Gerald A. Rosenthal

The Biological Effects and Mode of Action of L-Canavanine, a Structural Analogue of L-Arginine

Many of the 200 or so non-protein amino acids synthesized by higher plants are related structurally to the constituents of common proteins. L-Canavanine, the guanidinooxy structural analogue of L-arginine, is representative of this group. It has provided valuable insight into the biological effects and the mode of action of non-protein amino acids which act as analogues of the protein amino acids. The arginyl-tRNA synthetases of numerous canavanine-free species charge canavanine, and canavanine is subsequently incorporated into the nascent polypeptide chain. Production of canavanine-containing proteins ultimately can disrupt critical reactions of RNA and DNA metabolism as well as protein synthesis. Canavanine also affects regulatory and catalytic reactions of arginine metabolism, arginine uptake, formation of structural components, and other cellular processes. In these ways, canavanine alters essential biochemical reactions and becomes a potent antimetabolite of arginine in a wide spectrum of species. These deleterious properties of canavanine render it a highly toxic secondary plant constituent that probably functions as an allelochemic agent that deters the feeding activity of phytophagous insects and other herbivores.

Preparation and colorimetric analysis of l-Canavanine

Abstract Procedures for the preparation and colorimetric assay of l -canavanine, a structural analog of l -arginine, are presented.

L-Canavanine: a higher plant insecticidal allelochemical.

Summary.l-Canavanine, l-2-amino-4-(guanidinooxy)butyric acid, is a potentially toxic nonprotein amino acid of certain leguminous plants. Many species are prolific canavanine producers; they divert enormous nitrogen resource to the storage of this single natural product. Canavanine, a highly effective protective allelochemical, provides a formidable chemical barrier to predation and disease.The accumulated experimental evidence leaves little doubt that the key element in the ability of canavanine to function as an effective protective allelochemical is its subtle structural mimicry of arginine which makes it an effective substrate for amino acid activation and aminoacylation, and its marked diminution in basicity relative to arginine which mediates the production of structural aberrant, dysfunctional canavanyl proteins.The biological burdens of canavanyl protein formation by canavanine-treated Manduca sexta larvae were carried throughout their remaining life cycle. Protein-based sequestration of canavanine prevented turnover and clearance of the free amino acid, and undoubtedly contributed significantly to the antimetabolic character of this protective allelochemical.

The biochemical basis for the deleterious effects of l-canavanine

Abstract l -Canavanine, l -2-amino-4-(guanidinooxy)butyric acid, is a potentially toxic analogue of l -arginine. Canavanine-sensitive organisms activate and aminoacylate this non-protein amino acid and thereby create structurally aberrant, canavanine-containing proteins. Incorporation of canavanine into protein can alter the conformation and disrupt the function of the native macromolecule. Production of functionally impaired, canavanyl proteins affects developmental processes and contributes significantly to the expression of canavanines potent antimetabolic properties in insects.

Phototoxicity of the allelochemical, α-terthienyl, to larvae of Manduca sexta (L.) (Sphingidae)

Abstract α-Terthienyl, a common chemical constituent in the Asteraceae, was administered to fifth-stadium larvae (Day 1) of the tobacco hornworm, Manduca sexta (L.) (Sphingidae), by incorporation into artificial diet and by topical application. Toxicity toward larvae was elicite by simultaneous exposure to this allelochemical and uv-A irradiation (320–400 nm). Larval treatment with either α-terthienyl or uv-A irradiation alone caused no apparent adverse effects. A single, ingested dose of α-terthienyl (50 μg g larval weight −1 ), followed by irradiation for 4 hr, resulted in delayed and abnormal pupal formation with no subsequent adult emergence. Topical application of the plant compound (50 μg g −1 ) and uv-A irradiation led to tissue necrosis that affected both sclerotization and melanization of the pupal case in later development.

Non-protein amino acid-insect interactions—I. Growth effects and symptomology of l-canavanine consumption by tobacco hornworm, Manduca sexta (L.)

Abstract 1. 1. Incorporation of l -canavanine, a naturally occurring structural analogue of arginine, into the artificial diet of the tobacco hornworm larvae, Manduca sexta (L.), produced severely toxic effects. 2. 2. The toxic effects included enhanced larval mortality, decreased larval growth rates and malformed pupae and adults. 3. 3. Larval response to canavanine was concentration dependent at least over a range of 3–45 mM. 4. 4. Diets supplemented with arginine, ornithine, glycine and lysine were not toxic to fifth instar hornworms.

Combination therapy with 5-fluorouracil and L-canavanine: in vitro and in vivo studies.

L-Canavanine (CAV) is a potent L-arginine antagonist, produced by legumes such as the jack bean, Canavalia ensiformis. CAV is cytotoxic to MIA PaCa-2 human pancreatic cancer cells. We sought to determine whether CAVs efficacy as an anticancer agent might be increased in combination with 5-fluorouracil (5-FU), a pyrimidine antimetabolite with activity against solid tumors. Using optimal conditions for the expression of CAVs cytotoxicity against MIA PaCa-2 cells, CAV was more cytotoxic to the cells than 5-FU. The combination of both drugs at a fixed molar ratio of 1:1 exhibited synergistic effects in the cells as determined by combination index analysis. The combination of 5-FU:CAV was tested at a ratio of 5:1 and exhibited antagonism at lower effect levels, additivity at 50% effect levels and slight synergism at higher effect levels. A 10:1 combination of both drugs (5-FU:CAV) exhibited antagonistic effects at all levels. When the drugs were combined at a molar ratio of 20:1, increased antagonism was observed. When CAV (1.0 or 2.0 g/kg daily) and/or 5-FU (35 mg/kg daily) was administered to colonic tumor-bearing rats for five consecutive days, the antitumor activity of the drug combination was significantly greater than the combined effects of either drug alone. However, the body weight loss experienced by CAV-treated rats was increased in those rats exposed to a combination of both drugs. These studies using different tumors provide in vitro and in vivo evidence that combination therapy offers a viable means of improving CAVs intrinsic efficacy while decreasing the concentration of 5-FU required to produce the same cytotoxic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

L-Canavanine, a Dietary Nitrogen Source for the Seed Predator Caryedes brasiliensis (Bruchidae).

Larvae of the bruchid beetle Caryedes brasiliensis (Bruchidae) develop entirely within the seed of the neotropical legume Dioclea megacarpa. The seed contains an appreciable concentration of L-canavanine, a potent antimetabolite and structural analog of L-arginine. This bruchid beetle uses the nitrogen stored in this toxic allelochemical as an effective dietary nitrogen source for amino acid biosynthesis.

The preparation and colorimetric analysis of l-canaline

Abstract A method for the isolation of l -canavanine from the seed of jack bean, Canavalia ensiformis (L.) DC., is described. Procedures for the enzymatic preparation of l -canaline by hydrolysis of l -canavanine have been developed. A new colorimetric assay for canaline is presented.

Investigations of Canavanine Biochemistry in the Jack Bean Plant, Canavalia ensiiformis (L.) DC.

The canavanine content of developing leaves of jack bean, Canavalia ensiformis (L.) DC., increases during leaf development. The leaf possesses the enzymes required for synthesizing canavanine by a cyclic series of reactions analogous to the ornithine-urea cycle. This reaction series involves the sequential formation of canaline, O-ureidohomoserine, and canavaninosuccinic acid.