Partial purification and properties of willardiine and isowillardiine synthase activity from Pisum sativum

The enzymic activity responsible for synthesis of willardiine and isowillardiine in pea seedlings has been extracted and partially purified. Fresh tissue, pulverized in liquid-N₂, was extracted in a phosphate buffer (pH 7) and subjected to fractional precipitation with ammonium sulphate. After desalting on Sephadex G-25 and concentration by ultrafiltration, the fraction containing the activity was chromatographed sequentially on DEAE-Sepharose CL-6B, DEAE-cellulose (DE 52) and Sephadex G-200. Electrophoretic separation in polyacrylamide gels was also used. A 120-fold purification was achieved but at no stage was there any indication of a separation of willardiine synthase activity from that of isowillardiine synthase. Both activities paralleled one another when the enzymic preparation was progressively denatured by subjecting it to gradually increasing temperatures. Similarly, ageing at 4° and at ?196° resulted in a parallel loss of activity. Both synthase activities were maximal at 7.8–7.9and the pH optimum curves were of closely similar shape. From the results described, it is concluded that a single enzyme of relatively low MW (ca 50 000) is responsible for the synthesis of both uracilylalanines. Studies of the alanylation of uracil using a pyridoxal-metal ion model-enzyme system are described.     

Accumulation of 5-ribosyluracil (pseudouridine) within the tissues of Phaseolus vulgaris

Free 5-ribosyluracil (pseudouridine) accumulates within the acid-soluble fraction of germinating seeds and seedlings of Phaseolus vulgaris. Accumulation is significantly increased by erogenous uridine. Experiments with [¹⁴c]-labelled precursors indicate that UTP is an intermediate in the formation of this free 5-ribosyluracil.     

Purification and properties of sucrose-6-phosphatase from Pisum sativum shoots

Sucrose-6-phosphatase from pea shoots, which was purified to homogeneity, consists of two similar sub-units each with an MW of about 55 000. The pH optimum was at 6.8; the K_m for sucrose-6-phosphate was 250 μM and the K_m for magnesium was 175 μM. The enzyme was specific for sucrose-6-phosphate and was not inhibited by sucrose except at very high concentrations.     

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.     

Stereospecific reduction of progesterone by Pisum sativum

[4 -¹⁴C]-Progesterone was applied to the leaves of growing pea plants, Pisum sativum. After 3 weeks, about 50% of the administered steroid was reduced, about 20% being reduced to 5α-pregnane-3α,20β-diol as the major metabolite. The radioactivities of 5α-pregnane-3α,20α-diol and 5α-pregnane-3α,20β-diol after 3 weeks were more than twice those after one week. The following radioactive metabolises were also isolated: 5α-pregnane-3,20-dione; 20α-hydroxy-4- pregnen-3-one; 20β-hydroxy-4-pregnen-3-one; 3α-hydroxy-5α-pregnan-20-one; 3α-hydroxy-5β-pregnan-20-one; 3β-hydroxy- 5α-pregnan-20-one; 20β-hydroxy-5α-pregnan-3-one; 5α-pregnane-3β,20β-diol; and 5β-pregnane-3α,20β-diol. The radioactivities of the 5α-pregnane derivatives were considerably higher than those of the corresponding 5β-pregnane derivatives.     

Metabolism of abscisic acid and the occurrence of epi-dihydrophaseic acid in Phaseolus vulgaris

When (±)-abscisic acid-[2-¹⁴C] or (±)-abscisic acid-[4′-¹⁸O] was fed to bean (Phaseolus vulgaris) shoots, phaseic acid (PA) and dihydrophaseic acid (DPA) were the major metabolites, while epi-dihydrophaseic acid (epi-DPA) appeared as a minor metabolite. In the acidic fraction the amount of epi-DPA ranged from 18 to 42% of the DPA content, in the conjugated form from 50 to 200%. The content of endogenous epi-DPA amounted to only 1–2% of that of the DPA. These data indicate that the applied abscisic acid is not metabolised in a manner identical with that of the endogenous material. DPA and epi-DPA were shown to be formed separately from PA and could not be inter-converted either by the extraction conditions employed or when fed to bean shoots during short term experiments.     

Metabolism of 2-amino-3-phosphono[3-14C]propionic acid in cell-free preparations of rat liver

Incubation of 2-amino-3-phosphono[3-¹⁴C]propionic acid with cell-free preparations of rat liver yielded labelled 3-phosphonopyruvic acid, 2-phosphonoacetaldehyde, 2-aminoethylphosphonic acid and acetaldehyde. No radioactivity was found in phosphoenolpyruvate, pyruvic acid, alanine, and phosphonoacetic acid.When added to the cell-free preparations, 3-phosphonopyruvic acid trapped the radioactivity, resulting in decrease of incorporation of the radioactivity into 2-phosphonoacetaldehyde, 2-aminoethylphosphonic acid and acetaldehyde. Incorporation of the radioactivity into 2-aminoethylphosphonic acid and acetaldehyde was also decreased by 2-phosphonoacetaldehyde.Thus it appears that the main metabolic pathway of 2-amino-3-phosphonopropionic acid is deamination to produce 3-phosphonopyruvic acid which is, in turn, converted to 2-phosphonoacetaldehyde by decarboxylation, followed by both dephosphonylation and amination of the aldehyde to give acetaldehyde and 2-aminoethylphosphonic acid, respectively.     

Conversion of 4-androstene-3,17-dione to testosterone by Pisum sativum

4-Androstene-3,17-dione-[4-¹⁴C] was applied to the leaves of growing pea plants, Pisum sativum. Within a week, 28% of the administered steroid was specifically reduced to testosterone. Part of the testosterone was present in esterified form, and 5α-androstane-3β,17β-diol was also identified as a metabolite, but neither epitestosterone nor estrogens were detected.     

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.     

Degradation of [β-14C]hordenine in Hordeum vulgare plants

[β-¹⁴C]Hordenine is ultimately degraded by intact plants of Hordeum vulgare to C₆-C₁ intermediates that are incorporated into polymeric material.     

Exogenous [1-14C]lignoceric acid uptake by neurons,astrocytes and myelin,as compared to incorporation of [1-14C]palmitic and stearic acids

In order to compare the incorporation of several saturated fatty acids into the brain, radioactive palmitic, stearic and lignoceric acids were injected into mice. The radioactivity was measured in lipids from isolated neurons, astrocytes and myelin.Our data indicate that specific radioactivity of lignoceric acid after its injection was very high in neurons and astrocytes when comparing with serum lignoceric acid specific radioactivity: evidence of the uptake of exogenous lignoceric acid by brain cells and myelin is provided.The incorporation of exogenous palmitic acid into brain cells was much higher than the incorporation of exogenous stearic acid. We hypothesize that exogenous saturated fatty acid uptake is selective in relation with the acyl chain length and the intracerebral synthesis.     

Separation and properties of two ornithine carbamoyltransferases from Pisum sativum seedlings

Two ornithine carbamoyltransferases were separated from pea seedlings on DEAE-cellulose. The two enzymes have different pH-activity curves. Lineweaver-Burk plots for both enzymes are linear for carbamoyl phosphate and the Michaelis constants are of the same order of magnitude. The plot for ornithine was linear for one enzyme but a concave down for the other indicating negative cooperativity. The presence of two ornithine carbamoyltransferases is consistent with the presence of two pools for ornithine (one catabolic and the other anabolic previously suggested to exist in plant materials.     

In vivo conversion of [4-14C]sitosterol and [22,23-3H]sitosterol to stigmasterol in barley seedlings

Six-day-old barley seedlings were allowed to take up [4-¹⁴C]sitosterol and [22, 23-³H]sitosterol for 2.5 hr and the incorporation into the sterol fractions was determined after 0, 6, 12 and 24 hr. Sitosterol was readily incorporated into every sterol class. The ³H/¹⁴C ratio in the free forms dropped when compared with the ³H/¹⁴C ratio of the administered sitosterol. In the free sterol, radioactive stigmasterol, showing a ³H/¹⁴C ratio half that of the sitosterol ³H/¹⁴C ratio, was isolated and its radiochemical purity established by dilution with carrier material and crystallization to constant specific activity.     

Native gibberellins and the metabolism of [14C]gibberellin A53 and of [17-13C, 17-3H2]gibberellin A20 in tassels of Zea mays

The native hormones from tassels of maize (Zea mays) were re-investigated. The previous identification by GC/SIM of GA₁, GA₈ and GA₂₉ in normal tassels was confirmed by full GC/MS scans at the correct Kovats retention indices. In tassels of dwarf-1 mutants, GA₄₄,^?GA₁₉, GA₁₇, GA₂₀ and the 16,17-dihydro, 7β,16α,17-trihydroxy derivative of ent-kaurenoic acid were identified by GC/MS. Gibberellin A₁ was not found in the mutant tassels. [¹⁴C]Gibberellin A₅₃ was fed to tassels of the dwarf-5 mutant. In the ethyl acetate-soluble acidic fraction from the feeds, [¹⁴C]GA₄₄ was identified by GC/MS; [¹⁴C]GA₁₉ and [¹⁴C]GA₂₉ were identified by GC/SIM. The GA₂₉ is probably a metabolite of the feeds because the dwarf-5 mutant is known to control the step copalyl pyrophosphate to ent-kaurene in the maize GA-biosynthetic pathway and because GA₂₉ was not identified in a control experiment. The n-butanol fractions obtained from the feeds were shown, by GC/MS, to contain [¹⁴C]GA₅₃ after hydrolysis, suggesting that conjugated [¹⁴C]GA₅₃ is a major metabolite from GA₅₃ feeds. [17-¹³C, 17-³H₂]Gibberellin A₂₀ was fed to normal, dwarf-1 and dwarf-5 tassels. In each case, analysis of the purified ethyl acetate-soluble acidic extracts by GC/MS led to the identification of [¹³C]GA₂₉ and unmetabolized [¹³C]GA₂₀ in which no ¹³C-isotope dilution was observed.     

Metabolism of [14C]trans-zeatin and [14C]benzyladenine by letached yellow, green and variegated leaves of Schefflera

[8-¹⁴C]Benzyladenine (BA) and [8-¹⁴C] trans-zeatin (tZ) were fed through the petiole to mature, detached green, yellow and variegated leaves of Schefflera arboricola. Recovery of radioactivity from the plant material ranged between 4.2 and 22.1%. More radioactivity was recovered when tZ was applied compared to BA. Green leaves or the green parts of variegated leaves yielded more radioactivity than the yellow leaf material. BA was metabolized much faster than the endogenous cytokinin tZ. It would appear that while lower amounts of radioactivity were present in yellow leaves, as well as in yellow parts of variegated leaves, the rate of cytokinin metabolism was nevertheless faster. Metabolites that were formed to a greater extent in these yellow parts were the nucleotides of both cytokinins. Currently it is not known whether or not cytokinins influence chlorophyll and other pigment development in chimeric variegated leaves.     

Biosynthesis of nimbolide from [2-14C,(4R)4-3H1] mevalonic acid lactone in the leaves of Azadirachta indica

Nimbolide was biosynthesized from [2-¹⁴C, (4R)4-³H₁]mevalonic acid lactone in the leaves of Azadirachta indica. The nimbolide had a ³H:¹⁴C ratio of 3:5 which gives support to the suggestion of the involvement of a triterpenoid intermediate with a double bond at the Δ⁸⁽⁹)-position in the biosynthesis of nimbolide.