Lupinus Albus Seeds Research Articles

Purification and characterization of a tRNA nucleotidyltransferase from Lupinus albus and functional complementation of a yeast mutation by the corresponding cDNA

ATP (CTP):tRNA nucleotidyltransferase (EC 2.7.7.25) was purified to apparent homogeneity from a crude extract of Lupinus albus seeds. Purification was accomplished using a multistep protocol including ammonium sulfate fractionation and chromatography on anion-exchange, hydroxylapatite and affinity columns. The lupin enzyme exhibited a pH optimum and salt and ion requirements that were similar to those of tRNA nucleotidyltransferases from other sources. Oligonucleotides, based on partial amino acid sequence of the purified protein, were used to isolate the corresponding cDNA. The cDNA potentially encodes a protein of 560 amino acids with a predicted molecular mass of 64 164 Da in good agreement with the apparent molecular mass of the pure protein determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The size and predicted amino acid sequence of the lupin enzyme are more similar to the enzyme from yeast than from Escherichia coli with some blocks of amino acid sequence conserved among all three enzymes. Functionality of the lupin cDNA was shown by complementation of a temperature-sensitive mutation in the yeast tRNA nucleotidyltransferase gene. While the lupin cDNA compensated for the nucleocytoplasmic defect in the yeast mutant it did not enable the mutant strain to grow at the non-permissive temperature on a non-fermentable carbon source.

Molecular properties and thermal secretion of lupin seed acid phosphatase

An acid phosphatase with optimum pH at 6 was found in the total albumin extract of mature dry Lupinus albus seeds. Three groups of phosphatase isoenzymes were identified by IEF. The active bands in the IEF gel were run in 2D-SDS-PAGE under reducing conditions and found to contain one major ( ca 70%) polypeptide chain of M r 68 000 and one minor ( ca 30%) of 61 000. When lupin seeds were incubated in water for 3 hr at 60°, residual phosphatase activity was detectable in the medium suggesting that the enzyme was secreted together with other already identified polypeptides. The IEF and 2D-SDS-PAGE patterns of the constitutive and heat-secreted phosphatase(s) coincided.

(+)-15β-Hydroxy-17-oxolupanine a lupin alkaloid from the seeds of Lupinus albus

A new lupin alkaloid (+)-15β-hydroxy-17-oxolupanine was isolated from the ethanol extract of the seeds of Lupinus albus together with (+)-tetrahy

The Unusual Extracellular Localization of Conglutin γ in Germinating Lupinus albus Seeds Rules out its Role as a Storage Protein

Summary Antibodies specific to conglutin γ, a putative storage glycoprotein of Lupinus albus seeds, and to one of its constituent subunits devoid of carbohydrate, have been used to localize the protein in mature and germinating seeds by light and electron microscopy. Conglutin γ is located within the protein bodies of mature seeds, but after 2 days of germination labelling is also observed in the extracellular spaces of the cotyledonary tissue. By 8 days after germination, when most of the protein bodies and their electron dense contents have disappeared, conglutin γ is found predominantly within the intercellular spaces. In parallel experiments labelling of a typical lupin storage protein, a legumin-like globulin, was shown to be confined to the protein bodies in both mature and germinating seeds, but not to be localized within the intercellular spaces. On the basis of these findings the role of conglutin γ as a storage protein is questioned.

Preparation of rabbit polyclonal antibodies against lupin storage proteins

Agarose gel electrophoresis was used to separate three globular storage proteins from crude extracts of Lupinus albus seeds. Conglutin α, β, and γ purified by this technique were used as antigens to stimulate the production of antibodies in rabbits. The specificity of polyclonal antibodies produced was analysed by Western blot. Each antibody reacted with the specific antigen used to stimulate production of that antibody. In addition the anti-conglutin α preparation also showed binding to a region of the gel that comigrates with conglutin β. Key words: Lupin, conglutin, polyclonal antibody

Gibberellins A 82 and A 83 in seed of Lupinus albus

Extracts from seeds of Lupinus albus at 14, 22, 35 and 52 days after anthesis were separated into free gibberellins, ester conjugates and ether conjugates. Capillary GC-MS of the methylated and trimethylsilylated free gibberellin fractions showed the presence of the known gibberellins A 1, A 3, A 4, A 17, A 18, A 23 and A 43. In addition six new gibberellin-like compounds were detected that corresponded to the addition of the elements of water to gibberellins A 3, A 4, A 7, A 14, A 18, and A 34. Two of these components were identified by chemical syntheses as ent-3α,10β,17-trihydroxy-20-nor-16αHgibberellane-7,19-dioic acid 19,10-lactone and ent-3α,17-dihydroxy- 16αHgibberellane-7,19-dioic acid, which are accorded the gibberellin numbers A 82, and A 83, respectively. ent-3α,10β,16β,17-Tetrahydroxy-20-nor-16βHgibberellane-7,19-dioic acid 19,10-lactone was also identified by synthesis of the methyl ester. Similar analyses of the hydrolysed ether conjugate fractions showed the presence of the known gibberellins A 1, A 3, A 13, A 18 and A 43, the new gibberellin A 82 and the ‘hydrated’ gibberellins A 18 and A 34; the 15-ene isomers of gibberellins A 13 and A 43 and the 16ξ17-epoxide of gibberellin A 18 were also identified as probable artefacts. In the hydrolysed ester conjugate fractions the new gibberellin A 82 and the ‘hydrated’ gibberellin A 34 were detected. Gibberellin A 18 was by far the most abundant GA but quantitation of the GAs was not carried out.

Immunocytochemical localization of conglutin ? and legumin-like globulin in developing and mature seeds ofLupinus albus L.

The distribution of two seed proteins, namely conglutin γ and a legumin-like globulin, in developing and mature seeds ofLupinus albus L. has been examined by immunocytochemistry and the concomitant modifications of their constituent polypeptides followed by SDS-PAGE. Both proteins were found within vacuolar protein bodies in various tissues of the cotyledons, although with some differences in the distribution patterns. The legumin-like protein was found to be deposited within the large storage parenchyma cells of the cotyledons in a manner similar to that reported for other storage proteins; little or no immunolabelling was associated with the cotyledonary epidermal and vascular parenchyma cells. In contrast conglutin γ was present in all cell types.

Evidence for an arachidonic acid 5-, 8- and 15-lipoxygenase in Lupinus albus seeds

A lipoxygenase preparation was obtained from dried Lupinus albus seeds and was shown to differ from previously characterized soybean lipoxygenases in the positional specificity and pH characteristics of the dioxygenation reaction. The L. albus enzyme converted arachidonic acid into three HPETEs: 15-HPETE was the major product (60%) besides 5-HPETE (34%), and there were small amounts of 8-HPETE (6%). The enzyme had a pH optimum at 6.0 and was inactive at pH 9.O. The molecular weight of the enzyme was estimated at 71000, and p I was 5.35. The enzyme activity was inhibited strongly in the presence of lipoxygenase inhibitors, such as esculetin or BW 755 C.

Changes in lipoxygenase activity during seedling development of Lupinus albus

A lipoxygenase preparation was obtained from Lupinus albus seeds and was shown to differ from previously characterized lipoxygenase. This study describes changes in lipoxygenase activity during seedling development of Lupinus albus. The enzyme activity shows a decrease from 0–6 h postgermination (about 15%), is roughly constant or even rises slightly from 6–30 h and then shows a large increase between 30 and 48 h (about 50%). Enzymatically active proteins from 48 h-old seedlings were isolated and the increase of enzyme activity was mainly due to the presence of two components with maximum activity at pH 6 and pH 8.5, respectively. When arachidonic acid was used as substrate, the two enzymatic activities produce 15 HPETE. The increase in lipoxygenase activity during seedling development was inhibited by cycloheximide. Cordycepin appears to have no direct effect on lipoxygenase synthesis in vivo at the studied doses.

The legumin-like storage protein of Lupinus albus seeds

Legumin-like proteins of Lupinus albus seeds, consist of 12 S and 7 S species which are in part involved in an association-dissociation equilibrium that is shifted towards association by increasing the ionic strength of the medium. A consistent portion of the 7 S species does not associate to 12 S. The 12 S and the 7 S associating molecules have the same protomer composition. the 7 S non-associating molecules differ from them because they have less high M r acidic protomers. Partial proteolytic breakdown of these polypeptides appears to convert associable to non-associable molecules. The splitting protease is tightly associated to the legumin in the preparation. It is inhibited by sodium azide. The apparent M r of the heavy and the light species is 315 000 and 185 000 respectively. Also other parameters, such as diffusion coefficients, frictional ratios, Stokes' radii, are in good agreement with those of legumin-like storage proteins from other legume seeds. Binding of the purified legumin to a concanavalin A-Sepharose and incorporation of tritiated N-acetylglucosamine into legumin polypeptides during seed development have confirmed that lupin legumin is glycosylated. Only the acidic subunits appear to contain covalently linked carbohydrate.

Relationships between nitrogen, amino acids and storage proteins in Lupinus albus seeds

Lupinus albus seeds (20 samples from 10 different lines or cultivars) with protein contents distributed from 23.8 to 48.4% were analysed for their amino acid composition with great accuracy (from 6 hydrolysates per sample). Amino acid levels in seeds increased as linear functions of nitrogen content with correlation coefficients close to unit in all the lupin genotypes and phenotypes. Hence the composition of any lupin seed sample can be predicted from its nitrogen content. Amino acid in total seed protein varied as hyperbolic functions of nitrogen content. The same was true for nitrogen-to-protein conversion factors. By controst, the composition of storage proteins accumulated in seeds remained constant and independent of seed protein content.

Subunit composition of proteins from seeds of Lupinus albus

All the main globulins in the seeds ofLupinus Albus are oligomeric glycoproteins. Legumins (33%) consist of two similar protein molecules which contain protomers linked by disulphide bridges. They result from a partial proteolytic breakdown of an original polypeptide chain. Vicilins (44%) consist of four similar protein molecules with several protomers linked together by non-covalent bonds. Globulin 1 (6%) has a native M.W. of 199 kd and is formed by four 45.0 kd subunits consisting of two smaller protomers (28.0 and 16.0 kd) linked by -S-S- bonds. Globulin 9b (12.5%) has the lowest M.W. (44.0 kd) and is made up of three protomers, two of which are linked by disulphide bonds.

Properties of lupine proteins relevant to their nutritional performance

Composition, solubility, bound sugar content and quality, subunit composition and structure of the storage proteins of seeds ofLupinus albus are discussed. Aminoacid composition is also given for each protein, the various protein classes and the whole flour. These data allow for the characterization of the molecules of the various storage proteins and their contributions to the nutritional properties of the seed.In vitro digestibility (by mammalian endopeptidases) is reported and is less than for animal proteins. Possible causes, at the molecular level, for this behaviour and possible means to overcome these effects are examined. The relationships between the above data are considered in view of the nutritional performance of the proteins and of the genetical, agronomic and technological approaches most suited to improve the nutritional quality of lupine seeds as a protein source.

Unconventional protein sources. I. Lupinus albus.

Three different treatments were tested for their effectiveness in removing alkaloids, and producing protein products suitable for human consumption from Lupinus albus seeds. The treatments included: a) water leaching of the seeds, b) ammonium salt treatment of the seeds, and c) multistage extraction of the defatted meal. Results revealed that treatments a) and c) were highly effective in the removal of alkaloids. Protein contents of the resulting three products were about 62, 47 and 58%, respectively, compared to 39% in the seed. Amino acid analysis showed that the protein preparations lack sulphur amino acids, lysine, tryptophan and valine.

THE GROWTH ARREST CAUSED BY ANIMAL MUCOPROTEINS

Anti-growth activity of animal mucoproteins on germinating seeds ofLupinus albus was tested; the inhibition with plasma mucoprotein was statistically significant. The inhibition with urine mucoprotein according toAnderson was the most effective.

Relative Potency of Reductase in Dry, Wet and Germinated Lupinus albus Seeds

Relative Potency of Reductase in Dry, Wet and Germinated Lupinus albus Seeds

Effect of Deuterium Oxide on Action of Some Enzymes

Since the discovery of heavy water much speculation of very diverse character has been advanced in regard to its possible effects on life. The most extreme views are presented by those who claim, without adequate experimental proof, that deuterium oxide is a most powerful destructive physiological agent and those who, basing their conclusions on experiments made with deuterium oxide in dilute concentrations, state that its pharmacological effects are of negligible character., , The authors have recently discovered an interesting and striking contrast in the biophysical and biochemical behavior of H2O and D2O, which consists of a difference in velocity of reaction shown by certain animal and vegetable enzymes suspended therein. Studies were made on the behavior of muscle oxydase from the rat, of oxidative enzymes of fresh, blood-free brain tissue from the cat, and of reductase of finely ground seeds of Lupinus albus by the Thunberg method of determining the rapidity with which a standard buffered solution of methylene blue is decolorized in special vacuum tubes. Over 50 experiments were performed. The method employed is described in full elsewhere., A marked difference was found to exist between identical enzymes suspended in H2O and in the same kind of water to which small quantities of deuterium oxide had been added. This difference was demonstrated not only with concentrations of D2O, 1:100, but also with concentrations of 1:2000 and less. Further studies were made in connection with the influence of deuterium oxide on the activity of another enzyme, catalase. Catalase from fresh rat muscle and from Lupinus albus seeds in distilled water and in water to which small quantities of deuterium oxide were added also revealed a difference in the speed of evolution of oxygen gas from hydrogen peroxide in the presence of these enzymes.

Effects of Snake Venoms on Plants

With the exception of Mitchell1 and Salisbury,2 who made sporadic observations on the subject in nature, investigators have given very little attention to the effects of snake venoms on plants. In connection with phytopharmacological studies regarding the effect of various animal poisons on living seedlings, the writer made experiments with suspensions or solutions of snake venoms by dissolving small quantities of the dry scales in physiological saline. The method employed has been fully described.3, 4, 5 Seeds of Lupinus albus are soaked for 24 hours in water and then planted in finely divided sphagnum moss. When seedlings have germinated and borne roots from 30 to 40 mm. long, they are carefully selected, “matched,” and placed in upright, hard-glass test tubes containing a plant-physiological solution made up of equal parts of Shive's mixture and distilled water. The average growth of roots of 10 seedlings (in the dark at 20° C. for 24 hours) is determined. Dry scales of snake venoms were rubbed up with...

TEMPERATURE CHARACTERISTIC FOR THE ANAEROBIC PRODUCTION OF CO2 BY GERMINATING SEEDS OF LUPINUS ALBUS

The rate of anaerobic production of CO(2) by germinating seeds of Lupinus albus was studied as a function of temperature between 7.5 degrees and 18 degrees C. The mean value for the temperature characteristic was found to be 21,500+/- calories, which is slightly lower than that for the same process under aerobic conditions (23,500+/- calories). The values for the individual micro's in the two cases overlap considerably. The possible identity of the processes underlying the production of CO(2) aerobically and anaerobically is discussed.

THE EFFECTS OF CO AND LIGHT ON THE OXYGEN CONSUMPTION AND ON THE PRODUCTION OF CO2 BY GERMINATING SEEDS OF LUPINUS ALBUS

The consumption of oxygen by germinating seeds of Lupinus albus can be reversibly inhibited by CO to a maximum extent of 36 per cent with a mixture of 24 per cent O(2) and 76 per cent CO at 18 degrees , in darkness. This inhibition is completely abolished when the seed is illuminated. On returning to air, after a period in the CO-O(2) mixture, the rate of oxygen consumption is accelerated to as much as 68 per cent over what it had been previously, in air. The production of CO(2) is apparently not inhibited by CO. The bearing of these findings on the study of the rates of gas exchange of the seeds as a function of temperature is discussed.