L—canavanine, a nonprotien amino acid structurally similar to L—arginine, is potentially toxic to many insects. In northwestern Guanacaste Province in Costa Rica, most insect seed predators do not feed on seeds that contain canavanine. However, several larval Coleoptera have become specialists on canavanine—containing seeds. Three of these beetles were compared with a number of other insects with regard to their biochemical ability to deal with and utilize L—canavanine. Our analysis revealed that certain biochemical capacities required by canavanine—feeding insects may have existed prior to their exposure to dietary canavanine. See full-text article at JSTOR

Injection of L-canavanine, a naturally occurring arginine analogue, and of its metabolic derivative, L-canaline, induced almost continuous motor activity in adult tobacco hornworms, Manduca sexta (L.). Initially the moths flew normally, but after a time interval that depended both on the amino acid and on the dose (1-45/miol/g fresh weight) the moths became disorientated and muscle activity was less patterned. Canaline produced its initial effects 12-30 min after injection, whereas activity in response to canavanine began after a delay of i-z h. Canaline (derived from canavanine by an arginase-mediated hydrolytic cleavage) is probably the biologically active factor. Canaline did not affect axonal conduction of action potentials nor the activity of mechanoreceptors on the forewing. Canaline (22/miol/g fresh weight) prolonged the postsynaptic potential of flight muscle fibres, but after 20-40 min. the electrical activity of muscle fibres was normal. The results show that canaline alters the activity of the central nervous system of adult M. sexta, but its mode of action is unknown.

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 =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 ofammoniacal nitrogen released from canavanine. Third, it permits the carbon skeleton of canavanine, a secondary plant metabolite, to support vital primary metabolic reactions. L-Canavanine [L-2-amino-4-(guanidinooxy)butyric acid] is an L-arginine analog that occurs in at least 1500 legumes (1). This nonprotein amino acid can be the most abundant free amino acid of the plant (2). Arginase (L-arginine amidinohydrolase, EC 3.5.3.1) appears to be distributed universally in these canavanine-storing legumes (2). Since canaline is a product of arginase-mediated hydrolysis of L-canavanine, all of these legumes are a potential source of L-canaline [L-2-amino-4(aminooxy)butyric acid]. H2N-C(NH2)=N--O-CH2-CH2-CH(NH2)COOH L-canavanine H2N-O-CH2--CH2-CH(NH2)COOH L-canaline

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.

L-Canavanine, a potentially toxic antimetabolite of L-arginine that is stored by many leguminous plants, has demonstrative antineoplastic activity against a number of animal-bearing carcinomas and cancer cell lines. This investigation evaluated the natural abundance of this anti-cancer compound in commercially available sprouts, and in ten varieties of the seed of alfalfa, Medicago Sativa (L.). Canavanine abundance in commercially grown sprouts varied according to the source; the young plant stored appreciable canavanine that ranged from 1.3 to 2.4% of the dry matter. Alfalfa seeds were also rich in this nonprotein amino acid as the canavanine content varied from 1.4 to 1.8% of the dry matter. On average, the tested seeds contained 1.54 ± 0.03% canavanine. Alfalfa seed canavanine content was comparable to the levels found in the seeds of representative members of the genus Canavalia , which are amongst the more abundance sources of this antimetabolite.

L‐Canavanine (CAV) is a higher plant nonprotein amino acid and a potent L‐arginine antimetabolite. CAV can inhibit the proliferation of tumor cells in vitro and in vivo, but little is known regarding the molecular mechanisms mediating these effects. We demonstrated that the treatment of human lung adenocarcinoma A549 cells with CAV caused growth inhibition; G1 phase arrest is accompanied by accumulation of an incompletely phosphorylated form of the retinoblastoma protein, whose phosphorylation is necessary for cell cycle progression from G1 to S phase. In addition, CAV induces the expression of p53 and subsequent expression of a cyclin‐dependent kinase inhibitor, p21/WAF1. The p53–dependent induction of p21/WAF1 and the following dephosphorylation of the retinoblastoma protein by CAV could account for the observed CAV‐mediated G1 phase arrest.

A number of structural analogs of the antitumor compound, L-canavanine, [L-2-amino-4-(guanidinooxy)butyric acid], a delta-oxa analog of L-arginine, have been synthesized and their growth-inhibitory effects evaluated in cultured MIA-PaCa-2 pancreatic carcinoma cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results indicate that L-canavanine analogs in which the carbon chain-length and/or terminal guanidinooxy functional group has been modified elicit less growth inhibitory activity against these pancreatic cell lines than L-canavanine. On the other hand, several ester derivatives of L-canavanine have markedly enhanced growth inhibitory activity compared to L-canavanine. Thus, esterification of the carboxylic acid group constitutes an effective structural modification, which significantly amplifies the growth inhibitory properties of the parent compound against MIA-PaCa-2 cells. (C) 1997 Wiley-Liss, Inc.

A group of seven naturally-occurring, potentially toxic nonprotein amino acid antimetabolites, namely L-azetidine-2-carboxylic acid, L-homoarginine, L-canavanine, 5-hydroxy-L-lysine, L-albizzine, seleno-L-methionine, or 2-aminoethyl-L-cysteine were administered to terminal instar larvae of the tobacco hornworm, Manduca sexta [Sphingidae]. These potentially toxic secondary metabolites were provided concurrently with bacterial cell wall fragments that induce de novo-synthesis of lysozyme. Analysis of hemolymph lysozyme activity revealed that some of these compounds, such as 5-hydroxylysine, homoarginine, and albizzine, had no discernible effect on the activity of the induced lysozyme. On the other hand, providing canavanine, azetidine-2-carboxylic acid, selenomethionine, or 2-aminoethylcysteine, at the time of bacterial challenge, resulted in a significant loss of lysozyme activity.Concurrent administration of canavanine and 2-aminoethylcysteine markedly intensified their individual capacity to inhibit enzyme activity. Selenomethionine had a dramatic sparing effect on canavanine-mediated disruption of lysozyme activity. The individual deleterious effect of azetidine-2-carboxylic acid and canavanine were not exacerbated when these two toxicants were co-administered. (C) 1998 Elsevier Science Ltd. All rights reserved.

The ability of the several esters of L-canavanine, a potentially insecticidal natural product of leguminous plants, to enhance the potency of the parent compound was evaluated with the propyl, butyl, isobutyl, and octyl esters of L-canavanine. These compounds, administered either by a single parenteral injection or by dietary consumption, were tested for their intrinsic toxicity in studies conducted with terminal instar larvae of the tobacco hornworm Manduca sexta [Sphingidae]. Parenterally injected propyl or butyl esters were somewhat more toxic than canavanine, but the isobutyl and octyl esters were far more deleterious to larval growth and development. A single injection of the isobutyl or octyl ester (1.25 mg/g larva, molar equivalent of L-canavanine dose) was lethal to all the test animals before the end of the larval instar. Much of the toxicity of the octyl ester arguably was due to release of octyl alcohol, since the free alcohol is highly pernicious. In contrast, the isobutyl ester was far more toxic than isobutanol. A different picture emerged from chronic exposure to the tested esters via dietary consumption. By this administrative route, although all of the esters were marginally more toxic than canavanine, the tested compounds exhibited much less toxicity than occurred by parenteral injection. Little difference was noted in the relative growth-inhibiting activity between the esters, and all of the free alcohols exhibited little deleterious effect on larval development.

L-Canavanine, the principal nonprotein amino acid of certain leguminous plants, is a potent L-arginine antimetabolite. This natural product has demonstrative antineoplastic activity against a number of human cancers. Recent studies with MIAPaCa-2 and CFPAC have established canavanine's potential anticancer activity against these human pancreatic adenocarcinomas. Canavanine has promise as a lead compound in the development of a chemotherapeutic agent for the treatment of human pancreatic carcinoma, but it has not been adequately investigated. Greater study of canavanine and its derivatives is needed to fully realize the experimental and therapeutic value of this naturally-occurring nea-protein amino acid, and to obtain a chemotherapeutic agent of clinical value in treating human carcinomas.

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.

The tobacco budworm, Heliothis virescens (Noctuidae), a destructive insect pest, is remarkably resistant to L-canavanine, L-2-amino-4-(guanidinooxy)butyric acid, an arginine antimetabolite that is a potent insecticide for nonadapted species. H. virescens employs a constitutive enzyme of the larval gut, known trivially as canavanine hydrolase (CH), to catalyze an irreversible hydrolysis of L-canavanine to L-homoserine and hydroxyguanidine. As such, it represents a new type of hydrolase, one acting on oxygen-nitrogen bonds (EC 3.13.1.1). This enzyme has been isolated from the excised gut of H. virescens and purified to homogeneity; it exhibits an apparent Km value for L-canavanine of 1.1 mM and a turnover number of 21.1 micromol x min(-1)x micromol(-1). This enzyme has a mass of 285 kDa and is composed of two subunits with a mass of 50 kDa or 47.5 kDa. CH has a high degree of specificity for L-canavanine as it cannot function effectively with either L-2-amino-5-(guanidinooxy)pentanoate or L-2-amino-3-(guanidinooxy)propionate, the higher or lower homolog of L-canavanine, respectively. L-Canavanine derivatives such as methyl-L-canavanine, or L-canaline and O-ureido-L-homoserine, are not metabolized significantly by CH.

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 [L-2-amino-4-(aminooxy)butanoic acid] (L-CAN) and a family of eleven structurally related analogs were synthesized and evaluated for their inhibitory effect on PLP-dependent alanine aminotransferase (AlaAT) (EC 2.6.1.2) obtained from porcine heart. These congeners were selected to determine the stereochemical, aliphatic chain length, and aminooxy substitutional effects on L-CAN-mediated inhibition of AlaAT activity. L-CAN was the most effective inhibitor of the tested compounds; 10(-7) M L-CAN elicited a 55% reduction in AlaAT activity after a 5 min exposure. This deleterious effect results from the ability of L-CAN to react avidly with PLP moiety of the enzyme to form a stable, L-CAN-PLP oxime. In contrast, the methyl and ethyl esters of L-CAN reduced AlaAT activity by only 8% and 6%, respectively. While all of the L-enantiomeric forms of the tested compound were more potent AlaAT inhibitors than their corresponding D-stereoisomers, the D-enantiomers, particularly D-canaline, were active. Chain shortening or lengthening dramatically curtailed L-CAN-mediated loss in AlaAT activity, but the replacement of the alpha-amino group with a hydrogen was of little consequence in this regard. AlaAT was treated with L-CAN in the presence of free PLP to assess PLP capacity to protect AlaAT against 10(7) M L-CAN-dependent inactivation. L-CAN retained approximately two-thirds of its inhibitory ability in the presence of equimolar PLP, but AlaAT inhibition was reduced 90% by a 10-fold excess of PLP over L-CAN.

Larval tobacco budworm, Heliothis virescens [Noctuidae]are aggressive, generalist feeders that tolerate high levels of dietary l-canavanine, an insecticidal l-arginine antimetabolite; the LC50 for dietary canavanine is 300 mM. Arginine kinase (adenosine 5′ triphosphate: l-arginine phosphotransferase, EC 3.5.3.1), which mediates a phosphorylation of canavanine to yield the novel phosphagen, L-canavanine phosphate, was purified from larval gut. Kinetic parameters revealed an apparent Km value of 7.2 × 10−4 and 2.13 × 10−2M for arginine and canavanine, respectively. These Km values suggest that arginine kinase's affinity for canavanine is substantially less than its affinity for arginine. The ability of arginine kinase to metabolize canavanine accounts for the enhancement in canavanine catabolism in the presence of ATP observed in canavanine-treated larvae. Canavanine is a more effective substrate for arginine kinase than homoarginine, 2-amino-4-guanidinobutyric acid, and 2-amino-3-guanidinopropionic acid. However, all the arginine analogs were more active substrates for arginine kinase than homocanavanine and 2-amino-3-guanidinooxypropionic acid, the higher and lower canavanine analogs, respectively. When arginine kinase reacted with N-tris[hydroxymethyllmethylglycine (tricine) massive amounts of an unidentified, but ninhydrin-positive product was formed. The larvae showed no detectable loss in their ability to deal with dietary canavanine in the presence of tricine buffer.

The extraction and assay of antioxidant vitamins, such as carotenes, by traditional solvent extraction (TSE) require multiple steps, are time-consuming, and consume large amounts of organic solvent. Supercritical fluid extraction (SFE), a newer method for extracting natural products, is cost effective and eliminates toxic organic waste. The objective of this study was to optimize SFE of carotenoids from freeze-dried carrots and to compare this extraction to TSE. To optimize SFE for carotenoids, a factorial experiment was conducted. The factors assessed were the temperature of the sample chamber (30, 40, or 50 degrees C), the pressure of the extraction fluid (300, 400, or 500 atm), and cosolvent modification (5 or 10% ethanol). The optimum conditions for extraction of alpha-carotene and beta-carotene by SFE were 50 degrees C, 300 atm, and 10% cosolvent ethanol. Total provitamin A activity (alpha- plus beta-carotene) was greater in SFE than in TSE. The time required for SFE is 1 h as compared to 6 h for TSE. SFE is a reliable and improved method for extraction of carotenoids from carrot tissue.

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 CAV's 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 CAV's 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 CAV's intrinsic efficacy while decreasing the concentration of 5-FU required to produce the same cytotoxic effect.(ABSTRACT TRUNCATED AT 250 WORDS)