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Study on plant RNAases. Isolation and properties of several activities from Vicia faba root cells.

Vicia faba root cells contain several nucleolytic activities: phosphomonoesterase and phosphodiesterase (which however were not studied in details), one nuclease and four ribonucleases. These results were obtained by separating the extracted proteins into anionic and cationic species by chromatography on CM-cellulose at pH 5.5 and analysing each kind of proteins. Anionic species were subjected to chromatography on DEAE-cellulose which lead to isolation of one nuclease (A1) and two RNAases (A2, A3), the properties of which were studied. It was shown that the RNAases pH optima are near 6; A2 is more thermolabile than A3; both are endonucleases unable to attack double-stranded structure; studies with homopolymers, i.e. poly(A), poly(I), poly(C), poly(U), showed that their base specificities were analogous to that of already known plant RNAases. The cationic proteins, analysed with CM-cellulose, contain two RNAases (C1, C2). The pH optima were near 6 and 7, respectively; C1 is much more thermolabile than C2; both were endonucleases inactive on double-stranded structures. C1 and C2 hydrolysed poly(C) and poly(U) but not poly(A) and poly(U).

Cations

Proteomic characterization of acidic aqueous extracts from Vicia faba L. pod valves identifies chitinase as a major co-extracted protein macromolecule.

Naturally acidic aqueous extracts from Vicia faba L. pod valves are being explored as sustainable, L-DOPA-oriented plant preparations. Pod valves represent an underutilized processing by-product reported to contain L-DOPA, a compound widely used in Parkinson's disease therapy, while acidic aqueous media may help preserve its physicochemical stability. However, the protein macromolecules co-extracted from V. faba pod valves under these conditions remain poorly characterized. This information is relevant because persistent plant proteins may influence extract composition, stability, susceptibility to degradation, and downstream processing requirements. Here, we characterized co-extracted V. faba protein macromolecules in aqueous pod-valve extracts prepared in ultrapure water or naturally acidic media, including 2% Phyllanthus emblica, 5% Punica granatum, and 2% Ribes rubrum. Protein profiles were first evaluated by SDS-PAGE and subsequently analyzed by nanoflow liquid chromatography coupled to high-resolution tandem mass spectrometry (nLC-MS/MS). Protein identifications were complemented with Gene Ontology annotation and a descriptive semi-quantitative assessment of relative protein representation across extraction media. Chitinase was the most represented V. faba-assigned protein macromolecule across the extracts, with additional highly represented proteins including glucan endo-1,3-beta-D-glucosidase, pathogenesis-related proteins, and polyphenol oxidase A1. These co-extracted proteins are mainly associated with plant defense, stress responses, cell-wall remodeling, and oxidative processing, suggesting that they may be relevant for extract quality attributes during handling and storage. This study provides a compositional proteomic reference for the co-extracted protein macromolecules present in acidic aqueous extracts from V. faba pod valves, supporting future studies on extract stability, processing optimization, and the development of standardized plant-based preparations.

Vicia faba

Resolution of the light-harvesting chlorophyll a/b-protein of vicia faba chloroplasts into two different chlorophyll-protein complexes.

Thylakoids of Vicia faba chloroplasts disaggregated by sodium dodecyl sulfate were separated by means of different electrophoretic systems. Under the conditions of a high resolving gel system the chlorophyll containing zone previously termed chlorophyll-protein complex II or light-harvesting chlorophyll a/b-protein was found to be inhomogeneous. It represents a mixture of two distinct chlorophyll-proteins characterized by different spectral properties and different apoproteins. One chlorophyll-protein exhibits a chlorophyll a/b ratio of 0.9 and is associated with polypetides of 24,000 and 23,000 daltons. The 24,000 dalton band is proved to bind chlorophyll and has a light-harvesting function. The function of the 23,000 dalton band is unknown. The second chlorophyll-protein has a chlorophyll a/b ratio of 2.1 and an additional absorption maximum in the position of 637 nm. It is associated with only one polypeptide which has an apparent molecular weight of 23,000. The two 23,000 dalton polypeptides occurring in both complexes are not identical.

Chlorophyll

Analysis of the Vicia faba genome by use of restriction endonucleases.

DNA of the broad bean, Vicia faba, was cleaved by the restriction endonucleases endoR . EcoRI, endoR . HindIII, endoR . HincII, endoR . BamI, and endoR . BspRI. Separation in agarose gels of the resulting fragments revealed, in addition to the bulk DNA, an enzyme-specific pattern of bands composed of restriction fragments of 300 to more than 30,000 base pairs in length. Bulk DNA was characterized by an unusual size distribution which significantly deviated from that expected according to the random fragmentation theory. It is argued that the observed distribution is due to the high proportion of repetitive DNA within this species (approximately equal to 75%). In all digests, a class of high-molecular-weight restriction fragments of more than 30,000 base pairs in length was observed which comprised 5-8% of the genome. It showed hybridization with highly repetitive DNA (c0t less than or equal to 2 x 10(-2) M . s) and included a fraction (2-3% of the genome) highly resistant to the activity of all the enzymes tested. The buoyant density in CsCl of this resistant DNA was not different from that of the total DNA (36% dG + dC). In endoR . EcoRI digests, the high-molecular-weight fragment class contained, in addition to the resistant DNA, a fraction of relatively high buoyant density (calculated dG + dC content: 61%) containing cleavage sites for the other enzymes used.

DNA

Effect of raw field bean (Vicia faba) on amino-acid-degrading enzymes in rats and chicks.

The effects of Vicia faba diet on urinary nitrogenous compounds and on enzyme activities of pathways directly associated with amino acid metabolism were studied in rats and chicks. The urea and creatinine excretion of rats fed on V. faba was approximately 90% more than that of control rats. The V.-faba-fed rats had increased activities of liver arginase (EC 3.5.3.1), argininosuccinate synthetase (EC 6.3.4.5) and alanine aminotransferase (EC 2.6.1.2). The chicks fed on V. faba also showed increased activity of xanthine dehydrogenase (EC 1.2.3.2). The possible nature of these altered amino-acid-degrading enzyme activities is discussed.

Alanine Transaminase

RBE and OER values of negative pion beams from growth inhibition of Vicia faba roots.

Two pion beams of different momentum width have been used to expose meristems of Vicia Faba roots under aerobic and hypoxic conditions. The measurements of the resulting 10 days growth inhibition after exposures at various locations on the pion beam axes have been made and RBE and OER values evaluated for 50% effects compared to 60Co gamma-rays. The results have been related to the fractional doses from star products defined by telescope measurements of stopped pions along the same beams. It has been found that the RBE value increases with the fractional "star dose" up to a maximum after which the RBE decreases. The OER values, however, were found to decrease with increasing "star dose" fraction rather rapidly after which it was found to be independent of the "star dose" contribution.

Dose-Response Relationship, Radiation

The chemical characterization of favin, a lectin isolated from Vicia faba.

We have determined the subunit structure of the glucose- and mannose-binding lectin favin, from Vicia faba. The molecule is composed of two nonidentical polypeptide chains held together by noncovalent interactions. We have determined the complete amino acid sequence of the smaller alpha chain (Mr = 5,571) and shown that it is homologous to the alpha chain of the lectins from lentil and pea and to residues 72 to 120 of concanavalin A (Con A). The larger beta chain (Mr = 20,000) contains carbohydrate and is homologous to the beta chain of lentil, pea, soybean, peanut, and red kidney bean lectins and is homologous to a portion of the Con A molecule beginning at residue 122. Favin also contains a minor component, beta' (Mr = 18,700), that closely resembles the beta chain but lacks carbohydrate and may, on the basis of apparent molecular weight, lack some part of the COOH-terminal region of the polypeptide chain. Although favin is similar to Con A, it, like the lentil and pea lectins, appears to lack residues corresponding to positions 1 to 71 of Con A. Because these residues contribute significantly to the carbohydrate binding site of Con A, the lack of this region in the otherwise homologous lectin favin suggests that the carbohydrate binding site of favin differs from that of Con A or that the region represented by residues 1 to 71 of Con A is located in a different portion (i.e. in the beta chain) of the favin molecule.

Amino Acid Sequence

Affinity chromatography of human serum proteins using matrix bound lectins from Viscum album and Vicia faba.

The D-galactose specific lectin from Viscum album and the D-mannose specific lectin from Vicia faba react with serum proteins which contain the corresponding carboyhydrate moieties. By affinity chromatography of human serum using the combination of insolubilized lectins coupled to Sepharose it is possible to fractionate serum proteins in 3 groups: 1. proteins which react with both of the lectins; 2. proteins which react only with one of the lectins; 3. proteins which do not display any affinity for one of the two lectins.

Blood Proteins

Deoxyribonucleotide synthesis and DNA polymerase activity in plant cells (Vicia faba and Glycine max).

Enzymes of deoxyribonucleotide and DNA biosynthesis, which are little known in plants, were studied in root tips of germinating broad beans (Vicia faba) and in fast-growing cultures of soybean cells (Glycine max). The plant cells contain a ribonucleoside 5'-diphosphate reductase which is detected in vitro only during a limited period of growth, viz. 30--32 h after inhibition of Vicia seeds, and between the second and third day after inoculation of soybean cultures. In both species ribonucleotide reductase activity precedes maximum DNA synthesis. The reductases could be precipitated with ammonium sulfate but were not purified further due to the extremely low enzyme content of the plant extracts. Therefore the reductive pathway of deoxyribotide formation was also established in Vicia root tips by efficient labeling of the plant DNA with a ribonucleoside, [5-3H]cytidine, which reaches a maximum at the same time as the reductase activity measured in vitro. Cycloheximide inhibits this process, indicating the need for de novo enzyme induction. In contrast, DNA polymerase is present in the tissue throughout the entire development and rises only 2-fold in activity during the S phase. The soluble polymerases were partially characterized in both legume species and were found very similar to the DNA polymerase of pea seedlings. Ribonucleotide reductase is more likely a limiting component of DNA formation during the plant cell cycle than DNA polymerase.

DNA

Nutritional effects of field bean (Vicia faba L.) proteinase inhibitors fed to rats.

1. Weanling rats were fed on purified-casein diets containing active and inactive (autoclaved) field bean (Vicia faba L.) proteinase inhibitors (PI) at levels of 1.25, 2.5, 5.0 and 10 g/kg. Diets containing raw- and autoclaved-field-bean meal (FBM) were also given. The body-weight gain, protein efficiency ratio (PER); i.e. the ratio of the live weight gain in g/g of protein consumed and apparent nitrogen digestibility were determined 7, 14 and 21 d after the start of the trial. 2. Significant growth depression (P less than 0.001) was observed in rats given diets containing 2.5 g/kg PI (27--35%). In rats fed on raw FBM growth depression was severe after 7 d (57%). 3. Food intake was depressed at PI inclusion levels of 5 g/kg and higher between 14 and 21 d. 4. PER was depressed significanlty (33-44%) in rats given diets containing the active PI at all levels after 7 d. The protein utilization of all casein diets was higher than that of the FBM diets. 5. Apparent N digestibility was depressed in rats fed on diets containing 10 g/kg PI by 9 and 7% at 14 and 21 d respectively. The N digestibility of both raw and heated FBM was similar at approximately 84%. 6. It was concluded that the PI are not the main factors responsible for the growth depression observed when raw FBM is fed to rats.

Animals

Protein quality in cereals and pulses. 2. Influence of polyethyleneglycol on the nutritional availability of methionine in sorghum (Sorghum vulgar Pers.), field beans (Vicia faba L.) and barley.

1. Polyethyleneglycol (PEG 4000) was examined for its influence on relative nutritional value (RNV) and available methionine in sorghum (Sorghum vulgare Pers.), field beans (Vicia faba L.) and barley, as measured microbiologically with Streptococcus zymogenes. The results were assessed in relation to the content of tannins in the test samples. 2. In grain of hybrid sorghum the RNV averaged 87 (range 79--92) for six low-tannin varieties and 41 (30--53) for eleven high-tannin varieties. The corresponding available methionine values averaged 17.0 (15.7--18.9) and 8.9 (6.7--11.0) g/kg protein. Addition of PEG 4000 to the test samples increased the average RNV of the high-tannin varieties from 41 to 78, and the average available methionine content from 8.9 to 16.2 g/kg protein. 3. With seed of ten coloured flowered varieties of field beans, treatment with PEG gave a small but consistent increase in the available methionine content, which resulted from the inactivation of tannins in the testa. 4. In twenty-three samples of barley grain, treatment with PEG had no effect on the values obtained for available methionine. 5. Treatment of high-tannin sorghum grain with ammonia has been reported to inactivate the tannins and increase the nutritional value for rats and chicks. This finding was confirmed. The present study showed that ammonia and PEG 4000 were equally effective in enhancing the nutritional quality as measured in the microbiological tests.

Dietary Proteins

Protein quality in cereals and pulses. 1. Application of microbiological and other in vitro methods in the evaluation of rice (Oryza sativa L.), sorghum (Sorghum vulgare Pers.), barley and field beans (Vicia faba L.).

1. The Streptococcus zymogenes assay procedure was modified and used in the estimation of available methionine and relative nutritional value in rice (Oryza sativa L.), sorghum (Sorghum vulgare Pers.), barley and field beans (Vicia faba L.). The results were assessed in relation to the content of tannins and 'dye-binding lysine', and to published information on the nutritional quality of some of the test samples. 2. In grain of ten varieties of sorghum, for which other workers had reported a correlation (r -0.82; P less than 0.01) between tannin content and amino acid digestibility in chicks, the available methionine content ranged from 6.3 to 17.7 g/kg protein (nitrogen x 6.25) and was highly correlated with tannin content (r -0.97; P less than 0.001). The content of total methionine and dye-binding lysine varied little between varieties and was not related to tannin content. 3. In nine samples of rice the availability of methionine was uniformly high, in accordance with literature values for true digestibility of the N. 4. In field beans the presence of tammins in the seed coat was associated with a significant (P less than 0.001) reduction in the availability of methionine. In eleven tannin-free varieties the coefficient of availability averaged 0.791, compared with 0.685 in eleven tannin-containing varieties. The standard deviation associated with both values was 0.04. There was no such difference between the corresponding dye-binding lysine values. 5. Thirty-three samples of barley were examined, representing fourteen varieties, four of which had been grown at several widely different latitudes in England and Sweden. There was little difference in methionine content between samples. The average availability coefficient for eighteen samples grown in England was 0.995+/-0.044, and for fifteen varieties grown in Sweden it was 0.851+/-0.042. The difference was not associated with any difference in tannin content, which was uniformly low. The findings contradict published evidence for marked increase in tannin content with increase in geographical latitude of cultivation, associated with decrease in digestibility of N.

Biological Assay

Protein quality in cereals and pulses. 3. Bioassays with rats and chickens on sorghum (Sorghum vulgare Pers.), barley and field beans (Vicia faba L.). Influence of polyethylene glycol on digestibility on the protein in high-tannin grain.

1. Two preceding papers in this series describe the application of microbiological and other in vitro tests in the evaluation of sorghum (Sorghum vulgare Pers.), field beans (Vicia faba L.) and barley, and in assessing the influence of polyethylene glycol (PEG 4000) on the nutritional availability of the methionine. The present paper gives for comparison the results of bioassays on some of the same test samples. Net protein utilization (NPU) in rats was measured by the nitrogen balance method, and N digestibility in chickens by the ileal analysis procedure. 2. In rat tests on sorghum, N in grain of high-tannin varieties was poorly digested. Supplementation of the test diets with 0.1 g PEG 4000/g protein gave a large improvemnet, which was partly offset by an apparent decrease in biological value (BV). With chickens N digestibility was even lower, and was similarly improved with PEG 4000. Treatment of high-tannin grain with ammonia solution was also effective in improving N digestibility. 3. With low-tannin sorghum the amino acid digestibilities were uniformly high and were not affected by addition of PEG to the test diet. With high-tannin sorghums they were low and less uniform, and were much improved by PEG 4000. 4. With field beans, the influence of the seed-coat tannin on protein utilization was much less pronounced than with sorghum. In chickens there was a significant effect (P less than 0.05) of PEG 4000 on N idgestibility in a high-tannin variety. With rats the effect was smaller and not significant. 5. In four samples of barley. N digestibility was high (0.87--0.96) and was not further improved by PEG 4000. The BV of a high-lysine cultivar proved marginally inferior to that of a normal variety. Possible reasons for this are discussed. 6. Over all, the results were closely consistent with those from microbiological tests with Streptococcus zymogenes.

Animals