Major protein of resting rhizomes of Calystegia sepium (hedge bindweed) closely resembles plant RNases but has no enzymatic activity

The most abundant protein of resting rhizomes of Calystegia sepium (L.) R.Br. (hedge bindweed) has been isolated and its corresponding cDNA cloned. The native protein consists of a single polypeptide of 212 amino acid residues and occurs as a mixture of glycosylated and unglycosylated isoforms. Both forms are derived from the same preproprotein containing a signal peptide and a C-terminal propeptide. Analysis of the deduced amino acid sequence indicated that the C. sepium protein shows high sequence identity and structural similarity with plant RNases. However, no RNase activity could be detected in highly purified preparations of the protein. This apparent lack of activity results most probably from the replacement of a conserved His residue, which is essential for the catalytic activity of plant RNases. Our findings not only demonstrate the occurrence of a catalytically inactive variant of an S-like RNase, but also provide further evidence that genes encoding storage proteins may have evolved from genes encoding enzymes or other biologically active proteins.     

Purification, characterization and structural analysis of an abundant beta-1,3-glucanase from banana fruit.

An abundant, catalytically active beta-1,3-endoglucanase (EC 3.2.1. 39) has been isolated from the pulp of ripe bananas. Biochemical analysis of the purified protein, molecular modelling, and molecular cloning of the corresponding gene indicate that this banana enzyme closely resembles previously characterized plant beta-glucanases with respect to its amino-acid sequence, structure and biological activity. The results described in this paper demonstrate both the occurrence of an abundant active beta-1,3-endoglucanases in fruits and also readdress the question of the possible involvement of these enzymes in the ripening and/or softening process.     

Purification, characterization and immunolocalization of fimbrial protein from Porphyromonas (bacteroides) gingivalis.

Rapid and reproducible method is described here for the purification of the 43 kDa fimbrial protein from P. gingivalis by preferential fractionation in the presence of 1% SDS and 0.2M of a bivalent cation at pH 6.5. Homogeneity of the purified 43 kDa was confirmed by SDS-PAGE and Western blot analysis using monoclonal and polyclonal antibodies raised against this protein. Amino acid composition and the amino acid sequence of the first 30 amino acid residues of the purified fimbriae are consistent with the composition and sequence predicted from the cloned gene of the fimbrial subunit. Circular dichroism spectra shows high levels of beta-sheet structure. The purified 43 kDa polymer shows fimbriae-like morphology under the electron microscope. Ultrastructural localization of the 43 kDa protein by the immunogold technique revealed specific labeling of the fimbriae with a diameter of approximately 3.5 to 5.0 nm. Localization of this protein suggest that the 43 kDa component is a fimbrial subunit.     

Purification and characterization of extracellular beta-amylase of Bacillus megaterium B(6)

The extracellular beta-amylase from starch induced Bacillus megaterium B6 was purified to homogeneity in a very convenient way; through molecular sieving as demonstrated by the presence of a single band of protein in SDS-PAGE and single peak in gel scanning. The molecular mass of the purified enzyme (monomer) was found to be unusually high, around 105,000 Da. The pH and temperature optima of the purified beta-amylase were at 6.9 and 60 degrees C, respectively. Mn2+ and exogenous thiols were found to play a remarkable role in reactivation of thermally and chemically denatured enzyme. The purified enzyme could saccharify both pure and low quality starches, where maltose could be detected as the major end product.     

Purification, characterization, and immunolocalization of hydroxycinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)transferase from opium poppy

A development-specific and elicitor-inducible acyltransferase [hydroxycinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110)] that catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-CoA esters to hydroxyphenethylamines was purified 988-fold to apparent homogeneity from opium poppy (Papaver somniferum L.) cell-suspension cultures. The purification procedure, which resulted in a 6.8% yield, involved hydrophobic interaction and anion-exchange chromatography, followed by affinity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA) as eluent. Purified THT had an isoelectric point of 5.2, a native molecular mass of approximately 50 kDa, and consisted of two apparently identical 25-kDa subunits as determined by two-dimensional polyacrylamide gel electrophoresis. The purified enzyme was able to synthesize a variety of amides due to a relatively low specificity for cinnamoyl-CoA derivatives and hydroxyphenethylamines. The best substrates were feruloyl-CoA (VK _m ⁻¹13.4 mkat g⁻¹ M⁻¹) and tyramine (VK _m ⁻¹6.57 mkat g⁻¹ M⁻¹). The THT activity increased during development of opium poppy seedlings, occurred at high levels in roots and stems of mature plants, and was induced in cell-suspension cultures after treatment with a pathogen-derived elicitor. Immunoblot analysis using THT mouse polyclonal antibodies did not always show a correlation between THT polypeptide and enzyme activity levels. For example, despite low THT activity in leaves, an abundant 25-kDa immunoreactive polypeptide was detected. Immunohistochemical localization showed that THT polypeptides occur in cortical and xylem parenchyma, immature xylem vessel elements, root periderm, anthers, ovules, and the inner layer of the seed coat, but are most abundant in phloem sieve-tube members in roots, stems, leaves, and anther filaments. Received: 19 January 1999 / Accepted: 3 March 1999     

Purification and structural analysis of an abundant thaumatin-like protein from ripe banana fruit

 The pulp of ripe bananas (Musa acuminata) contains an abundant thaumatin-like protein (TLP). Characterization of the protein and molecular cloning of the corresponding gene from banana demonstrated that the native protein consists of a single polypeptide chain of 200 amino acid residues. Molecular modelling further revealed that the banana thaumatin-like protein (Ban-TLP) adopts an overall fold similar to that of thaumatin and thaumatin-like PR-5 proteins. Although the banana protein exhibits an electrostatically polarized surface, which is believed to be essential for the antifungal properties of TLPs, it is apparently devoid of antifungal activity towards pathogenic fungi. It exhibits a low but detectable in vitro endo-β-1,3-glucanase (EC 3.2.1.x) activity. As well as being present in fruits, Ban-TLP also occurs in root tips where its accumulation is enhanced by methyl jasmonate treatment of plants. Pulp of plantains (Musa acuminata) also contains a very similar TLP, which is even more abundant than its banana homologue. Our results demonstrate for the first time that fruit-specific (abundant) TLPs are not confined to dicots but occur also in fruits of monocot species. The possible role of the apparent widespread accumulation of fruit-specific TLPs is discussed. Received: 7 January 2000 / Accepted: 26 April 2000     

Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization

Euglena gracilis chloroplast leucyl-tRNA synthetase was purified to homogeneity by a series of steps including ammonium sulfate precipitation and chromatography on hydroxylapatite, DEAE-cellulose, Sepharose 6B, phosphocellulose, and Blue Dextran-Sepharose. The purified enzyme exhibits a specific activity of 1233 units/mg of protein, which is one of the highest specific activities obtained for an aminoacyl-tRNA synthetase prepared from plant cells. The enzyme has an apparent Km value of 8 x 10(-6) M for L-leucine, 1.3 x 10(-4) M for ATP, and 1.3 x 10(-6) M for tRNALeu. Chloroplast leucyl-tRNA synthetase appears to be a monomeric enzyme with a molecular weight of 100 000. The amino acid composition of chloroplast leucyl-tRNA synthetase has been determined. It is the first reported for a chloroplast aminoacyl-tRNA synthetase, and it reveals a relatively large proportion of apolar residues, as in the case of prokaryotic aminoacyl-tRNA synthetases.     

Purification, characterization, and immunolocalization of a thioredoxin reductase from adult Fasciola hepatica

Thioredoxin reductase (TrxR), an enzyme belonging to the flavoprotein family of pyridine nucleotide-disulfide oxidoreductases, was isolated from the deoxycholate-soluble extract of the common liver fluke, Fasciola hepatica. Purification to homogeneity of the 60-kDa enzyme from the adult worm was achieved by a combination of ammonium sulfate fractionation, anion exchange, and affinity chromatography on 2',5'-adenosine diphosphate-Sepharose. Using the 5,5'-dithiobis(2-nitrobenzoic acid) assay, the purified TrxR showed a specific activity of 7,117 U min(-1) mg(-1). The enzyme activity was completely inhibited by the presence of the gold compound aurothioglucose (IC50 = 120 nm), indicating that F. hepatica TrxR is a selenoenzyme. Also, the enzyme was capable of reducing disulfide bonds in insulin and was activated by the presence of the reduced form of flavin adenine dinucleotide, properties shared with mammalian TrxRs. Furthermore, the isolated enzyme showed very low glutaredoxin (Grx) activity (0.47 U mg(-1)), but no glutathione reductase activity was detected. Affinity-purified IgGs (20 microg ml(-1)) from the antisera produced against the purified TrxR inhibited its activity about 80% with respect to the control. The enzyme was immunolocalized in cells located within the parenchyma and in the testes, but it was not found in the tegument of the adult fluke.     

Purification and structural characterization of siderophore (corynebactin) from Corynebacterium diphtheriae

During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin.     

Purification, characterization, and structural analysis of a plant low-temperature-induced protein.

We have purified to near homogeneity a recombinant form of the protein BN28 (rBN28), expressed in response to low temperature in Brassica napus plants, and we have determined its solution structure. Antibodies raised against rBN28 were used to characterize the recombinant and native proteins. Similar to many other low-temperature-induced proteins, BN28 is extremely hydrophilic, such that it remains soluble following boiling. Immunoblot analysis of subcellular fractions indicated that BN28 was not strongly associated with cellular membranes and was localized exclusively within the soluble fraction of the cell. Contrary to predicted secondary structure that suggested significant helical content, circular dichroism analysis revealed that rBN28 existed in aqueous solution largely as a random coil. However, the helical propensity of the protein could be demonstrated in the presence of trifluoroethanol. Nuclear magnetic resonance analysis further showed that rBN28 was in fact completely unstructured (100% coil) in aqueous solution. Although it had earlier been speculated that BN28-like proteins from Arabidopsis thaliana might possess antifreeze protein activity (S. Kurkela and M. Franck [1990] Plant Mol Biol 15: 137-144), no such activity could be detected in ice recrystallization assays with rBN28.     

Purification,characterization, molecular cloning,and expression of novel members of jacalin-related lectins from rhizomes of the true fern Phlebodium aureum (L) J. Smith (Polypodiaceae)

A lectin was purified from rhizomes of the fern Phlebodium aureum by affinity chromatography on mannose-Sepharose. The lectin, designated P. aureum lectin (PAL), is composed of two identical subunits of approximately 15 kDa associated by noncovalent bonds. From a cDNA library and synthetic oligonucleotide probes based on a partial amino acid sequence, 5'- and 3'-rapid amplification of cDNA ends allowed the generation of two similar full-length cDNAs, termed PALa and PALb, each of which had an open reading frame of 438 bp encoding 146 amino acid residues. The two proteins share 88% sequence identity and showed structural similarity to jacalin-related lectins. PALa contained peptide sequences exactly matching those found in the isolated lectin. PALa and PALb were expressed in Escherichia coli using pET-22b(+) vector and purified by one-step affinity chromatography. Native and recombinant forms of PAL agglutinated rabbit erythrocytes and precipitated with yeast mannan, dextran, and the high mannose-containing glycoprotein invertase. The detailed carbohydrate-binding properties of the native and recombinant lectins were elucidated by agglutination inhibition assay, and native lectin was also studied by isothermal titration calorimetry. Based on the results of these assays, we conclude that this primitive vascular plant, like many higher plants, contains significant quantities of a mannose/glucose-binding protein in its storage tissue, whose binding specificity differs in detail from either legume mannose/glucose-binding lectins or monocot mannose-specific lectins. The identification of a jacalin-related lectin in a true fern reveals for the first time the widespread distribution and molecular evolution of this lectin family in the plant kingdom.     

Cloning and characterization of the beta-amylase gene from Bacillus polymyxa.

The gene for beta-amylase was isolated from Bacillus polymyxa by molecular cloning in B. subtilis. B. subtilis cells containing this gene express and secrete an amylase which resembles the B. polymyxa beta-amylase and barley beta-amylase in terms of the products it generates during carbohydrate hydrolysis. Starch hydrolysis with this beta-amylase produces maltose, not glucose, whereas maltotriose and cycloheptaose are resistant to the action of this beta-amylase. The enzyme has a molecular weight of approximately 68,000. Restriction endonuclease mapping demonstrated that the DNA inserted in pBD64 and containing the gene is approximately 3 kilobases in length.     

Purification and enzymic characterization of the cytoplasmic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum.

Cytoplasmic pyrophosphatase has been isolated from the thermoacidophilic archaebacterium Thermoplasma acidophilum. The enzyme was purified to electrophoretic homogeneity by combining ion-exchange and affinity-chromatographic separations. This soluble pyrophosphatase probably consists of six identical subunits, since SDS/PAGE gave an estimate of about 22 kDa for a single subunit and size-exclusion chromatography under non-denaturing conditions indicates a molecular mass of 110 +/- 5 kDa. The two most prominent catalytic features of this enzyme are the absolute requirement for divalent cations for catalytic action, Mg2+ conferring the highest activity, and the pronounced specificity for PPi. The catalytic behavior apparently follows simple Michaelis-Menten kinetics with a Km of about 7 microM for PPi and a specific activity of about 1200 U/mg at 56 degrees C. Surprisingly, maximum activity could be observed at 85 degrees C which is more than 20 degrees C above the temperature for optimal growth. Several cytoplasmic extracts of eubacteria and archaebacteria have been probed with a polyclonal antiserum raised against the purified archaebacterial protein. The only noticeable cross-reactivity could be detected with an extract from the methanogen Methanosarcina barkeri although this probably does not reflect the inferred phylogenetic relationship between methanogens and Thermoplasma acidophilum.     

Purification, characterization and functional analysis of an endo-arabinanase (AbnA) from Bacillus subtilis

Bacillus subtilis synthesizes at least one arabinanase encoded by the abnA gene that is able to degrade the polysaccharide arabinan. Here, we report the expression in Escherichia coli of the full-length abnA coding region with a His6-tag fused to the C-terminus. The recombinant protein was secreted to the periplasmic space and correctly processed by the E. coli signal peptidase. The substrate specificity of purified AbnA, the physico-chemical properties and kinetic parameters were determined. Functional analysis studies revealed Glu 215 as a key residue for AbnA hydrolytic activity and indicated that in addition to AbnA B. subtilis secretes other enzyme(s) able to degrade linear 1,5-alpha-l-arabinan.     

Cytochemical immunolocalization of the abundant pistil protein Sk2 in potato (Solanum tuberosum)

S_k2 protein is the most abundant member of the pistil-specific proteins of Solanum tuberosum. S_k2 protein has been localized by use of a polyclonal antibody (anti-S_k2) in the pistils of four clones of Solanum tuberosum. In the stigmas S_k2 protein accumulates to a high level in the cytoplasm of the internal secretory cells underlying the papillae one day prior to anthesis. In styles, the intercellular matrix of the transmitting tissue cells is intensely labelled by anti-S_k2. S_k2 protein is present in all four clones and shows the same labelling pattern. The possible role of the S_k2 protein in pollen tube growth is discussed.     

Purification, characterization, and gene analysis of cellulase (Cel8A) from Lysobacter sp. IB-9374

An enzyme that has both beta-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the beta-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to beta-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.     

Purification, structural characterization, cloning and immunocytochemical localization of chemoreception proteins from Schistocerca gregaria.

Soluble low-molecular-mass protein isoforms were purified from chemosensory organs (antennae, tarsi and labrum) of the desert locust Schistocerca gregaria. Five genes encoding proteins of this group were amplified by PCR from cDNAs of tarsi and sequenced. Their expression products are polypeptide chains of 109 amino acids showing 40-50% sequence identity with putative olfactory proteins from Drosophila melanogaster and Cactoblastis cactorum. Direct structural investigation on isoforms purified from chemosensory organs revealed the presence in the expression products of two of the genes cloned. Two additional protein isoforms were detected and their molecular structure exhaustively characterized. MS analysis of all isoforms demonstrated that the four cysteine residues conserved in the polypeptide chain were involved in disulfide bridges (Cys29-Cys38 and Cys57-Cys60) and indicated the absence of any additional post-translational modifications. Immunocytochemistry experiments, performed with rabbit antiserum raised against the protein isoform mixture, showed selective labelling of the outer lymph in contact sensilla of tarsi, maxillary palps and antennae. Other types of sensilla were not labelled, nor were the cuticle and dendrites of the sensory cells. No binding of radioactively labelled glucose or bicarbonate was detected, in disagreement with the hypothesis that this class of proteins is involved in the CO2-sensing cascade. Our experimental data suggest that the proteins described here could be involved in contact chemoreception in Orthoptera.     

Purification and structural characterization of insulin from a caecilian, Typhlonectes natans (Amphibia: Gymnophiona)

Despite the important position of amphibia in phylogeny, efforts at the structural characterization of amphibian neurohormonal peptides have largely been confined to the Anurans (frogs and toads). Insulin was purified from an extract of the pancreas of the caecilian, Typhlonectes natans. The primary structure of the peptide was established as:

A-chain: Gly-Ile-Val-Glu-Lys⁵-Cys-Cys-Leu-Ser-Thr¹⁰-Cys-Ser-Leu-Tyr-Glu¹⁵-Leu-Glu-Ser-Tyr-Cys²⁰-Asn

B-chain: Ile-Ala-Asn-Gln-His⁵-Leu-Cys-Gly-Ser-His¹⁰-Leu-Val-Glu-Ala-Leu¹⁵-Tyr-Leu-Val-Cys-Ala²⁰-Asp-Arg-Gly-Phe- Phe²⁵-Tyr-Thr-Pro-Lys-Ser³⁰

This amino acid sequence contains several unusual substitutions (Gln → Lys at A5, His → Leu at A8, Gln → Glu at A15, and Gly → Ala at B20) that are not present in other amphibian insulins. The structure of insulin appears to be less well conserved among the different orders of amphibia, compared with reptiles and birds.