Purification and characterization of arcelin seed protein from common bean

Arcelin, a seed protein originally discovered in wild bean accessions, was purified, characterized, and compared to phaseolin, the major seed protein of common bean, and to phytohemagglutinin (PHA), the major bean seed lectin. Arcelin and PHA has several characteristics in common. Both were glycoproteins having similar subunit M_r, deglycosylated M_r, and amino acid compositions. The two proteins were related antigenically and they had the same developmental timing of accumulation. Arcelin also had some hemagglutinating activity, a characteristic associated with lectins. However, several features distinguished arcelin from PHA. Arcelin had a more basic isoelectric point than PHA, greater numbers of basic amino acid residues, additional cysteine residues, and one methionine residue, which PHA lacks. Native PHA protein is a tetramer of subunits, and although a small component of native arcelin protein was also tetrameric, most of the arcelin preparation was dimeric. The hemagglutinating activity of arcelin was specific only for some pronase-treated erythrocytes. It did not agglutinate native erythrocytes, nor did it bind to thyroglobulin or fetuin affinity resins as did PHA. Although arcelin has lectin-like properties, we believe the distinctions between arcelin and PHA warrant the designation of arcelin as a unique bean seed protein.     

Phaseolin variability among wild and cultivated common beans (Phaseolus vulgaris) from Colombia

Phaseolin seed protein variability in a group of 8 wild and 77 cultivated common bean (Phaseolus vulgaris) accessions was determined using 1-dimensional SDS/ PAGE and 2-dimensional IEF-SDS/PAGE. Wild common bean accessions exhibited the 'CH' and 'B' patterns, previously undescribed among either wild or cultivated common beans. The cultivated genotypes showed (in decreasing frequency) the previously described 'S,' T,' and 'C phaseolin patterns as well as the new 'B' pattern similar to the pattern identified in a Colombian wild common bean accession. In the northeastern part of the Colombian bean-growing region, the cultivars exhibited almost exclusively an 'S' phaseolin type, while in the south-western part, the 'T' and 'C phaseolin cultivars were more frequent. Seed size analysis indicated that 'T' and 'C' phaseolin cultivars had larger seeds than 'S' and 'B' phaseolin cultivars. Our results suggest that Colombia is a meeting place for Andean and Middle American common bean germplasms, as well as a domestication center for the common bean.     

Bean lectins

Summary Seeds of forty bean cultivars having different lectin types based on two-dimensional isoelectric focusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF-SDS/PAGE) were analyzed for quantities of lectin, phaseolin and total protein. Significant differences were found among groups of cultivars with different lectin types for the quantity of lectin and phaseolin. Cultivars with more complex lectin types based on IEF-SDS/PAGE tended to have higher quantities of lectin and lower quantities of phaseolin than cultivars with simple lectin types. An association between lectin type and the quantity of lectin and phaseolin was found also in the seeds of F₂ plants that segregated in a Mendelian fashion for two lectin types. Seeds from plants with the complex lectin type had more lectin and less phaseolin than seeds from plants with the simple lectin type. Therefore, the genes controlling qualitative lectin variation also may influence the quantitative variation of lectin and phaseolin. The results of this study are related to other studies on the quantitative variation for seed proteins and to the possible molecular basis for variation in the quantity of lectins in beans.     

Glomerular lysozyme binding in protein-overload proteinuria

Binding of the cationic molecule lysozyme to the glomerular basement membrane and to the glomerular epithelial cell coat was investigated in the glomerulus of normal female Wistar rats and in rats in which heavy proteinuria was induced by the daily administration of 1 g of bovine serum albumin. In normal rats the binding of lysozyme to the anionic groups in the glomerular basement membrane and the cell coat had no effect on the ultrastructure of the glomerular epithelial cell, in particular the foot processes were unchanged. In the proteinuric rats the lysozyme-binding to the glomerular basement membrane and the epithelial cell coat was completely lost in the damaged glomeruli. In the apparently normal glomeruli present in these proteinuric animals binding was similar to that seen in normal rats. These results suggest that in protein-overload proteinuria there is a loss of glomerular anion and hence a reduction in the glomerular charge barrier. This may account, at least in part, for the increased glomerular leak of negatively charged serum albumin in this experimental model of proteinuria.     

PHOTOSYNTHESIS AND RESPIRATION OF ALPINE LICHENS

Photosynthetic and respiratory rates were studied in Cetraria islandica, C. nivalis, and Cladonia rangiferina in the alpine zone of Mt. Washington, New Hampshire. Measurements were made in the field using an infrared gas analyzer; light, temperature, and thallus water content were varied. In all species, considerable reduction in photosynthesis and respiration occurred with drying, more rapidly in photosynthesis than respiration. Optimal photosynthetic rates in all 3 species occurred at 15–20 C with light levels of 1,600 ft-c. Light compensation points ranged from 200 to 350 ft-c. Optimal respiratory rates were attained at 15 C in the 2 species of Cetraria and at 20 C in Cladonia. The data indicate that these wide-ranging, arctic-alpine and arctic-temperate lichens on Mt. Washington are quite well adapted to a moist, foggy environment with cool temperatures and low light levels, conditions which predominate in summer.     

Suppression of phaseolin and lectin in seeds of common bean, Phaseolus vulgaris L.: increased accumulation of 54 kDa polypeptides is not associated with higher seed methionine concentrations

Suppression of phaseolin and lectin accumulation in common bean resulted in higher concentrations of bean seed polypeptides with apparent molecular weights of 54 kDa and from 70 to 84 kDa on SDS-polyacrylamide gel electrophoresis. Polypeptides of 54 and 56 kDa segregated as products of different alleles. Genes for the 54/56 kDa bands and phaseolin were estimated to be 26.2±3.7 map units apart. The 54 kDa band phenotype manifested by SDS-PAGE consisted of from one to three polypeptides of 54 kDa MW on 2D gels, and the 56 kDa phenotype consisted of one polypeptide of 56 kDa plus two minor polypeptides of 54-54.5 kDa molecular weight. The pK_I of these polypeptides was approximately 5.25. The methionine content of the 54 kDa polypeptides of the cultivar Great Northern Star was 1.6±0.1 g/100 g protein, which was not statistically different from the value (1.5±0.1%) obtained for phaseolin isolated by the same procedure. F₂ seeds deficient for phaseolin and lectin contained as much total N per g as wild-type seeds and were not shrunken, but contained 50% more free amino acids. F₂ seeds from two of the three populations contained from 8 to 13% less methionine per mg total N.     

Genotypic variation in biological nitrogen fixation by common bean

Field experiments were performed in Austria, Brazil, Chile, Colombia, Guatemala, Mexico and Peru as part of an FAO/IAEA Co-ordinated Research Programme to investigate the nitrogen fixing potential of cultivars and breeding lines of common bean (Phaseolus vulgaris L.). Each experiment included approximately 20 bean genotypes which were compared using the ¹⁵N isotope dilution method. Great differences in nitrogen fixation were observed between and within experiments, with average values of 35% N derived from atmosphere (% Ndfa) and highest values of 70% Ndfa being observed. These values which were larger than had been reported previously for common bean, were observed only when environmental factors were favorable. Therefore, common bean lines are available, which can support high biological nitrogen fixation. These can be used either directly as cultivars for production or in breeding programmes to enhance nitrogen fixation in other cultivars.     

Protein Synthesis and Accumulation in Bean Cotyledons during Growth

Analysis of total protein, of specific proteins by gel electrophoresis and immunoelectrophoresis, and of protein synthetic activity in vitro confirmed that intense protein synthesis and accumulation occurred as the French bean (Phaseolus vulgaris L). seed grew from 12 to 20 millimeters. These techniques showed that there was no globulin-1 (G1) fraction (requiring high salt for solubility) present in 6-millimeter seeds, and only very small amounts were synthesized in seeds less than 9 millimeters long. The 7- to 9-millimeter stages represent a 2-day transition period over which genetic information for the G1 protein becomes actively expressed, accounting for at least 50% of all protein synthesized in this tissue during the following 14 days. At maturity, the electrophoretic analysis confirmed that G1 globulin was the major storage protein, representing some 50% of the dry seed protein. Cell-free protein synthesis assays, including immunoprecipitation of the in vitro products, clearly showed G1 polypeptides to be among the polysome-directed products.     

Purification and characterization of an (Na++K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain

Highly purified lamb kidney (Na⁺+K⁺)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na⁺+K⁺)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (γ1 and γ2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that γ1 is an aggregated form of γ2. The total yield was 0.8–1.5 mol of γ component per mol of catalytic subunit recovered. This indicates that the γ component is present in stoichiometric amounts in the (Na⁺+K⁺)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids.     

Bean lectins

Summary Single seeds of over 100 bean cultivars were analyzed by two-dimensional electrophoresis. The cultivars could be classified into eight groups by virtue of their G2/albumin electrophoretic patterns: T_G2, S_G2, V_G2, Pr_G2, B_G2, M_G2, P_G2, and Pi_G2, The polypeptide compositions of these types were largely inter-related having particular polypeptides in common. It was possible to correlate the G2/albumin patterns with agglutinating activity of cow and rabbit blood cells as measured by the agglutination ratio (minimum concentration of extract required to agglutinate cow blood cells: minimum concentration of extract required to agglutinate rabbit blood cells). The active lectin polypeptides were identified by extracting lectins from agglutinated erythrocytes and by comparing the qualitative similarities and differences of the G2/albumin patterns and their agglutination activities. A reference catalogue of over 100 bean cultivars giving their phaseolin and G2/albumin electrophoretic patterns, and agglutination ratios is presented.