Lipoxygenase from Medicago sativa: Purification on hydroxyapatite

Three lipid-oxidizing fractions obtained from an alfalfa extract by stepwise elution from a CM-cellulose column were further purified on hydroxyapatite columns. Although substantial separation between lipoxygenase and peroxidase activities was achieved, only one of the protein fractions migrated as a single band on polyacrylamide gel electrophoresis and exhibited the properties of pure lipoxygenase,     

Growth and betaine formation in Medicago sativa tissue cultures

Static and suspension tissue cultures of Medicago sativa were initiated from sterile seedlings, excised roots and aerial portions of the seedlings. Flask suspension cultures containing chlorophyll were also established. The various tissues were harvested after 30 days, growth rates were determined, and the tissue was later lyophilized. The lyophilized tissues from various batches were extracted separately with 70% EtOH. The betaine mixture was isolated from these extracts and from seeds of the plant as their tetraphenylborate salts. The betaines were separated from the latter complex via ion exchange and column chromatography and were identified by standard procedures. All of the tissues, regardless of origin, produced choline. Only chlorophyllous tissue biosynthesized stachydrine.     

Variable sensitivity of Trichoderma viride to Medicago sativa saponins

Eight saponins were isolated from alfalfa roots (Medicago sativa). The sensitivity of Trichoderma viride to the saponin varied with the individual saponin isolate. Seven isolates appeared to contain the aglycone, medicagenic acid, and while the other did not, it inhibited the growth of the fungus at higher concentrations than the other isolates. One pair and a triplet of saponins with divergent R_fs evoked near identical biological responses suggesting structural similarity toxic to T. viride.     

Free amino acids in Crotalaria seeds

The free amino acids of seeds of 163 species of Crotalaria have been identified. Their pattern of distribution is compared with recent classifications of the genus and the possible ecological significance of their presence is discussed. Attention is drawn to the occurrence of toxic amino acids.     

Free amino acids in the seeds of Acacia species

Free amino acids in the seeds of 80 species of Acacia have been determined. In addition to confirming the known difference in seed amino acid ‘patterns’ shown by species of the series Gummiferae on the one hand and species of the series Phyllodineae and Botryocephalae on the other, we have found that species of the series Vulgares Benth. show two additional ‘patterns’. One of these is common to some of the Afro-Asian species of the series which have been analysed and the other is common to species from America and Africa. One of the ‘marker’ amino acids of the Afro-Asian group is the neurotoxic lathyrogen α-amino-β-oxalylaminopropionic acid. The possible taxonomic and phylogenetic significance of these results is discussed, with special reference to five species A. albida, A. confusa, A. heterophylla, A. coulteri and A. kauaiensis.     

Co-occurrence of nicotianamine and avenic acids in Avena sativa and Oryza sativa

An amino acid derivative isolated from seedlings of Avena sativa and Oryza sativa, along with avenic acid A and its derivatives which possess a chelating ability with iron ions, has been shown to be nicotianamine. The co-occurrence of nicotianamine and avenic acids in the same plant, as well as their structural similarity, reveals their close biosynthetic relationship.     

Keto and imino acids in Delonix regia flowers

Floral parts and buds of Delonix regia contain α-ketoglutaric acid, oxaloacetic acid, pyruvic acid and glyoxylic acid. Proline and hydroxyproline biosynthesis has been demonstrated. The calyx and androecium accumulate glyoxylic acid, in amounts greater than those reported for other plants.     

2(S),3(S)-3-hydroxy-4-methyleneglutamic Acid from Gleditsia caspica

A new natural product, 2(S),3(S)-3-hydroxy-4-methyleneglutamic acid (G3) has been isolated from seeds of Gleditsia caspica. The structure has been established by chemical and spectroscopic methods. Catalytic reduction of G3 yields 2(S),4(S)-4-methylglutamic acid and a new amino acid, 2(S),3(S),4(S)-3-hydroxy-4-methylglutamic acid. Ozonolysis of G3 followed by oxidation gives 2(S),3(R)-3-hydroxyaspartic acid. The S- (or l-) configurations at C₂ in G3 and in 2(S),3(S),4(S)-3-hydroxy-4-methyglutamic acid and the S-configurations at C₃ for G3 and 2(S),3(S),4(S)-3-hydroxy-4-methylglutamic acid and at C₄ for 2(S),3(S),4(S)-3-hydroxy-4-methylglutamic acid are inferred from the configurations at C₂ in 2(_S),4(_S)-4-methylglutamic acid and at C₂ and C₃ in 2(S),3(R)-3-hydroxyaspartic acid. The seeds also contain appreciable quantities of 2(S),3(S),4(R)-3-hydroxy-4-methylglutami c acid (G1) and 2(S),4(R)-4-methylglutamic acid.     

Flavonoids and phenolic acids in adenostoma, a dominant genus of the californian chaparral

The flavonoid and phenolic acid pattern of Adenostoma fasciculatum and A. sparsifolium, two dominant, endemic species of the Californian chaparral, was analysed qualitatively and quantitatively. Adenostoma sparsifolium was found to secrete large amounts of lipophilic, unusually substituted flavonoids onto the leaf surface; A. fasciculatum produces five hydrophilic flavonol 3-O-glycosides of kaempferol, quercetin and isorhamnetin. The phenolic acid pattern differed quantitatively but not qualitatively between the species. The amounts of phenolic acids that could be detected within the leaves, leaf litter and soil beneath the shrubs seem too small to explain allelopathic effects as the main reason for the dominance of the two species.     

Non-protein amino acids of Acacia species and their effect on the feeding of the acridids Anacridium melanorhodon and Locusta migratoria

The leaves of Acacia species have been found to contain homoarginine, pipecolic acid and 4-hydroxy-pipecolic acid. The nymphs of the tree locust Anacridium melanorhodon, which feed on the leaves of Acacia species, were not inhibited from feeding on palatable media containing concentrations of these amino acids equivalent to, or greater than, those found in the leaves. The graminivorous Locusta migratoria was more sensitive to these compounds, inhibitory effects being observed at concentrations comparable to those found in the leaves. The inhibitory effects of mixtures of homoarginine and pipecolic acid were additive in A. melanorhodon but not in L. migratoria. Three of the non-protein amino acids found in the seeds of Acacia species, 2,3-diaminopropionic acid, 2-amino-3-acetylaminopropionic acid and 2-amino-3-oxalylaminopropionic acid, were more effective inhibitors of feeding in Anacridium than were the leaf amino acids.     

Biosynthesis of pterocarpan and isoflavan phytoalexins in Medicago sativa: the biochemical interconversion of pterocarpans and 2′-hydroxyisoflavans

Feeding experiments have shown that 2′-7-dihydroxy-4′-methoxy-isoflavone-[Me-¹⁴C] and -isoflavanone-[Me-¹⁴C] are efficient precursors of the phytoalexins demethylhomopterocarpin, sativan and vesitol in CuCl₂-treated lucerne (Medicago sativa) seedlings. Demethylhomopterocarpin-[Me-¹⁴C] was also incorporated into sativan and vestitol, and vestitol-[Me-¹⁴C] was incorporated into demethylhomopterocarpin and sativan. Thus, the pterocarpan demethylhomopterocarpin and the 2′-hydroxy-isoflavan vestitol are interconvertible in M. sativa, but incorporation data, and the results of kinetic feeding experiments with l-phenylalanine-[U-¹⁴C] suggest that these compounds are synthesized simultaneously from a common intermediate, which could be involved in the interconversion. A carbonium ion, derived from an isoflavanol, a likely intermediate in the biosynthetic reductive sequence from 2′,7-dihydroxy-4′-methoxy-isoflavone and -isoflavanone, is proposed as this common intermediate. 7-Hydroxy-2′,4′-dimethoxyisoflavone-[4′-Me-¹⁴C] was a very poor precursor of all three phytoalexins. Sativan, then, is most probably derived by methylation of vestitol. The incorporation of vestitol-[Me-¹⁴C] into demethylhomopterocarpin, but not into maackiain, pterocarpan phytoalexins of red clover (Trifolium pratense), is also demonstrated.     

Free and bound lipids of Neurospora crassa

Extraction of light and dark grown cells of Neurospora crassa with chloroform-methanol gave a free lipid extract in which the relative amounts and compositions of sterols, fatty acid and carotenoid fractions were determined. Further extraction of the cells with methanolic potassium hydroxide solution liberated a bound lipid fraction from the cells. The levels of the bound lipid fraction were much lower than those of the free lipids but analysis showed that the composition was similar to that of the free lipids.     

Biosynthesis of pterocarpan,isoflavan and coumestan metabolites of Medicago sativa: chalcone,isoflavone and isoflavanone precursors

Comparative feeding experiments in CuCl₂,- and UV-treated lucerne (Medicago sativa) seedlings have shown that 2′,4,4′-trihydroxychalcone-[carbonyl-¹⁴C] and formononetin-[Me-¹⁴C] but not 2′,4′-dihydroxy-4-methoxychalcone-[carbonyl- ¹⁴C] or daidzein-[4-¹⁴C] were incorporated into the phytoalexins demethylhomopterocarpin, sativan and vestitol, and also into 9-O-methylcoumestrol. The synthesis of 9-O-methylcoumestrol is greatly stimulated by this abiotic treatment but coumestrol production is not noticeably affected. Daidzein and the trihydroxychalcone were precursors of coumestrol. The results are interpreted in favour of a mechanism in which methylation is an integral part of the aryl migration process associated with the biosynthesisof 4′-methoxyisoflavonoids. Formononetin, 2′,7-dihydroxy-4′-methoxyisoflavone-[Me-¹⁴C], 7-hydroxy-4′-methoxyisoflavanone-[Me-¹⁴C] and 2′,7-dihydroxy-4′-methoxyisoflavanone-[Me-¹⁴C] were all excellent precursors of demethylhomopterocarpin, sativan, vestitol and 9-O-methylcoumestrol, and thus a metabolic grid may be involved in their biosynthetic origin.     

Resin acids from two amazonian species of Hymenaea

The major diterpene acid constituents of two Amazonian species of Hymenaea have been isolated and identified. A new resin acid, guamáic acid, is described.     

The seed globulins of Lupinus albus

The seed globulins of Lupinus albus were extracted and 12 ditterent proteins were separated: four of them correspond to vicilins and two to legumin     

Carotenoids in petals of Medicago falcata

Petals in three strains of M. falcata L. contain lutein 50–52%, lutein-5,6-epoxide 16–18%, chrysanthemaxanthin 9–10%, flavoxanthin 7–8%, and in smaller amounts β-carotene, ζ-carotene, a hydroxy-α-carotene-like pigment, two neoxanthins, auroxanthin and a flavoxanthin-like pigment. Almost 98% of all carotenoids were xanthophylls esterified with fatty acids. Uniformity of major carotenoid content in the three strains is consistent with their all belonging to the same species.     

Synthesis of α- and β-N-oxalyl-l-α,β-diaminopropionic acids and their isolation from seeds of Lathyrus sativus

The α- and β-N-oxalyl derivatives of l-α,β-diaminopropionic acid have been chemically synthesized and also isolated from seed extracts of Lathyrus sativus. Chemical and physical properties of the natural and synthetic isomers were in good agreement. The toxicity of the α-isomer to chicks was evaluated and compared with that of the β-isomer.     

Biosynthesis of pterocarpan,isoflavan and coumestan metabolites of Medicago sativa: The role of an isoflav-3-ene

Feeding experiments in CUCl₂- and UV-treated lucerne (Medicago sativa) seedlings have shown that demethylhomopterocarpin- [6a-³H, Me- ¹⁴C] is incorporated into vestitol and sativan without any loss of ³H label, and vestitol-[3-³H, Me- ¹⁴C] is similarly incorporated into demethylhomopterocarpin and sativan with retention of the ³H: ¹⁴C ratio. Thus, an isoflav-3-ene intermediate in the interconversion of demethylhomopterocarpin and vestitol is excluded. 7,2′-Dihydroxy-4′-methoxyisoflav-3-ene- [Me-¹⁴C] was not incorporated into the three phytoalexins, but was an excellent precursor of 9-O-methylcoumestrol, as also was 7,2′-dihydroxy-4′-methoxyisoflav-3-en-2-one- [Me-¹⁴C]. A biosynthetic pathway to coumestans via isoflav-3-enes and 3-arylcoumarins is proposed. A metabolic scheme in M. sativa interrelating eight classes of naturally occurring isoflavonoids is presented.