Purification of multiple pea ferredoxins by chromatography at high ionic strength on unsubstituted Sepharose 4B.

At high concentrations of ammonium sulfate (3--4 M) pea ferredoxin (which is soluble under these conditions) can be adsorbed to Sepharose 4B, either by column chromatography or by batchwise treatment. A reverse (3 M to 1 M) ammonium sulfate gradient results in the elution of three peaks of ferredoxin. The spectral ratio A420/A280 of 0.47--0.54 indicates that each peak of ferredoxin is highly purified by this single step. A further gel filtration removes residual high molecular weight contaminants from the ferredoxin. The spectrum of the purified pea ferredoxin is typical of other plant ferredoxins in having absorbance peaks at 276 nm, 330 nm, 422 nm and 465 nm. Other chromatographic matrices are capable of adsorbing ferredoxin. Sepharose and Sephacryl wee the best adsorbents while Sephadex and cellulose adsorbed ferredoxin less tenaciously. The polyacrylamide-based resins Biogel P-4 and P-200 did not adsorb ferredoxin at high ionic strength.

Isolation and characterization of the trypsin inhibitors from winged bean seed (Psophocarpus tetragonolobus (L) Dc.).

The trypsin inhibitors from winged bean seed were isolated by affinity chromatography on trypsin-Sepharose 4B and the components fractionated by chromatography on SP-Sephadex C-25 and Sephadex G-100. The major components, inhibitors 2 and 3 were found to be homogeneous proteins with molecular weights of about 20,000. The inhibitors stoichiometrically inhibited bovine trypsin in the molar ratio of 1 : 1 whereas the inhibition of bovine alpha-chymotrypsin was weak and non-stoichiometric. Amino acid analysis indicated that both the inhibitors contain four cysteine residues and are rich in aspartic acid, glutamic acid, glycine, valine and leucine; however, inhibitor 3 lacks histidine and methionine while inhibitor 2 contains one histidine and three methionines. A minor trypsin inhibitor fraction was also isolated which contained at least three proteins with a molecular weight of about 10,000 and a high content of half-cystine.

A Zn2+ binding constituent of fababeans.

Binding of Zn2+ by an aqueous extract of fababeans was estimated spectrophotometrically. Treatment of the extract with phytase removed virtually all of the phytate and about 82% of the Zn2+ binding. Gel permeation chromatography of the extract showed that the Zn2+ binding factor eluted at the same volume as phytate. The major Zn2+ binding constituent of the bean extract appears to be phytate.

A precise localization of cardiolipin in plant cells.

Cardiolipin and cytochrome aa3 contents of isolated plant cells (sycamore cells) and their purified mitochondria were measured. Since the cardiolipin/cytochrome aa3 ratio was the same in the intact cells and in the isolated mitochondria it was strongly suggested that cardiolipin is present only in the mitochondria. Furthermore, outer and inner mitochondria membranes of purified sycamore cells and mung bean hypocotyl mitochondria were separated and it was shown that cardiolipin is localized in the inner mitochondrial membrane.

The glycosyl moiety of lectin from sainfoin (Onobrychis viciifolia, Scop.).

A lectin isolated from the seeds of sainfoin (Onobrychis viciifolia, Scop. var Eski) has been shown to be a glycoprotein containing 2.6% (w/w) neutral carbohydrate and 1.6% (w/w) glucosamine (Hapner, K.D. and Robbins, J.E. (1979) Biochim. Biophys. Acta 580, 186--197) A homogeneous glycopeptide accounting for 70% of the original glycoprotein carbohydrate was isolated from pronase digests of the lectin by gel filtration chromatography. Gas-liquid chromatographic and amino acid analyses showed the glycosyl portion to contain glucosamine, mannose, xylose and fucose in molar ratio to glycopeptide of 1.8 : 1.8 : 0.7 : 0.9. The amino acid sequence was determined as H2N-Ser-Asn(glycosyl)-glu-Thr-COOH. The glycosyl moiety was attached to the peptide through N-glycosidic linkage between asparagine and glucosamine.

Evidence for the presence of a [2Fe-2S] ferredoxin in bean sprouts.

An iron-sulfur protein with properties similar to those of ferredoxins found in the leaves of higher plants has been isolated from bean sprouts--a non-photosynthetic plant tissue. The bean sprout protein has a molecular mass of 12.5 kDa and appears to contain a single [2Fe-2S] cluster. The absorbance and circular dichroism spectra of the bean sprout protein resemble those of spinach leaf ferredoxin and the bean sprout protein can replace spinach ferredoxin as an electron donor for NADP+ reduction, nitrite reduction and thioredoxin reduction by spinach leaf enzymes. Although the reduced bean sprout protein (Em = -440 mV) is a slightly stronger reductant than spinach ferredoxin and appears to be less acidic than spinach ferredoxin, the two proteins are similar enough so that the bean sprout protein is recognized by an antibody raised against spinach ferredoxin.

Specificity and stability of the chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L) Dc).

The specificity of the winged bean chymotrypsin inhibitor is restricted to the chymotrypsins (EC 3.4.21.1 and EC 3.4.21.2). Trypsins (EC 3.4.21.4), elastase (EC 3.4.21.11), subtilisins (EC 3.4.21.14), proteinase K (EC 3.4.21.14) and Pronase (EC 3.4.24.4) are not inhibited. The inhibitor reacts with two molecules of chymotrypsin to form a stable complex (Mr approx. 70 0000) which was isolated by gel filtration on Sephadex G-100. When mixed with substrate, the interaction of the inhibitor with alpha-chymotrypsin is characterized by substrate-induced dissociation of the complex. In contrast, the interaction with chymotrypsin B is quantitative with no substrate-induced dissociation. The inhibitor reacts with alpha-chymotrypsin to form a 1 : 2 molar complex at all ratios of [I]/[E]; however, the interaction with chymotrypsin B is characterized by the formation of initially of a 1 : 1 molar complex at [I] greater than [E] followed by the formation of the 1 : 2 molar complex at [I] less than 2[E]; an intermediate species of Mr approx. 48 000 was demonstrated by gel filtration on Sephadex G-100. The inhibitor is stable over the pH range 2.0-11.5 and to heating up to 70 degrees C at pH 4.1 and 8.0, and up to 90 degrees C at pH 3.0. The inhibitor resists denaturation in 8.0 M urea at pH 8.0 and 4.0, is stable in 0.12 M beta-mercaptoethanol at pH 8.0; however, reduction in 8.0 M urea results in a loss of inhibitory activity. The inhibitor resists digestion with pepsin at pH 2.0, being only slowly degraded over a period of 7 days with an equimolar amount of pepsin.

Isolation and properties of a lectin from sainfoin (Onobrychis viciifolia, Scop.).

A glycoprotein capable of binding simple carbohydrates and causing hemagglutination has been isolated from seeds of the legume plant sainfoin (Onobrychis viciifolia, Scop. var Eski). The phytolectin was prepared by affinity chromatography of pH 7.0 sodium phosphate extracts on columns of Sepharose-4B containing covalently attached D-mannose. Molecular weight determinations showed the lectin to be a dimer consisting of 26 000 dalton, non-covalently associated monomers. Amino acid analyses indicated high amounts of aspartate, glutamate, threonine and serine which accounted for 41% of all amino acids. One residue of cysteine was present and methionine was totally absent. The lectin contained 2.6% (w/w) neutral carbohydrate and two residues of N-acetylglucosamine/monomer. Carbohydrate-binding specificity was directed toward D-mannose and D-glucose and their alpha-glycosidic derivatives. The purified protein agglutinated cat erythrocytes at 5 micrograms/ml. Antiserum to seed lectin showed a single common immunoprecipitation line in Ouchterlony double diffusion against both the seed and root antigen. Lectin isolated from sainfoin seedling roots showed molecular weight, amino acid and carbohydrate values similar to that of the seed lectin.

Physical studies on three lectins from the seeds of Abrus precatorius.

The physical properties of three lectins from the seeds of the Abrus precatorius plant, abrin C, abrin A and the Abrus agglutinin, were studied. All three exhibited similar circular dichroic (CD) spectra in the near-ultraviolet having negative maxima at 286 and 293 nm. In addition, D-galactose induced similar conformational alterations in the three proteins as observed through changes in the near-ultraviolet CD from 280 to 295 nm. The near-ultraviolet CD spectrum of the toxic subunit of abrin C was very different from that of the parent molecule. The fluorescence emission spectra of the three proteins were also studied. All exhibited fluorescence near 335 nm which is quenched 9% by galactose. Iodide quenching of fluorescence using the Stern-Volmer analysis indicated different tryptophan accessibilities in the presence and absence of D-galactose for the Abrus agglutinin. The results suggest that there is a saccharide-induced conformational change which buries several partially exposed tryptophan residues. A comparable analysis of the closely related Ricinus agglutinin revealed that its tryptophan residues are more buried than those of the Abrus agglutinin and, unlike the Abrus agglutinin, there was no saccharide-induced change in tryptophan accessibility.

Trifolin: a Rhizobium recognition protein from white clover.

A protein agglutinin, trifoliin, was purified from white clover seeds and seedling roots. Trifoliin specifically agglutinates the symbiont of clover, Rhizobium trifolii, at concentrations as low as 0.2 microgram protein/ml, and binds to the surface of encapsulated R. trifolii 0403. This clover protein has a subunit with Mr approximately 50 000, an isoelectric point of 7.3, and contains carbohydrate. Antibody to purified trifoliin binds to the root hair region of 24-h-old clover seedlings, but does not bind to alfalfa, birdsfoot trefoil or joint vetch. The highest concentration of trifoliin on a clover root is present at sites where material in the capsule of R. trifolii binds. 2-Deoxy-D-glucose elutes trifoliin from intact clover-seedling roots, suggesting that this protein is anchored to root cell walls through its carbohydrate binding sites. We propose that trifoliin on the root hair surface plays an important role in the recognition of R. trifolii by clover.

Studies on lectins. XLIII. Isolation and characterization of the lectin from restharrow roots (Ononis hircina Jacq.).

From 1 kg of dried Ononis hircina Jacq. roots 36 mg of a lectin were isolated by affinity chromatography on O-beta-lactosyl polyacrylamide gel. The lectin is homogeneous as judged by ultracentrifugal analysis (S20,W=6.2 S), polyacrylamide disc electrophoresis at pH 8.6 or 4.5, gel filtration on thin layers of Sephadex G-200 (Mr = 110 000) and dodecyl sulfate electrophoresis (Mr of subunits 31 000, both in presence and absence of mercaptoethanol) and disc dodecyl sulfate electrophoresis (pH 9.5). The lectin contains much aspartic and glutamic acids, serine and threonine and also 7.2% of neutral sugar. It is relatively specific for human type O erythrocytes that are agglutinated at a minimal lectin concentration 0.3 microgram/ml. The erythroagglutinating activity is not stimulated by Ca2+, Zn2+, Mg2+, Mn2+, Co2+ or Ni2+ salts; it is inhibited most effectively by N-acetyl-D-galactosamine and a number of D-galactose derivatives. Dissociation constants of several lectin . sugar complexes were estimated by affinity electrophoresis. The lectin is not mitogenic in rabbit lymph node lymphocytes.

A characterization of abrin A from the seeds of the Abrus precatorius plant.

Abrin A was purified from the seeds of the Abrus precatorius plant and its physical and biological properties were studied. The biological properties of abrin A were found to be similar to the better studied Abrus protein, abrin C, in that it is toxic to cell-free protein synthesis and binds D-galactose. Abrin A contains carbohydrate moieties including both neutral and amine sugars but no metals, similar to the other two Abrus proteins (abrin C and the Abrus agglutinin). Amino acid compositions of the subunits of abrin A indicated that it consists of two different subunits of comparable size. Furthermore, one of the subunits showed microheterogeneity suggesting that abrin A is a mixture of isolectins. A comparative study of abrin A and abrin C based on compositions and tryptic maps reveals them to be closely related. The evidence suggests that the two abrins may have the same mechanisms of toxic action. Far-ultraviolet circular dichroic studies of abrin A show it to contain 47% beta-pleated sheet and 10% alpha-helix, again similar to the other two Abrus proteins.

Complete assignment of the 1H nuclear magnetic resonance spectrum of French bean plastocyanin. Sequential resonance assignments, secondary structure and global fold.

Sequence-specific proton nuclear magnetic resonance (n.m.r.) assignments for all 99 amino acid residues of French bean Cu(I) plastocyanin are described. The assignments were made using standard sequential assignment procedures and were greatly facilitated by the availability of complete spin system assignments. The characteristic short NOE connectivities between backbone protons, the values of 3JHN alpha, and the locations of slowly exchanging backbone amide protons, identify and define the elements of regular secondary structure. Eight well-defined beta-strands, a small helical segment and eight tight turns can be identified unambiguously. On the basis of a very extensive set of inter-strand NOE connectivities, the beta-strands can be packed into two distinct beta-sheets. Over 80% of the residues in the protein can be assigned to some regular element of secondary structure. The n.m.r. data is sufficient to define the chain folding topology, which is that of a Greek key beta-barrel, and provides a qualitative description of the global fold. The overall structure of French bean plastocyanin in solution is very similar to that of poplar plastocyanin in crystals. Significant local differences are, however, observed, particularly in the loops connecting some of the beta-strands.

Complete assignment of the 1H nuclear magnetic resonance spectrum of French bean plastocyanin. Application of an integrated approach to spin system identification in proteins.

The identification of the spin systems that comprise the 1H nuclear magnetic resonance spectrum of French bean Cu(I) plastocyanin (Mr 10,600) has been made using an approach that integrates a wide range of two-dimensional nuclear magnetic resonance experiments. A very large percentage of these assignments has been obtained in spectra acquired from 1H2O solution using a backbone amide-based strategy. The spin systems of 91 of the 99 residues have been assigned to the appropriate amino acid, thereby providing an ample basis for obtaining sequence-specific assignments, as described in the accompanying paper.

Crystallization and preliminary X-ray studies of two isolectins from the seeds of Lathyrus ochrus.

Two isolectins from the seeds of Lathyrus ochrus, LOL I and LOL II, which specifically bind N-acetyllactosamine, have been crystallized using the hanging-drop method and the interface diffusion method, respectively. In the case of LOL I, 2-methylpentane-2,4-diol, polyethylene-glycol 400 or ammonium sulphate have been used as precipitating agents. The best crystals of LOL I were grown at room temperature from a solution of 40% (v/v) methylpentane diol, 50 mM-Hepes at pH 7.5. LOL II crystals have been grown at room temperature from a solution of 32% (v/v) methylpentane diol, 50 mM-2-(N-morpholino)-ethanesulphonic acid at pH 5.5. X-ray examination of the LOL I and LOL II crystals shows that both are monoclinic, space group P2(1). Their cell dimensions are: LOL I, a = 56.4 A, b = 138.8 A, c = 62.9 A, beta = 91 degrees; and LOL II, a = 54.8 A, b = 71.4 A, c = 105.5 A, beta = 105 degrees. Density measurements of the crystals of LOL I indicate that there are two molecules per asymetric unit (Vm = 2.07 A3/dalton). LOL I crystals diffract strongly up to at least 1.8 resolution. Putative crystals of complexes of LOL I with various glycosides were obtained through co-crystallization under the conditions used for the native protein.

Polypeptide composition of higher plant photosystem I complex. Identification of psaI, psaJ and psaK gene products.

High resolution gel electrophoresis of the native photosystem I complex retaining light-harvesting chlorophyll complex revealed the presence of three low-molecular-mass proteins of 7, 4.1 and 3.9 kDa in spinach, and 6.8, 4.4 and 4.1 kDa in pea, in addition to the other well-characterized higher-molecular-mass components. Upon further detergent treatment to deplete light-harvesting chlorophyll complex, the 7 kDa and 4.1 kDa proteins were removed from the photosystem I core complex of spinach, while the 3.9 kDa protein was retained. N-terminal sequencing demonstrated that the 4.1 kDa proteins from both spinach and pea correspond to the gene product of ORF42/44 in chloroplast genome of liverwort and higher plants, which was previously hypothesized as a photosystem I gene (psaJ) based on sequence homology with the cyanobacterial photosystem I component of 4.1 kDa [(1989) FEBS Lett. 253, 257-263]. N-terminal sequence of the spinach 3.9 kDa and pea 4.4 kDa proteins fitted with chloroplast ORF36/40 (psaI) although no homologue has been found in cyanobacteria. The spinach 7 kDa and pea 6.8 kDa proteins correspond to the nuclear-encoded psaK product and significantly matched with the N-terminal sequence of the cyanobacterial 6.5 kDa subunit. The evolutional conservation of the psaJ and psaK seems to suggest their intrinsic role(s) in photosystem I.

N-terminal amino acid sequence analysis of the subunits of pea photosystem I.

Six 'core' subunits of pea photosystem I have been isolated and their N-terminal amino acid sequences determined by gas-phase or solid-phase sequencing. On average more than thirty residues were determined from the N-terminus of each polypeptide. This sequence analysis has revealed three polypeptides with charged N-terminal regions (21, 17 and 11 kDa subunits), one polypeptide with a predominantly hydrophobic N-terminal region (9 kDa subunit), one polypeptide which is cysteine-rich (8 kDa subunit) and one which is alanine-rich (13 kDa subunit).

Relationship between polyphenol intake and blood glucose response of normal and diabetic individuals.

Five leguminous and eight nonleguminous foods were analyzed for polyphenol concentration by the Prussian Blue and the Folin Denis methods and correlated with blood glucose response (glycemic index) in normal or diabetic volunteers. Polyphenol concentrations and intakes per 50 g available carbohydrate portions were higher in the leguminous foods than those in the nonleguminous foods. In both normal and diabetic individuals, a negative correlation was observed between glycemic index and the concentration or total intake of polyphenols. Polyphenols, especially the large polymeric type or condensed tannins, appear to be responsible in part for the reduced glycemic response to carbohydrate foods and in part to lower blood glucose response to legumes compared with cereal products.

Factors affecting the rate of hydrolysis of starch in legumes.

In an attempt to understand the mechanism for the extremely slow rate of digestion and absorption of carbohydrate from legumes, we have examined a number of factors which could potentially affect the process in vitro. The rate of hydrolysis of legume starch in vitro was not affected by the presence of fat (as either butter or an emulsion). However, it was significantly increased in commercially available canned bean preparations, suggesting that the high temperatures used in the canning process may alter the availability of starch in legumes. In vitro starch hydrolysis rate was also significantly increased by grinding legumes finely prior to cooking. Finally, the slow rate of digestion and absorption of legume carbohydrate does not appear to be due to viscosity since a) increasing the shaking rate of viscous mixture of either red kidney beans or lentils from 0 to 120 oscillations per minute did not affect the hydrolysis rate, and b) a thick viscous mixture of either of these legumes did not retard the diffusion of free glucose from a dialysis sac into the dialysate.

Cell structure and starch nature as key determinants of the digestion rate of starch in legume.

The factors responsible for the slow digestibility of starch in leguminous seeds have been studied by examining microscopically the cooked seeds after various treatments and by measuring starch digestion in vitro. Starch in leguminous seeds is entrapped in parenchyma cells and swells only partially during cooking. The alpha-amylase cannot easily penetrate within the gelatinized starch granules due to steric hindrance and the physical nature of the leguminous starch. Disruption of the cells, especially before cooking increases the susceptibility of starch to alpha-amylase digestion.