The relationship of the pyridine nucleotide cycle to ricinine biosynthesis in Ricinus Communis

Quinolinic acid-6-¹⁴C feeding experiments were performed using Ricinus communis L. plants to determine the efrect of selected inhibitors on the incorporation of this precursor into the alkaloid, ricinine, and into the pyridine nucleotide cycle intermediates. Administration of azaserine and azaleucine resulted in a decrease in the incorporation into ricinine and an increase in the radioactivity remaining in quinolinic acid. Administration of excess ricinine and ethionine yielded similar results. This mutual inhibition indicated an interdependency between the conversion of quinolinic acid to ricinine and to the pyridine nucleotide cycle intermediates.     

Homoserine dehydrogenase in Pisum sativum and Ricinus communis

Homoserine dehydrogenase was extracted from Ricinus communis and Pisum sativum. The kinetic parameters of the forward and reverse reactions were determined. In the forward reaction only the enzyme from Ricinus is inhibited by threonine. The response to K⁺ is different for the enzyme from the two sources.     

Alkaloid biosynthesis in Croton linearis

The ability of Croton linearis to form crotonosine from linearisine has been demonstrated; the extent of this conversion appears to vary according to the sex of the plant.     

Biosynthesis of crotsparine,crotsparinine and sparsiflorine

The biosynthesis of crotsparine, crotsparinine and sparsiflorine in Croton sparsiflorus has been studied using racemic [Ar-³H]-coclaurine, isococlaurine and norcoclaurine. Tyrosine and coclaurine are shown to be precursors of all three alkaloids.     

Biosynthesis of securinine,the main alkaloid of Securinega suffruticosa

Biosynthesis of securinine was studied by incorporation experiments in Securinega suffruticosa. Among presumed precursors tested, lysine, cadaverine, and tyrosine showed the highest incorporation into securinine. Degradation experiments revealed that cadaverine-[1,5-¹⁴C] labelled specifically the piperidine ring of securinine and the radioactivity from dl-tyrosine-[2-¹⁴C] was introduced into the C-11 lactone carbonyl. Experiments with L-tyrosine-[U-¹⁴C] and L-tyrosine-[3′,5′-³H; U-¹⁴C] prove that the remaining C₆Sz.sbnd;C₂ moiety is derived from the aromatic ring and the C-2 and C-3 or tyrosine.     

Characterization and partial purification of three glycosidases from castor bean endosperm

α-Mannosidase and β-N-acetylhexosaminidase, which could function in the cleavage of glycosidic linkages in the native Ricinus communis lectins, and β-galactosidase were purified some 100-fold from the endosperm tissue of castor bean seedlings. The procedure used ammonium sulphate precipitation followed by chromatography on CM-cellulose, hydroxyapatite and Sephacryl S-300 to separate the three activities. All three glycosidases were present, with the lectins, in the protein bodies of dry seed and increased in activity during the time that lectins are broken down in the vacuoles. The enzymes show optimal activity in the range pH 3–5.5. The α-mannosidase had a K_m of 0.77 mM for p- nitrophenyl-α-D-mannopyranoside. The β-galactosidase showed a K_m of 1.39 mM for p-nitrophenyl-β-D-galactopyranoside. The β-N-acetylhexasominidase had a K_m of 0.47 mM for p-nitrophenyl-N-acetyl-β-N-glucosamide and a K_m of 0.33 mM for p-nitrophenyl-N-acetyl-β-D-galactosamide. Effects of competitive inhibitors and cations were described.     

Endosperms and embryos of mature dry castor bean seeds contain active ribosomes

Mature dry endosperms and embryos of castor bean contain ribosomes which are capable of catalysing protein synthesis in vitro. This observation contradicts previous reports that ribosomes are absent from mature dry endosperms. On hydration of the mature seeds the polyribosome content of both embryos and endosperms increases. These polyribosomes are lost on subsequent dehydration but, again, ribosomes are conserved. REFERENCES     

Comparison of homoserine dehydrogenase from different plant sources

Homoserine dehydrogenase (HSD) was partially purified from castor bean, pea and wheat seedlings. The enzyme from pea had a MW of 75 000 and no sensitivity to threonine when measured in the direction of homoserine formation (forward reaction). The enzyme purified from castor bean had a MW of 290 000–350 000 and exhibited an almost complete inhibition by 1 mM threonine. Furthermore, this enzyme exhibited a polymeric nature as shown by polyacrylamide electrophoresis of the desensitized preparation and by SDS electrophoresis of the native enzyme. In wheat two isoenzymes were separated by gel filtration on Sephadex G 200. The fast-moving fraction (HSD I) was completely inhibited by threonine and exhibited a MW of 280 000, while the slow-moving fraction (HSD II) was insensitive to threonine and had a MW of 75 000. The sensitive enzyme from wheat and castor bean showed an almost absolute requirement for K⁺. The enzyme from pea and the insensitive form from wheat did not show a requirement for K⁺. For the wheat enzyme the effect of several amino acids and the main kinetic constants were studied.     

Mixed function oxidases from germinating castor bean endosperm

Homogenates from germinating castor bean endosperm were fractionated by sucrose density gradient centrifugation and examined for mixed function oxidase activity. Activity of cinnamic acid 4-hydroxylase and p-chloro-N-methylaniline N-demethylase was highest in the endoplasmic reticulum fraction. Activity of both enzymes is dependent on NADPH and on molecular oxygen; both activities are inhibited by carbon monoxide. When challenged with a number of potential inhibitors the enzymes responded in ways fairly typical of mixed function oxidases from other plants and animals. The N-demethylase appears to be specific for N-methylarylamines. In the absence of NADPH, cumene hydroperoxide is able to support N-demethylation. The mechanistic significance of this activity is discussed.     

Phloem sap exudation in Ricinus communis: elastic responses and anatomical implications

Abstract. Ricinus communis plants have an unusually high capacity to exude considerable quantities of phloem sap from bark incisions. We have used Ricinus as an experimental system to study different aspects of sap exudation. Dimensional changes in the bark, monitored by a displacement transducer, showed that pressure release in the sieve tubes was accompanied by elastic shrinkage. The rate of exudation was also controlled by the degree of pressurization and elastic properties of the sieve tubes. A displacement transducer was used to measure the elastic modulus (ɛ) of phloem samples by immersing them in a range of different osmotica. The cells had a low elastic modulus (ɛ= 1.62 ± 0.41 MPa at full turgor). ɛ of phloem tissue in massage pretreated bark, from which exudation was enhanced, was not significantly different from that of unmassaged bark in contrast with the suggestion of Lee (1981). However, anatomical studies showed that massage pretreatment has a stimulating influence on the cambial cell division, which increased the phloem tissue cross-section up to 160%. The newly-formed sieve tubes were 'spliced' into existing ones in the unmassaged zone to re-establish vascular continuity. Plants with a greater capacity to exude phloem sap from a given stem location had a greater cross-sectional area of sieve tubes in the vicinity.
The significance of observations with respect to other sap exudation phenomenon is discussed. The importance of the present work in understanding the technique of palm tapping, on which the palm sugar industry depends, is also considered.     

δ-N-Methylornithine: a natural constituent of Atropa belladonna

δ-N-Methylornithine, a tropane alkaloid precursor, is shown for the first time to be a natural plant constituent; it was isolated in radioactive form after feeding [5-¹⁴C]- and [5-³H]ornithine to Atropa belladonna. This finding supports the deduced role of δ-N-methylornithine in tropane alkaloid biosynthesis.     

Glutamate dehydrogenase isoenzymes in Ricinus communis seedlings

Castor bean seedling glutamate dehydrogenase isoenzymes are not artifacts. The isoenzymes have different salting out properties and they utilize NAPD to differing extents, but they have the same isoelectric point of pH 6·2. Tissue specific patterns occur but the patterns are the same between genotypes. The GDH isoenzymes are probably of functional significance in castor bean seedlings.     

Structure and biosynthesis of malloprenols from Mallotus japonicus

The mallo prenol isolated from the leaves of Mallotus japonicus was elucidated to be a mixture of (2Z,6Z, 10Z, 14Z, 18Z, 22Z, 26E, 30E, 34E)-3,7,11,15,19,23,27,31,35,39-decamethyl-2,6,10,14,18,22,26,30,34,38-tetracontadecaen-1-ol and its C₄₅- and C₅₅-homologues and not the previously reported structure. The malloprenols were demonstrated to be biosynthesized by successive cis condensation of isoprene residues with (2E, 6E, 10E)-geranylgeranyl pyrophosphate.     

Biosynthesis of ent-kauranes: Evidence for a C-17, C-16 hydrogen 1,2-shift

The incorporation of ent-kaurenoic acid into the derived hydroxy acid and dihydroxy acid by Beyeria calycina has been studied. The biosynthesis of the hydroxy acid involves a hydrogen 1,2-shift from the C-17 position of ent-kaurenoic acid.     

Biosynthesis of papaverine

Tracer experiments have shown that in Papaver somniferum papaverine arises from (−)-norreticuline via norlaudanidine and norlaudanosine.     

Metabolism of [2-3H]- and [6-3H]-nicotinic acid in intact Nicotiana tabacum plants

Earlier observations of Dawson on the relative incorporation of [2-³H]- and [6-³H]-nicotinic acid into nicotine have been confirmed in intact Nicotiana tabacum plants. All the tritium in the nicotine derived from [2-³H]-nicotinic acid was located at C-2 of the pyridine ring. However the radioactive nicotine derived from [6-³H]-nicotinic acid was not labelled specifically at C-6 with tritium. By carrying out feeding experiments with [6-¹⁴-C, 2-³H]- and [6-¹⁴C, ³H]-nicotinic acids, it was established that there was very little loss of tritium from C-2 and C-6 of nicotinic acid during 5 days of metabolism in the tobacco plant.     

Casbene: An anti-fungal diterpene produced in cell-free extracts of Ricinus communis seedlings

The capacity of cell-free extracts of 2·5-day-old castor bean seedlings for synthesis of casbene from mevalonic acid were compared for seedings which had been germinated under sterile conditions and seedlings which were intentionally exposed to fungal cultures. Extracts from seedlings exposed to cultures of Rhizopus stolonifer, Aspergillus niger or Fusarium moniliforme produced much higher levels of casbene than extracts from sterile controls; the initial rates of casbene synthesis were 20–40 times higher in the extracts of seedlings that had been exposed to fungus. Although some variation in the capacity for synthesis of other diterpene hydrocarbons from mevalonic acid was seen in the two types of extracts, no consistent or striking stimulation in the synthesis of any of these was noted under these conditions of exposure of the seedlings to fungi. The potato-dectrose agar used as a fungal growth medium did not itself evoke the increase in casbene synthesis. Intact mycelia and cell-free extracts of mycelia of Rhizopus stolonifer gave no indication of diterpene biosynthesis from mevalonic acid. Purified casbene at concentrations of 10 μg ml⁻¹ or greater retarded the development of A. niger on potato-dextrose medium. Casbene was also found to inhibit the endogenous and gibberellic acid-stimulated growth of leaf sheaths of the dwarf-5 mutant of Zea mays and of the growth of the K-12 strain of Escherichia coli on glucose-minerals medium. It is suggested that casbene may serve the castor bean plant as a phytoalexin.     

Diverse transporters for neutral amino acids in Ricinus communis L. seedlings

Uptake mechanisms for neutral amino acids were investigated by expression of mRNA isolated from seedlings of Ricinus communis L. in Xenopus laevis oocytes. After injection of mRNA from root, hypocotyl and cotyledon currents elicited by saccharose and neutral amino acids ranged from 0.3 nA up to 2 nA depending on the respective substrate and the source of mRNA. These currents were due to expression of low affinity uptake mechanisms and the KM values found for amino acid induced charge flow range from 1 to 2 mM. The abundance and/or the specificity of the expressed mechanisms differ in the various tissues. Currents of similar magnitude were recorded for alanine and glutamine with mRNA isolated from root, hypocotyl and cotyledons. Serine and proline induced currents after injection of mRNA from hypocotyl and roots, in case of α-aminoisobutyric acid (AIB) induced currents were generally small with mRNA from all tissues tested. In addition, differential sensitivity of glutamine and AIB uptake in the high affinity range was evident towards the amino acid analogue 2-chloro-aminophenoxybutyric acid which indicated an additional set of carriers operating in the micromolar concentration range. The results suggest that multiple transporters for neutral amino acids exist in various tissues of the plant differing in specificity of charge flow and in sensitivity towards the inhibitor 2-chloro-aminophenoxybutyric acid. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biosynthesis of gramine in Phalaris arundinacea

The administration of L-tryptophan-[3-¹⁴C] to Phalaris arundinacea L. (Vantage strain) for 9 days resulted in the formation of radioactive gramine (8.2% absolute incorporation). A systematic degradation of the alkaloid indicated that essentially all its activity was located on the methylene group, indicating that its biosynthesis is the same as that occurring in Hordeum species and Lupinus hartwegii.