Isoflavonoids from Phaseolus coccineus

After treatment with CuCl₂, the following isoflavonoids have been isolated from the runner bean, Phaseolus coccineus: daidzein, genistein, isoprunetin, 2′-hydroxygenistein, phaseoluteone, 2′-hydroxydihydrodaidzein, isoferreirin, kievitone, cyclokievitone, glycinol, phaseollidin, phaseollin, demethylvestitol, phaseollinisoflavan, 2′-hydroxyisoprunetin and 7,4′-dihydroxy-5,2′-dimethoxyisoflavanone. The latter two compounds are novel natural products.     

Lectin variability in Phaseolus coccineus

Lectin variability within Phaseolus coccineus is revealed by non-denatured electrophoretic patterns and immunological labelling of total seed protein extracts, showing that the different cultivars and wild varieties studied can be classified into three main categories according to the number of isolectins (three, two or one) present in each extract. Attempts in the purification of these isolectins were performed on three different affinity systems in which ligands were thyroglobulin (known to purify the P. vulgaris isolectins), pig red cell membrane ghosts (stroma) or antibodies against the P. vulgaris cv. Contender E₂L₂ isolectin. The P. coccineus isolectins exhibit varied affinities towards thyroglobulin and stroma, the cathodic and anodic (pH 4.5) isolectins being respectively retained by the two systems, whereas the antibody affinity system is the only one able to purify the totality of the isolectins present in an extract.     

Polyoxygenated ent-kauranes and water-soluble conjugates in seed of Phaseolus coccineus

C-α and C-β, previously isolated from seed of Phaseolus coccineus, are shown respectively to be the bis-O-isopropylidene and the 16,17-mono-O-isopropylidene derivatives of ent-6α,7α,16β,17-tetrahydroxykauranoic acid. By GC-MS characterization of the products of acidic, basic and enzymatic hydrolysis, water soluble conjugates of the following compounds have been shown to occur in P. coccineus seed: GA₈, GA₁₇, GA₂₀, GA₂₈, ent-6α,7α,13-trihydroxykaurenoic acid, ent-6α,7α,17-trihydroxy-16β-kauranoic acid, ent-6α,7α,16β,17-tetrahydroxykauranoic acid, 7β,13-dihydroxykaurenolide and abscisic acid.     

Kaurene and kaurenol biosynthesis in cell-free system of Phaseolus coccineus suspensor

It is shown that suspensor tissue of Phaseolus coccineus can biosynthesize ent-kaur-16-ene and ent-kaur-16-en-19β-ol, two key precursors in the biosynthesis of gibberellins.     

Cell wall glycoproteins and polysaccharides of parenchyma of Phaseolus coccineus

Fractionation of the cell wall material of parenchyma of mature runner beans with and without chlorite-HOAc treatment, clearly showed that at least two main types of wall proteins were present. One relatively rich in hydroxyproline (HP) associated with α′-cellulose, from which most (90%) of it could be readily liberated by chlorite-HOAc treatment and the other relatively poor in HP associated with hemicellulose A. The chlorite HOAC solubilized “glycoprotein” contained a high proportion of arabinose and galactose. It was purified by PhOH-H₂O fractionation and the molar ratios of HP, arabinose, galactose, xylose, rhamnose, glucose and uronic acid in the purified glycoprotein (“glycoprotein X”) were 1:2·6:2·4:0·2:0·2:0·1:0·3. The principal amino acids of glycoprotein X were HP (43·5 mol%), serine and proline which together comprised 66 mol% of the total. These results suggest that the HP-rich wall glycoprotein is associated with cellulose microfibrils and approximates in conformation to polyhydroxyproline carrying arabinose and galactose oligosaccharide side chains.     

Isolation of legumin-like protein from Phaseolus aureus and Phaseolus vulgaris

An 11S seed globulin has been isolated from Phaseolus aureus and P. vulgaris by zonal isoelectric precipitation and the MWs of the constituent subunits determined. The protein of P. vulgaris occurs in the protein body fraction and its chemical composition, including the N-terminal amino acids and amino acid composition has been determined. The similarity between the 11S globulin of the two Phaseolus spp. and legumin from other leguines is discussed.     

Protein-polysaccharide linkages in glycoproteins from Phaseolus vulgaris

Serine and hydroxyproline participate in protein-polysaccharide linkages in hydroxyproline-poor glycoproteins from Phaseolus vulgaris cv Pinto. Most substituted hydroxyproline residues contain arabinose, galactose and glucose, but some have arabinose only. Serine residues contain arabinose, galactose and glucose.     

Attempts to demonstrate incorporation of labelled precursors into acetylcholine by Phaseolus vulgaris seedlings

The labelling of acetylcholine was investigated in bean seedlings (Phaseolus vulgaris) and hypocotyl hooks. Labelling could only be found after red light irradiation but not in the dark. Acetate was a much better precursor than choline. Glucose gave no detectable amounts of acetylcholine labelling during 24 hr of incubation. The rate of acetylcholine labelling was higher in whole seedlings than in hypocotyl hook preparations.     

Comparison of globulin proteins from Phaseolus vulgaris with those from Vicia faba

Extraction of maturing Phaseolus vulgaris seeds with an ascorbic acid—NaCI medium facilitated the preparation of two globulin fractions which wer     

GC-MS identification of endogenous gibberellins and gibberellin conjugates as their permethylated derivatives

A convenient method of preparing permethyl derivatives of GAs and GA-glucosides is described using NaH and MeI in DMF. The permethyl derivatives of the glucosides give sharp GC peaks and their MS provide information for the identification of the GA and carbohydrate moieties. The detection and characterization of endogenous GAs and GA-glycosides in pods of Phaseolus coccineus by GC-MS of permethylated extracts are described. Permethylation of carbohydrates, IAA, ABA and cytokinins by the same method is described.     

Catabolism of sulphoquinovosyl diacylglycerol by an enzyme preparation from Phaseolus multiflorus

An enzyme which will deacylate sulphoquinovosyl diacylglycerol (SQDG) has been partially purified from the leaves of runner bean (Phaseolus multiflorus). No monoacyl intermediate was observed and the acyl hydrolase was more active towards unsaturated molecular species of SQDG than towards saturated species. The major peak of activity of SQDG acyl hydrolase, separated on both DEAE-cellulose and Sephadex columns, also contained galactolipid acyl hydrolase activity. The distribution of these activities together with substrate competition and inhibitor experiments indicated that at least part of the SQDG acyl hydrolase activity was due to an enzyme that also hydrolysed galactolipids.     

Arabinogalactan from Phaseolus atropurpureus leaves

Water-soluble polysaccharide material comprising d-galactose (53·0%), l-arabinose (33·2%) and d-glucuronic acid (13·8%) has been isolated from the leaves of Phaseolus atropurpureus. Acid hydrolysis, periodate oxidation and methylation have indicated a highly branched structure. The principal interglycosidic linkages have been tentatively identified as 1,3- and 1,6-linked d-galactopyranose and 1,3-linked l-arabinofuranose residues. In synthesising polysaccharide with these structural features, P. atropurpureus differs from other legumes such as soybean, lucerne and Centrosema.     

Starch synthesis in isolated Phaseolus vulgaris chloroplasts

Isolated bean (Phaseolus vulgaris) chloroplasts were used to investigate the mode of synthesis of transitory amylose and amylopectin from ADP-glucose. Pulse chase experiments showed that labelled glucose in amylose decreased when chased with cold substrate as compared to controls. A significant portion of this decrease appeared in the amylopectin fraction indicating that amylopectin was formed from amylose. However, time course experiments showed that the rate of amytopectin synthesis is higher than that of amylose at the early stages of incubation, suggesting a certain degree of independent synthesis of the two fractions. High concentration of citrate increased the rate of amylopectin synthesis.     

Conversion of gibberellin A14 to other gibberellins in seedlings of dwarf Pisum sativum

Gibberellin A₁₄-[17-³H] applied to seedlings of dark grown dwarf pea (Pisum sativum L. cy. Meteor) was converted to GA₁, GA₈, GA₁₈, GA₂₃, GA₂₈, and GA₃₈. The sequence of interconversion of GA₁₄→ GA₁₈ → GA₃₈ → GA₂₃ → GA₁ → GA₈ is indicated. Identifications were made by gas-liquid radiochromatography using three liquid stationary phases.     

Primary metabolites of Phaseolus vulgaris nodules

More ethanol soluble material (carbohydrate and amino nitrogen) was found in both host cell and bacteroid components of Phaseolus vulgaris nodules from plants grown at 28 W/m² than from plants grown at 7 W/m². The range of compounds identified was similar at the two irradiances. On feeding ¹⁴CO₂ to the plant tops at either irradiance the labelling patterns of carbohydrates and organic acids in the nodule host cells and bacteroids suggested that any or all of the following substances could be donated by the host to the bacteroids for general metabolism: sucrose, fructose, glucose, an unidentified carbohydrate, malic acid and an organic acid co-chromatographing with 6-phosphogluconate. Distribution and labelling patterns of nodule amino compounds were consistent with the hypothesis that ammonia is the primary product of nitrogen fixation within bacteroids, and that this ammonia is transported to host cells for assimilation, initially into glutamine and glutamate.     

Properties of acyl hydrolase enzymes from Phaseolus multiflorus leaves

The properties of acyl hydrolase enzymes purified from the leaves of Phaseolus multiflorus have been studied. Hydrolase I which deacylates phosphatidylcholine and oleoylglycerol had a pH optimum towards phosphatidylcholine of 5.3. Hydrolase II which deacylates glycosylglycerides and oleoylglycerol showed pH optima of 7.3 (monogalactosyldiglyceride, MGDG) and 4.3 (sulphoquinovosyldiglyceride, SQDG). Both enzymes showed activity peaks towards oleoylglycerol at pH 6.8 and 8.8. Unesterified fatty acids and Triton X-100 inhibited the rate of SQDG hydrolysis while bovine serum albumin increased activity. An apparent K_m for SQDG of 0.15 mM was found. Hydrolase II catalysed transmethylation of liberated fatty acids during the hydrolysis of oleoylglycerol when methanol was included in the assay system. A number of salts inhibited SQDG hydrolysis but their effect on oleoylglycerol was less consistent. The position of ester cleavage of oleoylglycerol was determined by the use of H₂¹⁸O. Cell-free extracts from P. multiflorus leaves degraded SQDG as far as sulphoquinovose.     

Purification of acyl hydrolase enzymes from the leaves of Phaseolus multiflorus

Acyl hydrolase activities have been purified from the leaves of Phaseolus multiflorus. The purification procedure involved heat treatment, DEAE-cellulose chromatography, Sephadex G-100 filtration and hexyl agarose chromatography. The elution pattern from hexyl agarose columns together with substrate competition experiments indicated the presence of two hydrolase enzymes. The first could hydrolyse oleoylglycerol and phosphatidylcholine while the second would deacylate glycosylglycerides and oleoylglycerol. Overall purification of both enzymes was ca 70-fold and the MW of the glycosylglyceride-hydrolysing enzyme was in the range 70–78000.     

Structural analysis of the xyloglucan from Phaseolus coccineus cell-walls using cellulase-derived oligosaccharides

Oligosaccharides, obtained by digestion of a xyloglucan from the cell walls of Phaseolus coccineus with cellulase, have been isolated by gel filtration. Oligosaccharides containing 2–6 residues accounted for ～57% of the hydrolysate, with larger oligosaccharides (d.p. ～10) and partially degraded xyloglucan accounting for ～32% of the polymer. The major glycosidic linkages were determined by methylation analysis. Methylated penta- and hexa-saccharide alditols were isolated by reverse-phase h.p.l.c. and characterised by e.i.-m.s. and f.a.b.-m.s. Methylated derivatives of the di-, tri-, and tetra-saccharide alditols were examined by g.l.c.-m.s. in the e.i. and c.i. modes. A structure based on these results is proposed.     

Metabolism of phaseollin by Septoria nodorum and other non-pathogens of Phaseolus vulgaris

Phaseollin is metabolised by cultures of Septoria nodorum, a non-pathogen of bean, into cis and trans isomers of 12,13-dihydrodihydroxyphaseollin. These products are much less fungitoxic than phaseollin which suggests that the capacity to detoxify phytoalexins is not confined to pathogenic fungi.     

A comparison of the thermal stability and substrate binding constants of prolyl-tRNA synthetase from Phaseolus aureus and Delonix regia

Pro-tRNA synthetase from P. aureus and D. regia was protected against thermal denaturation by various substrates; the kinetics of this protection was investigated. The affinity of substrates for each synthetase was studied by a thermal inactivation technique. In the presence of ATP, Pro and several Pro-analogues were bound to each enzyme more efficiently than when ATP was absent. The efficiency of imino acid analogue binding, relative to that of Pro, was greater when ATP was absent. Pyrrolidine and 3-pyrroline were able to bind to the enzyme only in the presence of ATP. The ratio of the ATP/Pro binding constants for the Delonix enzyme was greater than that for the Phaseolus enzyme. Values for several thermodynamic parameters involved in substrate binding were determined for each synthetase. The results are discussed in relation to the order of substrate binding and the known differences in substrate specificity between the enzymes from P. aureus and D. regia.