L-Canaline Detoxification: A Seed Predator's Biochemical Mechanism.

The seeds of the Neotropical legume, Dioclea megacarpa, the sole food source for developing larvae of the bruchid beetle, Caryedes brasiliensis, contain about 13 percent L-canavanine (dry weight). Canavanine detoxification and utilization produces L-canaline, a potent neurotoxic and insecticidal amino acid. This seed predator has developed a unique biochemical mechanism for degrading canaline by reductive deamination to form homoserine and ammonia. In this way, canaline is detoxified; canavanine's stored nitrogen is more fully utilized and its carbon skeleton is conserved.

Degradation and detoxification of canavanine by a specialized seed predator.

Larvae of the bruchid beetle Caryedes brasiliensis feed exclusively on seeds of the Neotropical legume Dioclea megacarpa, which contains 13 percent L-canavanine by dry weight. L-Canavanine, a nonprotein amino acid analog of L-arginine, exhibits potent insecticidal properties. Most of the seed nitrogen is sequestered in canavanine, and bruchid beetle larvae do not simply excrete this toxic compound. Instead, these larvae possess extraordinarily high urease activity, which facilitates the conversion of canavanine to ammonia through urea. In this way, canavanine is effectively detoxified and a supply of nitrogen for fixation into organic linkage is ensured.

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.

A novel means for dealing with L-canavanine, a toxic metabolite.

L-canavanine is a highly toxic L-arginine analog found in some leguminous seeds. Larvae of the bruchid beetle Caryedes brasiliensis, collected in Costa Rica, subsist solely on tissues of the mature seed of Dioclea megacarpa, which contains more than 8 percent L-canavanine by dry weight. The arginyl-tRNA synthetase of the bruchid beetle larvae discriminates between L-arginine and L-canavanine, and canavanyl proteins are not synthesized. In this way, bruchid beetle larvae avoid an adverse biochemical effect of L-canavanine.

Inhibition of the growth of human pancreatic cancer cells by the arginine antimetabolite L-canavanine.

L-Canavanine (CAV), the L-2-amino-4-guanidinooxy structural analogue of L-arginine (ARG), is a potent ARG antagonist which occurs in the jack bean, Canavalia ensiformis. This ARG antimetabolite is active against L1210 murine leukemia and a solid colonic tumor in the rat. Our initial studies using a microtiter assay show that CAV exhibits a 50% inhibitory concentration of approximately 2 mM against the human pancreatic adenocarcinoma cell line, MIA PaCa-2, when these cells are grown in Dulbecco's modified Eagle's medium containing 0.4 mM ARG. When the ARG concentration is reduced to 0.4 microM, the 50% inhibitory concentration for CAV falls precipitously to 0.01 mM. The pronounced increase in the ability of CAV to inhibit MIA PaCa-2 cell growth at the lower ARG concentration may result from enhanced CAV competition with ARG for incorporation into newly synthesized cellular proteins. At 0.4 microM ARG, 30 mM CAV almost completely inhibits cell growth by 6 h. In contrast, with 0.4 mM ARG, complete inhibition does not occur until after 48 h. A dramatic reversal of growth inhibition caused by a very high concentration of CAV was observed when cells treated with CAV were replenished with a high concentration of ARG. Our results suggest that CAV has real potential as a lead compound for the development of analogues with enhanced activity against human pancreatic cancer.

Growth inhibition of a rat colon tumor by L-canavanine.

The effects of L-canavanine, a higher plant nonprotein amino acid, on the growth of a rat colon carcinoma were assessed. The 1 and 10% lethal dose values following a single s.c. injection in Fischer rats were 4.75 and 5.57 g/kg, respectively. Rats received s.c. injections of a 10% (w/v) tumor cell suspension. When the tumors reached a size of 500 to 1000 mm3, the rats received canavanine, 2.0 g/kg or 3.0 g/kg s.c. daily for 5 or daily for 9 days. Control animals received a 0.9% NaCl solution. Administration of canavanine, 2.0 g/kg for 5 days produced a treated versus control of 23%; the treated versus control for 9 days was 14%. The 3.0-g/kg dosing regimen resulted in a treated versus control value of -13% after 5 days and -8% after 9 days. The negative values indicated regression of the tumor. The reduction in tumor volume, expressed as the percentage of regression, was 22% in animals receiving canavanine, 3.0 g/kg daily for 5 days and 60% in the 3.0-g/kg-daily-for-9-days treatment group. Cumulative toxicity caused death in 2 of 5 animals in the 3.0-g/kg-for-9-days treatment group; the average weight loss was 31%. The 3.0-g/kg-for-5-days treatment also produced undesirable cumulative toxicity as indicated by a weight loss of 19%. Cumulative toxicity was reduced greatly when canavanine was administered at a dose level of 2.0 g/kg for 5 days (weight loss of 13%). Analysis of the relationship of caloric deprivation to tumor growth reduction established that canavanine-mediated curtailment of tumor growth was not caused by reduced food intake and its associated loss in body weight. Histological examination of tissues from rats receiving canavanine, 2.0 or 3.0 g/kg daily for 5 or 9 days failed to reveal lesions in any of the examined tissues, except for varying degrees of pancreatic acinar atrophy. All other tissues appeared normal. The white and red blood cell values of canavanine-treated rats were also normal following 1, 3, or 6 injections of canavanine, 2.0 or 3.0 g/kg. The results indicated that canavanine induced marked growth inhibition of the rat colon carcinoma. Our experiments also disclosed that further studies must be conducted to optimize the dosing schedule to enhance drug efficacy and to reduce its cumulative toxicity.

L-homoarginine studies provide insight into the antimetabolic properties of L-canavanine.

A method for the chemical synthesis of L-homoarginine, based on the guanidination of L-lysine with O-methylisourea, has been developed; this procedure provides radiochemically pure L-[guanidino-14C]homoarginine in high yield. Radiolabeled homoarginine is incorporated readily into the newly synthesized hemolymphic proteins of larvae of the tobacco hornworm, Manduca sexta without adversely affecting larval growth and development. This finding stands in sharp contrast to the effect of L-canavanine, another L-arginine analog, which is markedly deleterious to these larvae. Homoarginine is incorporated into M. sexta lysozyme, and the antibacterial proteins of the fly, Phormia terranovae with impunity. In contrast, the comparable canavanine-containing enzymes are inhibited severely. Experimental evidence is presented that the innocuous nature of homoarginine results from the elevated pKa value of its guanidino group which arguably exceeds even that of arginine. As a result, homoarginine does not disrupt essential residue interactions. In contrast canavanine, which is much less basic than arginine, does adversely affect R group interactions forming the requisite three-dimensional conformation of the protein.

L-canaline: a potent antimetabolite and anti-cancer agent from leguminous plants.

L-Canaline, the L-2-amino-4-(aminooxy)butyric acid structural analog of L-ornithine' is a powerful antimetabolite stored in many leguminous plants. This nonprotein amino acid reacts vigorously with the pyridoxal phosphate moiety of vitamin B6-containing enzymes to form a covalently-bound oxime that inactivates, often irreversibly, the enzyme. Canaline is not only capable of inhibiting ornithine-dependent enzymic activity, but it also can function as a lysine antagonist. Recently, this natural product was found to possess significant antineoplastic in vitro activity against human pancreatic cancer cells.

L-Canavanine: a higher plant insecticidal allelochemical.

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.

Investigations of Canavanine Biochemistry in the Jack Bean Plant, Canavalia ensiformis (L.) DC: II. Canavanine Biosynthesis in the Developing Plant.

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.

Investigations of Canavanine Biochemistry in the Jack Bean Plant, Canavalia ensiformia (L.) DC: I. Canavanine Utilization in the Developing Plant.

An ontogenetic study of the canavanine and soluble protein pools in the developing jack bean plant, Canavalia ensiformis (L.) DC., was conducted. Evidence was presented which clearly established the conversion of canavanine to canaline and urea as the principal pathway of canavanine utilization. The catabolic reactions of certain bacteria involving the formation of guanidine or hydroxyguanidine from canavanine are not operative in the cotyledons of jack bean. Evidence was obtained which indicates that a second, minor reaction is functioning in canavanine degradation.

An Ontogenetic Study of Canavanine Formation in the Fruit of Jack Bean, Canavalia ensiformis (L.) DC.

An ontogenetic study of canavanine formation in the fruit of jack bean, Canavalia ensiformis (L.) DC. was conducted. Evidence was presented to show that the ovary wall is the reservoir for seed canavanine. The testa possesses sufficient canavanine to account for the continued elevation in seed canavanine after the pod senesces. The seed canavanine concentration is not constant inasmuch as the canavanine content per milligram dry weight or soluble protein increases abruptly with seed growth and levels off only with the onset of fruit ripening.

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 acts 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 precesses. 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.

A mechanism of L-canaline toxicity.

L-Canaline, a highly toxic structural analogue of L-ornithine, reacted with pyridoxal phosphate to form a stable, ninhydrin-positive complex. NMR analysis revealed the involvement of a Schiff's base in complex formation. Interaction of L-canaline with L-tyrosine decarboxylase, a known B6-containing enzyme, curtailed significantly enzyme-mediated decarboxylation. Spectral scans provided evidence that the loss in catalytic activity is associated with the reaction of canaline with the pyridoxal phosphate moiety of the enzyme. Thus, the marked antimetabolic properties of canaline are due in part of its ability to form a Schiff's-base-containing complex with the B6 moiety of the enzyme.

l-Canavanine Metabolism in Jack Bean, Canavalia ensiformis (L.) DC. (Leguminosae).

l-Canavanine, a highly toxic arginine antimetabolite, is the principal nonprotein amino acid of many leguminous plants. Labeled-precursor feeding studies, conducted primarily with [(14)C]carbamoyl phosphate, and utilization of the seedlings of jack bean, Canavalia ensiformis (L.) DC. (Leguminosae), have provided evidence for l-canavanine biosynthesis from l-canaline via O-ureido-l-homoserine. This reaction pathway appears to constitute an important in vivo route of canavanine production. Canavanine cleavage to canaline may represent a degradative phase of canavanine metabolism distinct from the anabolic reactions described above. Thus, while these reactions of canavanine metabolism bear analogy to the mammalian Krebs-Henseleit ornithine-urea cycle, no evidence has been obtained at present for the reutilization of canaline in ureidohomoserine formation.

Metabolism of l-Canavanine and l-Canaline in Leguminous Plants.

Massive accumulation of l-canavanine, the 2-amino-4-(guanidinooxy)butyric acid structural analog of l-arginine, occurs in many legumes. Accumulation of large amounts of this nonprotein amino acid results in large part from canavanine's protective efficacy; it forms an effective chemical barrier to predation, disease, and even competition with other plants. Diversion of metabolic resources for the synthesis and storage of appreciable canavanine does not place an inordinate burden on the plant. Catabolism of this nonprotein amino acid provides respiratory carbon, generates essential primary metabolites, and ammoniacal nitrogen for the developing plant.

Biochemical insight into insecticidal properties ofL-Canavanine, a higher plant protective allelochemical.

L-Canavanine manifests potent insecticidal properties in a canavanine-sensitive insect such as the tobacco hornworm,Manduca sexta (L.) (Sphingidae). Investigations of the biochemical basis for the antimetabolic properties of this arginine analog reveal that it is activated and aminoacylated by arginyl tRNA synthetase and incorporated into the nascent polypeptide chain. This creates structurally aberrant, canavanine-containing proteins that can possess altered physicochemical properties. Evidence is presented in studies with the tobacco hornworm; the canavanine-adapted bruchid beetle,Caryedes brasiliensis (Bruchidae) and the weevil,Sternechus tuberculatus (Curculionidae); as well as the canavanine-resistant larvae ofHeliothis virescens [Noctuidae] to support the contention that formation of aberrant, canavanyl proteins produce deleterious biological effects and is a significant basis for canavanine's antimetabolic properties.

Purification and characterization of the higher plant enzyme L-canaline reductase.

A newly discovered enzyme, L-canaline reductase (NADPH:L-canaline oxidoreductase, EC 1.6.6-), has been isolated and purified from 10-day-old leaves of the jack bean Canavalia ensiformis (Leguminosae). This higher plant is representative of a large number of legumes that synthesize L-canavanine, an important nitrogen-storing nonprotein amino acid. Canavanine-storing legumes contain arginase, which hydrolyzes L-canavanine to form the toxic metabolite L-canaline. Canaline reductase, having a mass of approximately 167 kDa and composed of 82-kDa dimers, catalyzes a NADPH-dependent reductive cleavage of L-canaline to L-homoserine and ammonia. This is the only enzyme known to use reduced NADP to cleave an O-N bond. Canaline reductase performs at least three important functions for canavanine-synthesizing legumes. First, it detoxifies canaline. Second, it increases by one-half the overall yield of ammoniacal nitrogen released from canavanine. Third, it permits the carbon skeleton of canavanine, a secondary plant metabolite, to support vital primary metabolic reactions.