active fractions pooled. The pooled fractions were mixed with 1 ml

Results and discussion

The myrosinase from freeze-dried aphids was purified in five steps (Table 1). Myrosinase was precipitated at 40–60% saturation with ammonium sulphate with no appreciable activity present in any other fractions. The gel filtration step (Table 1) yielded a four-fold purification while affinity chromatography on Concanavilin A removed glycosylated proteins resulting in further purification. Aphid myrosinase did not bind to the lectin concanavalin A indicating that either the protein is not

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