Chapter 9
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ß-Glucosidase (Linamarase) of the Larvae of the Moth Zygaena trifolii and Its Inhibition by Some Alkaline Earth Metal Ions Adolf Nahrstedt and Elisabeth Mueller Institut für Pharmazeutische Biologie und Phytochemie, Westf., WilhelmsUniversität, D-4400 Münster, Germany
ß-Glucosidases involved in the metabolism of endogeneous secondary constituents have rarely been found in insects. One example is the ß -glucosidase(linamarase) of the larvae of Zygaena trifolii (Zygaenidae, Lepidoptera) which is a glycoprotein and co-occurs with its substrates, the cyanogenic glucosides linamarin and lotaustralin, in the hemo lymph of the insect. In vitro experiments showed a noncompetitive or mixed type inhibition of the linamarase by the alkaline earth metal ions Mg and Ca . When fully activated by chelating agents the linamarase causes strong cyanogenesis (liberation of HCN) in the hemolymph. Lowering the pH from its physiological value of 6.2 in a non-chelating buffer to 3.6 causes also almost full cyanogenesis. Mg and Ca ions occur at 18 and 7 mM, respectively, in the hemolymph; the inhibitor constants (K ) are 5 m M for Mg and 80 m M for Ca . The data suggest that both ions are natural inhibitors of the linamarase in the intact insect. ++
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Although B-glucosidases capable to catalyze the hydrolyzation of glycosidic seconda ry metabolites to aglykones and glucose are well known from many plants (7-5) and microorganisms (4) their occurrence and characterization in insects are rarely docu mented (5). Most of such B-glucosidases have been obtained from the intestinal tract of several insects; they possess in vitro activities towards cellobiose (indicating cellulose degrading activity), or towards salicin, helicin, amygdalin and arbutin, which are usually used to demonstrate B-glucosidase activity; but they are seldom the natural substrates originating from the food, by sequestration or by de novo-synthesis within the insect (5-8). Especially xylophagous insects have B-glucosidases that hydrolyze cellulose (cellobiose) (5,9) for energy supply. Subspecies of Papilio glaucus (Lepidoptera) adapted to feed on plants rich in phenolics contain higher activity of 1,4-B-glucosidase to degrade salicin and salicortin more efficiently than do
0097-6156/93/0533-0132$06.00/0 © 1993 American Chemical Society
Esen; ß-Glucosidases ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
9. NAHRSTEDT & MUELLER
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non-adapted populations (10). Other insects are able to select their food plant in that they hydrolyze glycosidic allelochemicals of their host as it was observed for the peachtree borers (77) which metabolize the cyanogenic glucoside prunasin; this activity was also inducible during feeding on an amygdalin containing diet (77). Such examples demonstrate the advantage of endogeneous B-glucosidases for adapted insects making them able to metabolize exogeneous more or less toxic metabolites for their own benefit. For the "normal" non-adapted feeder, however, numerous plant glycosides possess antifeedant, oviposition deterrent or toxic activities towards herbivorous insects produced by their corresponding aglykones as has been observed for glucosinolates (72), cyanogenic glycosides (75) or cardiac glycosides (14). It is, however, not clear how far exogeneous B-glucosidases of the host or endogeneous Bglucosidases of the herbivore contribute to the liberation of the respective aglykone. Only a few reports deal with B-glucosidases involved in the metabolism of endogeneous secondary metabolites of insects. Cockroaches (Blatta orientalis, Periplaneta americana) produce a milky secretion in their left colleterial gland that contains the 4-O-glucoside of 3,4-dihydroxybenzoic acid, a polyphenol oxidase and a structural protein. The right gland, however, produces a clear secretion containing a B-glucosidase activity. When both secretions are mixed free protocatechuic (3,4dihydroxybenzoic) acid is released from its glucoside and is used for the production of sclerotin after oxidation and reaction with the supplied protein to form the ootheca (75). Some members of the Diptera (16,17) and Lepidoptera (18-21) produce B-Dglucopyranosyl-O-L-tyrosine that is primarily synthesized during the late larval feeding period and accumulates in highest levels prior to ecdysis; tyrosine is released from its glucoside and used to produce quinones and catechols to tan the new cuticle; in Manduca sexta hydrolysis is catalyzed by a B-glucosidase, that is preferentially localized in the fat body and is under the control of 20-hydroxyecdysone (22). Cyanogenesis in Zygaena Species A l l hitherto investigated species of the lepidopteran moth family Zygaenidae, several species of the butterfly family Nymphalidae and some of the Lycaenidae are cyano genic (for review see 25 and 24). With the exception of Acraea horta (Nymphalidae) (25) all species investigated so far contain the cyanogenic glucosides linamarin and lotaustralin or linamarin alone (Figure 1). One of the best investigated species of the cyanogenic Lepidoptera is Zygaena trifolii (Esper 1783) whose larvae were used for many experiments. It was shown that linamarin and lotaustralin are not only synthesized de novo by the larvae starting from valine and isoleucine as biogenetic precursors (24) in close similarity to the pathway used by higher plants (26), but it was also demonstrated that the larvae are able to sequester both glucosides from their host, Lotus corniculatus (Fabaceae), which also accumulates linamarin and lotaustralin (27). When the larvae are injured hydrogen cyanide is liberated indicating the activity of degrading enzymes such as B-glucosidases and, eventually, hydroxynitrile lyases (HNLs) as is well known from cyanogenic higher plants (5): When hydrolyzed by a B-glucosidase the substrates linamarin and lotaustralin are converted to the corresponding cyanohydrins (hydroxynitriles) acetone cyanohydrin and methylethyl
Esen; ß-Glucosidases ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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/9-GLUCOSIDASES: BIOCHEMISTRY AND MOLECULAR BIOLOGY
(D H
H 0 2
3 R - H linamarin R - C H lotaustralin
(2)
3
[1} 2)
0-glucosIdase hydroxynltrile lyase
+ HCN vn-t
Figure 1. Degradation of linamarin and lotaustralin catalyzed by a B-glucosidase and a hydroxynitrile lyase, so called cyanogenesis.
ketone cyanohydrin; both decompose spontaneously at pH values above ca. 5.5 to give acetone, methylethyl ketone and hydrogen cyanide (Figure 1). Thus, for cyanogenesis a B-glucosidase is sufficient; HNLs, however, accelerate the final step of cyanogenesis up to 20-fold as observed for the H N L of Hevea brasiliensis (Euphorbiaceae) (28) and the H N L of Zygaena trifolii (29). When testing the different tissues of a dissected larva of Z. trifolii, using the endogeneous substrate linamarin, it became evident that both enzyme activities are present preferentially in the hemolymph whereas other organs contain small or negligible amounts of activity (Table 1). Thus, the larvae possess the entire cyanogenic system as do cyanogenic higher plants. Both enzymes, the B-glucosidase (30) and the hydroxynitrile lyase (29, Mueller and Nahrstedt in preparation) have been isolated from the hemolymph, purified and characterized.
Table I. Distribution of Cyanoglyucosides, B-Cyanoalanine (B-CNA), Activity of Linamarase, Hydroxynitrile Lyase (HNL) and B-Cyanoalanine Synthase (8CAS) in a Larval Body of Zygaena trifolii
Organ Hemolymph Integument Secretion Fat body Gut epithel Gut content Malpighii Silk gland
Cyanoglucosides 33.3% 65.8% 19.6% 0.25%
