Characterization and comparison of arcelin seed protein variants from common bean

Four variants of arcelin, an insecticidal seed storage protein of bean, Phaseolus vulgaris L., were investigated. Each variant (arcelin-1, -2, -3, and -4) was purified, and solubilities and M_rs were determined. For arcelins-1, -2, and -4, the isoelectric points, hemagglutinating activities, immunological cross-reactivities, and N-terminal amino acid sequences were determined. On the basis of native and denatured M_rs, the variants were classified as being composed of dimer protein (arcelin-2), tetramer protein (arcelins-3 and -4), or both dimer and tetramer proteins (arcelin-1). Although the dimer proteins (arcelins-1d and -2) could be distinguished by M_rs and isoelectric points, they were identical for their first 37 N-terminal amino acids and had similar immunological cross-reactions, and bean lines containing these variants had a DNA restriction fragment in common. The tetramer proteins arcelin-1t and arcelin-4 also could be distinguished from each other based on M_rs and isoelectric points; however, they had similar immunological cross-reactions and they were 77 to 93% identical for N-terminal amino acid composition. The similarities among arcelin variants, phytohemagglutinin, and a bean α-amylase inhibitor suggest that they are all encoded by related members of a lectin gene family.     

Altering protein composition by genetically removing phaseolin from common bean seeds containing arcelin or phytohemagglutinin

 Arcelin seed proteins of common bean (Phaseolus vulgaris L.) confer resistance to bruchid pests, and in vitro results suggested that greater resistance could be achieved by increasing the concentration of arcelin. We created backcross lines having arcelin alleles (SMARC lines), or alleles for the related protein phytohemagglutinin (PHA) (SMPHA lines), and a null allele for phaseolin to determine if seeds lacking phaseolin would contain increased quantities of arcelin or PHA proteins. To test the affects of genetically removing phaseolin, SMARC and SMPHA lines were derived as pairs of phaseolin-containing and phaseolin-null lines. Parental, SMARC, and SMPHA lines were grown in a replicated greenhouse trial and measured for days-to-flower, days-to-maturity, seed weight, and for quantities of phaseolin, arcelin dimer, PHA, and total proteins. There were no differences between pairs of phaseolin and phaseolin-null lines for days-to-flower, seed weight or total protein, and inconsistent differences for days-to-maturity. Arcelin concentrations were significantly increased in two of four pairs of SMARC lines, and PHA concentration was significantly greater in four of five pairs of SMPHA lines. These or other changes in the seed protein composition in phaseolin null lines may improve resistance to bruchids. Received: 2 April 1997 / Accepted: 20 May 1997     

Purification and characterization of a protein phosphatase from winged bean.

A protein phosphatase (WbPP) has been purified from the soluble fraction of the winged bean (Psophocarpus tetragonolobus) shoot extract. The preparation is essentially homogenous as shown by the constant specific activity of the enzyme across the peak fractions, eluted from the thiophosphorylated histone-Sepharose affinity column, the last step of purification and by single protein bands on polyacrylamide gel electrophoresis (PAGE) in the presence as well as absence of denaturating agents. The monomeric nature of WbPP is revealed by an M(r) of 92,000 and 85,000, respectively, as estimated by SDS-PAGE and gel permeation chromatography under non-denaturating conditions. Autophosphorylated calmodulin-like domain protein kinase (P-WbCDPKI) [Saha, P., & Singh, M. (1995). Biochem. J., 305, 205] and phosphohistone H1 (P-hisH1), prepared by using the other homologous CDPK, i.e. WbCDPKII [Ganguly, S., & Singh, M. (1998). Phytochemistry, 48(1), 61], are good substrates of the purified enzyme, while P-hisH1 and phosphocasein prepared by using heterologous cAMP-dependent protein kinase, are respectively very poor and totally inactive as substrate. WbPP is adjudged to be a protein phosphoserine phosphatase since phosphoserine is the only phosphorylated amino acid residue detected in our earlier analysis of P-WbCDPKI and P-hisH1. The enzyme is strongly stimulated by a combination of Mg2+ and Ca2+, without being dependent on either of them and is also unaffected by calmodulin and fluphenazine. Orthovanadate strongly inhibits the enzyme while okadaic acid is a poor inhibitor.     

Purification and characterisation of a novel papain inhibitor from common bean (Phaseolus vulgaris) seed

A proteinaceous inhibitor of papain was purified to apparent homogeneity from mature seeds of common bean ( Phaseolus vulgaris L.). After four chromatographic steps, the papain inhibitor was purified 219‐fold with 12% recovery. On the basis of papain inhibitory activity, cystatins have been estimated to account for about 0.1% of the total protein content of mature common bean seeds. The purified protein, as other plant cystatins, is an acidic protein, heat stable and insensitive to reducing agents. Its molecular mass is about 37 kDa as judged by size exclusion chromatography and SDS‐PAGE. Moreover it is immunologically related to oryzacystatins, since it is recognised by a specific oryzacystatin I antiserum. Based on its biochemical properties the papain inhibitor described here belongs to the phytocystatin family. Papain inhibitory assays carried out during seed development showed that bean cystatin is active since early maturation stages. Our results suggest that, in common bean seed, cysteine proteinase inhibitors are important during seed development with a putative role in the control and regulation of endogenous thiol protease activity.     

Post-translational processing of two alpha-amylase inhibitors and an arcelin from the common bean, Phaseolus vulgaris

Mass spectrometric methods were used to investigate the proteolytic processing and glycopeptide structures of three seed defensive proteins from Phaseolus vulgaris. The proteins were the alpha-amylase inhibitors alphaAI-1 and alphaAI-2 and arcelin-5, all of which are related to the seed lectins, PHA-E and PHA-L. The mass data showed that the proteolytic cleavage required for activation of the amylase inhibitors is followed by loss of the terminal Asn residue in alphaAI-1, and in all three proteins, seven or more residues were clipped from the C-termini, in the manner of the seed lectins. In most instances, individual glycoforms could be assigned at each Asn site, due to the unique masses of the plant glycopeptides. It was found that alphaAI-1 and alphaAI-2 differed significantly in their glycosylation patterns, despite their high sequence homology. These data complement the previous X-ray studies of the alpha1-amylase inhibitor and arcelin, where many of the C-terminal residues and glycopeptide residues could not be observed.     

Genetic mapping of microsatellite markers around the arcelin bruchid resistance locus in common bean

The deployment in common beans (Phaseolus vulgaris L.) of arcelin-based bruchid resistance could help reduce post-harvest storage losses to the Mexican bean weevil [(Zabrotes subfasciatus (Boheman)]. Arcelin is a member of the arcelin-phytohemagglutinin-α-amylase inhibitor (APA) family of seed proteins, which has been extensively studied but not widely used in bean breeding programs. The purpose of this study was to evaluate microsatellite markers for genetic analysis of arcelin-based bruchid resistance and to determine the orientation of markers and the rate of recombination around the APA locus. A total of 10 previously developed microsatellites and 22 newly developed markers based on a sequenced BAC from the APA locus were screened for polymorphism and of these 15 were mapped with an F₂ population of 157 individuals resulting from a susceptible × resistant cross of SEQ1006 × RAZ106 that segregated for both the arcelin 1 allele and resistance to the bruchid, Z. subfasciatus. Microsatellites derived from APA gene sequences were linked within 0.8 cM of each other and were placed relative to the rest of the b04 linkage group. In a comparison of genetic to physical distance on the BAC sequence, recombination was found to be moderate with a ratio of 125 kb/cM, but repressed within the APA locus itself. Several markers were predicted to be very effective for genetic studies or marker-assisted selection, based on their significant associations with bruchid resistance and on low adult insect emergence and positions flanking the arcelin and phytohemagglutinin genes.     

Bruchid resistance of common bean lines having an altered seed protein composition

 Arcelin seed proteins of common bean (Phaseolus vulgaris L.) are toxic to one of the most damaging pests of bean seeds, Zabrotes subfasciatus (Boheman), but they appear to have little effect on another important bean pest, Acanthoscelides obtectus (Say), when introduced into standard cultivars by backcrossing. With the goal of increasing arcelin concentration to improve resistance, we modified seed-protein composition by introducing a null allele for the major seed protein, phaseolin, into lines (SMARC1, 2 and 4) or three phytohemagglutinin types (SMPHA lines). These lines were tested for resistance to both insects by measuring percentage insect emergence (%E) and days-to-adult emergence (DAE). For SMARC lines, arcelin type was the most important factor in resistance levels, with SMARC1 lines being most resistant, SMARC2 lines intermediate, and SMARC4 lines the least resistant to both bruchids. Additionally, the absence of phaseolin was a significant factor in the resistance of SMARC lines to A. obtectus. SMARC1 lines without phaseolin had half the percentage insect emergence of lines with phaseolin. SMARC1 lines with an altered seed composition had the highest levels of resistance to both bruchids of any large-seeded line reported to-date. Received: 2 April 1997 / Accepted: 20 May 1997     

Iron accumulation in seed of common bean

The effect of soil and genotype on iron concentration [Fe] in common bean (Phaseolus vulgaris L.) seed was studied in the greenhouse. Liming an acid soil increased soil pH from 6.0 to 7.3 but had no effect on seed [Fe] of three bean genotypes (Voyager, T39, UI911) from the Middle American gene pool in North Dakota. However, liming decreased seed-manganese concentration [Mn]. The influence of FeEDDHA on Fe accumulation in seed of the three bean genotypes, grown on acid (pH=6.0) and naturally calcareous (pH=8.2) soils, was also studied in North Dakota. Seed from the acid soil contained 25% higher [Fe] than seed from the calcareous soil. FeEDDHA increased seed [Fe] only on the calcareous soil, but reduced seed [Mn] on both soils. Voyager seed, characterized by a relatively low [Fe] in the seed coat, had a higher seed [Fe] than the other two genotypes. The hypothesis that high seed [Fe] is characterized by a low seed-coat [Fe] was next investigated. Voyager, T39 and 10 diverse Latin American genotypes from the Middle American gene pool were grown on a soil (pH=7.0) with Andic properties in Mexico in the presence and absence of FeEDTA. FeEDTA increased seed [Fe]. Seed of Voyager and a Mexican genotype (Bayo 400) had the highest seed [Fe]. However, Bayo 400, unlike Voyager, contained a high percentage of its seed Fe in the seed coat. Consequently, a high seed [Fe] genotype does not necessarily have a low seed-coat [Fe]. Both soil and genotype affect Fe accumulation in bean seed.     

Purification and characterization of leucine aminopeptidase from kidney bean cotyledons

A leucine aminopeptidase (EC 3,4,11.1) was purified from cotyledons of resting kidney beans ( Phaseolus vulgaris L. cv. Processor) by acidic extraction, ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, Mono Q HPLC and Superose HPLC columns. The yield of the 317-fold purified enzyme was 9%. On gel filtrations on Sephacryl S-300 and Superose HPLC the elution volumes of the enzyme corresponded to an M, of 360 000. The enzyme gave one band on native gel electrophoresis and an electrophoretic titration in an immobilized pH gradient gave a single curve with a pI of 4.8. Two bands were observed in an SDS-gel electrophoresis with M_r values of 58 000 and 60 000 both with and without reduction by 2-mercaptoethanol, indicating that subunits of the enzyme are not linked by disulphide bridges. The purified enzyme most rapidly liberated Leu and Ala of the N-termini of di-and oligopeptides, optimally at pH 9.0 ± 0.5. The enzyme was stable in the presence of glycerol, dithiothreitol and Mg²⁺, while the latter also had an activating effect. Bestatin inhibited the enzyme competitively with Leu-Gly-Gly with a Kⁱ-value of 1.5 nM . These observations indicate that the purified aminopeptidase from the cotyledons of resting kidney beans corresponds to the cytosolic leucine aminopeptidase of mammalian tissues (EC 3.4, 11.1). The high enzyme activity observed suggests that this aminopeptidase has an important role in the production of free amino acids during germination.     

Purification and partial characterization of an aminopeptidase from mung bean cotyledons

An aminopeptidase (EC 3.4.11.-) was purified to homogeneity, as judged by SDS-PAGE. from mung bean ( Vigna radiata ) cotyledons. The molecular mass of this peptidase was estimated as 75 kDa by gel filtration. When an oligopeptide consisting of 5 amino acid residues was used as substrate, amino acids were released in the order of the N-terminal sequence of the oligopeptide chain. This enzyme apparently requires free sulfhydryl for its activity, as judged by the effects of various proteinase inhibitors. Among aminoacyl- p -nitroanilides examined for the availability as substrates of the enzyme, p -nitroanilides with hydrophobic amino acids were preferred substrates. According to western immunoblot profiles, the enzyme level in cotyledons was high at the early stage of imbibition and declined rapidly after germination.     

Purification and characterization of tonoplast ATPase from etiolated mung bean seedlings

The tonoplast ATPase from etiolated seedlings of Vigna radiata L. (mung bean) was isolated using a two-step detergent solubilization modified from Mandala and Taiz (S Mandala, L Taiz [1985] Plant Physiol 78: 327-333). After ultracentrifugation on 10 to 28% sucrose gradient, the ATPase showed a 31.6-fold purification over the initial specific activity of the starting tonoplast-enriched membranes. The purified ATPase used Mg²⁺-ATP as the preferred substrate. The tonoplast ATPase was isolated in a form with characteristics similar to that on its native membrane environment. Analysis by SDS-PAGE revealed two prominent bands with molecular weights of 78,000 (α subunit) and 64,000 (β subunit). The intensity of Coomassie blue staining showed a 1:1 stoichiometry for α and β subunits. The amino acid composition of α and β subunits also confirmed the suggested stoichiometry of the subunit composition of the tonoplast ATPase. Moreover, radiation inactivation analysis yielded a functional size of 414 ± 24 and 405 ± 25 kilodaltons for soluble and membrane bound tonoplast ATPases, respectively. It is possible that the functioning tonoplast ATPase may be in a form of αβ-heteromultimer.     

Purification of the major UsnRNPs from broad bean nuclear extracts and characterization of their protein constituents.

Small nuclear ribonucleoprotein particles containing the five major nucleoplasmic snRNAs U1, U2, U4, U5 and U6 as well as two smaller sized snRNAs were purified from broad bean nuclear extracts by anti-m3G, monoclonal antibody, immunoaffinity chromatography. We have so far defined 13 polypeptides of approximate mol. wts. of 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 18.5 kd, 25 kd (double band), 30 kd, 31 kd, 35 kd, 36 kd and 54 kd. Upon fractionation of the UsnRNPs by anion exchange chromatography, essentially pure U5 snRNPs were obtained, containing the 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 35 kd and 36 kd polypeptides. These may therefore represent the common snRNP polypeptides and which may also be present in the other snRNPs. By immunoblotting studies, using anti-Sm sera and mouse monoclonal antibodies we show that the 35 kd and 36 kd proteins are immunologically related to the mammalian common B/B' proteins. The broad bean 16 kd and 17 kd proteins appear to share structural elements with the mammalian D protein. The three proteins of mol. wts. 11 kd, 11.5 kd and 12.5 kd probably represent the broad bean polypeptides E, F, and G. Cross-reactivity of proteins of mol. wts of 30 kd and 31 kd with Anti-(U1/U2)RNP antibodies suggests that they may represent the broad bean A and B" polypeptides. The 54 kd protein and the 18.5 kd protein could be candidates for the U1 specific 70 k and C polypeptides. Our results demonstrate a strong similarity between the overall structure of broad bean and mammalian snRNPs.     

Purification and characterization of rho-crystallin from Japanese common bullfrog lens

In a previous paper, we reported that the partial amino acid sequence (225 residues) from the COOH terminus of rho-crystallin from European common frog lens shows 77% similarity to that of prostaglandin (PG) F synthetase, an aldo-keto reductase, from bovine lung (Watanabe, K., Fujii, Y., Nakayama, K., Ohkubo, H., Kuramitsu, S., Kagamiyama, H., Nakanishi, S., and Hayaishi, O. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 11-15). Here rho-crystallin was purified to apparent homogeneity from the eye lens of the Japanese common bullfrog (Rana catesbeiana) by four sequential chromatographies using Sephadex G-100, Red Sepharose, and dual Mono S. Two types of rho-crystallin, RHO-I and RHO-II, named according to their elution order from a Mono S column, are essentially identical in terms of immunochemical properties, amino acid composition, and partial amino acid sequence. But the NH2-terminal Thr of RHO-I is blocked with an acyl group, while that of RHO-II is free. Both crystallins as well as PGF synthetase are monomeric proteins with a molecular weight of about 35,000 and they have the ability to bind NADPH with a stoichiometry of 0.75 mol of cofactor/mol of protein. Although rho-crystallin does not cross-react with antibody against PGF synthetase, the NH2-terminal amino acid sequence (107 residues) of rho-crystallin shows 77% similarity to that of the enzyme. However, PGD2, PGE2, 9,10-phenanthrenequinone, p-nitrobenzaldehyde, DL-glyceraldehyde, D-glucuronic acid, D-glucose, D-xylose, menadione, p-nitroacetophenone, dihydroxyacetone, succinic semialdehyde, phenylglyoxal, and testosterone were not substrates for these crystallins. PGH2 9,11-endoperoxide reductase activities of RHO-I and RHO-II were 1.3 and 1.0 milliunits/mg of protein, respectively, which are only about 2% of that of bovine lung PGF synthetase. These results indicate that the rho-crystallins RHO-I and RHO-II belong to a group of aldo-keto reductases based on primary structure, molecular properties, and NADPH-binding ability, but show only low PGH2 9,11-endoperoxide reductase activity.     

Purification and characterization of tubulin from mung bean (Vigna radiata)

Tubulin has been purified from mung bean seedling by Zn²⁺-induced polymerization. Both α- and β-subunits of mung bean tubulin are different from those of brain tubulin in electrophoretic mobility, colchicine binding and peptide map. Heterogeneity of mung bean tubulin has also been documented suggesting diversification of tubulin despite its conserved nature in general.     

Purification and characterization of the common yolk protein,vitellin, from the estuarine amphipod Leptocheirus plumulosus

Vitellin is a major yolk protein that plays a significant role in the embryonic development of crustacean embryos. This protein was rapidly purified from embryos of the estuarine amphipod, Leptocheirus plumulosus, by subjecting the crude protein homogenate to high affinity column chromatography. SDS-PAGE revealed a single band with an approximate molecular weight of 200,000 daltons. Vitellin was characterized by SDS-PAGE techniques and amino acid composition analysis. L. plumulosus vitellin is a lipoglycophosphoprotein with serine, glutamic acid/glutamine, alanine, and aspartic acid/asparagine accounting for almost 66% of all amino acid residues. Polyclonal antibodies were raised against L. plumulosus vitellin and antibody reactivity was verified by dot-blotting and immuno-fluorescence confocal microscopy. These antibodies are specific for purified vitellin and show little cross-reactivity with other embryonic proteins.     

Protein markers and seed size variation in common bean segregating populations

Selection and random genetic drift are the two main forces affecting allele frequencies in common bean breeding programs. Therefore, knowledge on allele frequency changes attributable to these forces is of fundamental importance for breeders. The changes in frequencies of alleles of biochemical markers were examined in F₂ to F₇ populations derived from crosses between cultivated Mesoamerican and Andean common bean accessions (Phaseolus vulgaris L.). Biochemical markers included the seed proteins phaseolin, lectin and other seed polypeptides, and six isozymes. The Schaffer’s test detected a high significant linear trend of the 63% of the polymorphic loci studied, meaning that directional selection was acting on those loci. Associations between seed size traits, phaseolin seed-storage protein and isozyme markers were detected based on the comparisons of the progeny genotypic means. In the interracial populations the intermediate form PhaH/T, b6, and Rbcs ⁹⁸ alleles had a positive effect on seed size. In the inter-gene pool populations, a higher transmission of Mesoamerican alleles in all loci was showed, although the Andean alleles Pha^T, Skdh ¹⁰⁰ , Rbcs ⁹⁸ , and Diap ¹⁰⁰ showed positive effects on seed weight. Our results suggest that phaseolin and other seed proteins markers are linked to loci affecting seed size. These markers have good potential for improving the results of the selection and should be considered as a strategy for germplasm enhancement and to avoid the reduced performance of the inter-gene pool populations.     

Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyls

1-Aminocyclopropane-1-carboxylate (ACC) synthase, EC 4.4.1.14, was purified to homogeneity from etiolated mung bean hypocotyl segments. This was made possible by the ability to elevate the enzyme level markedly through hormone treatments and by stabilization of the enzyme with high phosphate concentrations. The four-step procedure resulted in 1050-fold purification with 25% yield, and consisted of stepwise elution from hydroxylapatite, chromatography on phenyl-Sepharose CL-4B, gradient elution from hydroxylapatite, and fast protein liquid chromatography (FPLC) on a MonoQ anion-exchange column. FPLC-purified ACC synthase migrated as a single band of Mr 65,000 on denaturing polyacrylamide gel electrophoresis. The molecular weight of native enzyme by Bio-Gel A-0.5 M chromatography was 125,000, indicating that the enzyme probably exists as a dimer of identical 65,000 Mr subunits. The mung bean ACC synthase exhibited a pH optimum of 8.0 for activity and a Km for S-adenosylmethionine (AdoMet) of 55 microM at 30 degrees C. It exhibited an Arrhenius activation energy of 12 kcal mol-1 degree-1 and was inactivated at temperatures in excess of 40 degrees C. The specific activity for pure ACC synthase was 21 mumol of ACC formed/mg protein/h when determined under optimal conditions with 400 microM AdoMet.