Purification of ecdysone oxidase and 3-dehydroecdysone 3α-reductase from the cotton leafworm,Spodoptera littoralis

The 3-epimerization of ecdysteroids (insect moulting hormones) is an inactivation pathway of the hormones that has been reported to occur in midgut cytosol of Lepidoptera. The pathway involves ecdysone oxidase-catalysed conversion of ecdysone into 3-dehydroecdysone, which is then irreversibly reduced to 3-epiecdysone by 3DE 3α-reductase. In this study, ecdysone oxidase and 3DE 3α-reductase from the cotton leafworm, S. littoralis, have been purified by extensive chromatography together with electrophoresis on native gels. Gel filtration suggested that the native ecdysone oxidase might be a trimer with apparent molecular mass of approximately 190 kDa, since the apparent molecular mass of the oxidase subunit was determined to be 64 kDa by SDS-PAGE. Two forms of 3DE 3α-reductase were observed during the purification, the 26 kDa form reductase has been purified to homogeneity and the second form of the reductase identified as a 51 kDa protein. The former reductase may be a trimer with apparent molecular mass of 76 kDa, whilst the latter was suggested to be a monomer by gel filtration. Chromatographic behaviour suggested that the 26 kDa form of the reductase has a lower pI value and a higher degree of hydrophobicity than that of the 51 kDa reductase. Substrate specificity and the tissue distribution of these enzymes are discussed.     

Isolation of an N-acetyl-D-glucosamine specific lectin from the rhizomes of Arundo donax with antiproliferative activity

A lectin with antiproliferative activity towards human cancer cell lines and mitogenic towards human peripheral blood mononuclear cells was purified from the rhizomes of Arundo donax (Linn.) by affinity chromatography on N-acetyl-d-glucosamine linked to epoxy-activated sepharose-6B. The pure preparation apparently yielded a single band of approximately 15 kDa on SDS-PAGE, pH 8.3, under both reducing and non-reducing conditions. The molecular mass of native lectin was 32 kDa as determined by gel filtration chromatography. This showed the lectin to be a dimer, with subunits not held together by disulphide linkages. The A. donax lectin (ADL) agglutinated rabbit erythrocytes and the agglutination was inhibited by N-acetyl-d-glucosamine and its di- and trimer. The lectin was thermostable upto 55 degrees C and showed optimum activity in the range of pH 7.0-9.0 and comprised of 2.1% carbohydrate content.     

Induction of oxidative stress in leukocytes by an Enterobacter cloacae toxin able to form oligomers and binding to proteins

A leukotoxic and hemolytic toxin was purified from cultures of Enterobacter cloacae. Stimulation of oxidative stress was observed and the production of reactive oxidant species was measured in leukocytes treated with toxin by means of nitroblue tetrazolium and chemiluminescence assays. Molecular weight of toxin was estimated by chromatography and SDS-PAGE. Two protean peaks with toxic activity were found in Sephadex G-100 (P1, 42.0 kDa; and P2, 13.3 kDa). The relative amounts between the peaks (P1/P2 = 0.36) changed when 2-mercaptoethanol was employed (P1/P2 = 0.59). When Sephadex G-200 chromatography was performed, a protean peak of Ve = 113 mL (100 kDa) was found; its was dissociated with 3 M urea in toxic proteins of lower mass: 42, 27, and 13.3 kDa. SDS-PAGE (15%) showed a single toxin band of purified monomer (13.3 kDa), but electrophoresis of a 42-kDa toxin with urea presented three bands of trimer, dimer, and monomer. An increase of casein hydrolysate and albumin molecular weight was observed by chromatography after incubation with toxin due to the binding of both proteins with toxin.     

Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.     

Duality monomer-dimer of the pheromone-binding protein from Bombyx mori

The analysis of a recombinant pheromone-binding protein from the silkworm moth, Bombyx mori, by native gel electrophoresis with Coomassie staining showed one single band with a molecular mass consistent with a monomer. A slow migrating band, detected in the recombinant and native samples by a polyclonal antibody, was indistinguishable from the monomer in the mass spectrum fragmentation pattern and chromatographic behavior. Flow injection analyses of the protein by mass spectrometry in the negative mode showed fragments of a dimer. The dimeric form was also supported by estimation of the molecular mass by gel filtration at basic pH. A cross-linked dimer coeluted with the noncovalent dimer on a gel filtration column. The molecular mass of the protein changed in a pH-dependent way with a dramatic transition from dimer to monomer between pH 6 and 4.5. A low pH induced not only dissociation of the dimer, but also a conformational change in the protein. In marked contrast to denaturation with guanidinium chloride, the emission maxima of tryptophan was not significantly changed at low pH. BmPBP is thus a dimer at slightly acid, neutral, and basic pH, which dissociates and then undergoes conformational change at low pH.     

Constitutive HSP70: oligomerization and its dependence on ATP binding.

The constitutive HSP70 purified from CHO cells, which indicated a single band in SDS-polyacrylamide gel electrophoresis, showed multiple bands in native-polyacrylamide gel electrophoresis. These results indicate that the protein may exist in oligomeric forms. After crosslinking the oligomers with glutaraldehyde, SDS-polyacrylamide gel electrophoresis showed three protein bands of molecular weight 70 kDa, 153 kDa, and 200 kDa corresponded to monomer, dimer, and trimer, respectively. The relative amount of oligomeric forms was dependent upon ATP concentrations: it increased upon hydrolysis of ATP or decreased upon incubation with high concentrations of ATP (1-10 mM). Autoradiographic analysis of the native polyacrylamide gel electrophoresis of HSP70 following incubation with [gamma-32P]ATP revealed that ATP bound to only monomer. These results suggest that the equilibrium between oligomeric forms is dependent on ATP concentrations. Nonetheless, during heat shock, both monomer and oligomer might be indistinguishably associated with some proteins, probably denatured proteins.     

One interferon gamma receptor binds one interferon gamma dimer

We investigated the stoichiometry of the interferon gamma and interferon gamma receptor interaction, using recombinant interferon gamma and recombinant soluble interferon gamma receptor, applying chemical cross-linking and chromatographic techniques, and analyzing the resulting products in denaturing polyacrylamide gels. Interferon gamma cross-linked to itself produced a major band of an apparent molecular mass of 34 kDa, which suggests that it exists as a dimer in physiological buffer and which agrees with published data. Soluble interferon gamma receptor cross-linked to itself produced mainly a 28-kDa band, suggesting that the interferon gamma receptor exists as a monomer. Interferon gamma cross-linked to the soluble interferon gamma receptor resulted in the formation of two main products of apparent molecular masses of 60 and 44 kDa. The predominant 60-kDa band resulted from the cross-linking of one interferon gamma dimer (34 kDa) to one interferon gamma receptor molecule (27 kDa). The 44-kDa band was formed by the cross-linking of one interferon gamma molecule to one interferon gamma receptor. Kinetic studies showed that the cross-linking of interferon gamma dimer to the soluble receptor proceeds through the intermediate formed by cross-linking one molecule of the interferon gamma dimer to the receptor. Reducing and dissociating agents inhibited complex formation. When chromatographed on Sephadex G-100, interferon gamma was eluted as a protein of 34-kDa molecular mass, the soluble interferon gamma receptor as a protein of 40 kDa, and their mixture was eluted in one peak corresponding to an apparent molecular mass of 73 kDa. Sodium dodecyl sulfate-polyacrylamide gel analysis of the eluted mixture showed the presence of both interferon gamma and interferon gamma receptor at a ratio of 2:1. The found results suggest that the interferon gamma receptor binds interferon gamma as a dimer.     

Acetolactate synthase from Brassica napus: Immunological characterization and quaternary structure of the native enzyme

Acetolactate synthase (ALS, AHAS; EC 4. 1. 3. 18) from Brassica napus has been partially purified and characterized using polyclonal antibodies. Following denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blot analysis, 65 and 66 kDa ALS subunit polypeptides were immunologically detected, along with a novel 36 kDa polypeptide which cross-reacted with the anti-ALS antibody. Partial peptide sequencing of the 36 kDa peptide revealed significant similarity to plant aldolase proteins. ALS activity from stromal extracts fractionated by gel filtration chromatography as a single species of estimated molecular mass of 124 kDa, while comparative sedimentation coefficient in glycerol gradients indicated a corresponding molecular mass of 132 kDa. The results suggest that the native enzyme is a dimer of 65 and/or 66 kDa subunits. Anion exchange chromatography resolved two classes of ALS activity of equal native molecular weight, but which exhibited different properties with respect to subunit structure, sensitivity to inhibition by chlorsulfuron and feedback inhibition by branched chain amino acids.     

Cysteinyl-tRNA synthetase from Saccharomyces cerevisiae. Purification, characterization and assignment to the genomic sequence YNL247w

Cysteinyl-tRNA synthetase (CRS) from Saccharomyces cerevisiae was purified 2300-fold with a yield of 33%, to high-specific activity (k_cal4.3 s⁻¹ at 25°C for the aminoacylation of yeast tRNA^Cys). SDS-PAGE revealed a single polypeptide corresponding to a molecular mass of 86 kDa. Polyclonal antibodies to the purified protein inactivated CRS activity and detected only one polypeptide of 86 kDa, the native extract subjected to SDS-PAGE followed by immunoblotting. In contrast to bacterial CRS which is a monomer of about 50 kDa, the native yeast enzyme behaved as a dimer, as assessed by gel filtration and cross-linking. Its subunit molecular mass is in good agreement with the value of 87.5 kDa calculated for the protein encoded by the yeast genomic sequence YNL247w. The latter was previously tentatively assigned to CRS, based on limited sequence similarities to the corresponding enzyme from other sources. Determination of the amino acid sequence of internal polypeptides derived from the purified yeast enzyme confirmed this assignment. Alignment of the primary sequences of prokaryotic and yeast CRS reveals that the larger size of the latter is accounted for mostly by several insertions within the sequence.     

Purification and partial characterization of Oenococcus oeni exoprotease

The exoprotease from Oenococcus oeni produced in stress conditions was purified to homogeneity in two steps, a 14-fold increase of specific activity and a 44% recovery of proteinase activity. The molecular mass was estimated to be 33.1 kDa by gel filtration and 17 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggest that the enzyme is a dimer consisting of two identical subunits. Optimal conditions for activity on grape juice were 25 degrees C and a pH of 4.5. Incubation at 70 degrees C, 15 min, destroyed proteolytic activity. The SDS-PAGE profile shows that the enzyme was able to degrade the grape juice proteins at a significantly high rate. The activity at low pH and pepstatin A inhibition indicate that this enzyme is an aspartic protease. The protease activity increases at acidic pH suggesting that it could be involved in the wine elaboration.     

Purification and characterization of a lectin from wild sunflower (Helianthus tuberosus L.) tubers

A lectin (HTTL) was isolated from Helianthus tuberosus L. (wild sunflower) tubers using ion-exchange chromatography, gel filtration, and affinity chromatography. The lectin agglutinated both untreated and trypsin-treated rabbit erythrocytes and did not agglutinate human blood cells of groups A, B, and O. The gel filtration showed the native molecular mass of 72 kDa and subunit molecular masses of 17 and 18.5 kDa on 12% SDS-PAGE. The lectin activity was inhibited by D-mannose. The tetrameric protein revealed a unique characteristic by forming a broad zone of protein in native PAGE at pH 8.3, which dissociated into seven subunits of varying e/m ratios on acid gel at pH 4.3. These seven bands revealed two polypeptide species of molecular masses 17 and 18.5 kDa on 12% SDS-PAGE, as in the case of the native protein. The result indicated that of the seven subunits, three were homotetramers of 17 kDa, one was a homotetramer of 18.5 kDa, and three were heterotetramers of 17 and 18.5 kDa. The lectin was thermostable with broad pH optima (pH 4-8) and had no requirement for divalent metal cations for its activity. The amino acid composition showed that the lectin contained higher amounts of glycine, alanine, and lysine, but no methionine. The sugar content was estimated to be 5.3% mannose equivalent. The HTTL was mitogenic to mouse spleen (total) cells at 25 microg/ml concentration. The lectin showed characteristics different from those of the earlier reported H. tuberosus tuber lectins and hence opens up a new avenue to investigate the structure-function relationship of lectin in Helianthus species.     

Purification and characterisation of adenosine nucleosidase from Coffea arabica young leaves

An adenosine nucleosidase (ANase) (EC 3.2.2.7) was purified from young leaves of Coffea arabica L. cv. Catimor. A sequence of fractionating steps was used starting with ammonium sulphate salting-out, followed by anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme was purified 5804-fold and a specific activity of 8333 nkat mg-1 protein was measured. The native enzyme is a homodimer with an apparent molecular weight of 72 kDa estimated by gel filtration and each monomer has a molecular weight of 34.6 kDa, estimated by SDS-PAGE. The enzyme showed maximum activity at pH 6.0 in citrate-phosphate buffer (50 mM). The calculated Km is 6.3 microM and Vmax 9.8 nKat.     

Mass spectrometry analysis of HIV-1 Vif reveals an increase in ordered structure upon oligomerization in regions necessary for viral infectivity

HIV-1 Vif, an accessory protein in the viral genome, performs an important role in viral pathogenesis by facilitating the degradation of APOBEC3G, an endogenous cellular inhibitor of HIV-1 replication. In this study, intrinsically disordered regions are predicted in HIV-1 Vif using sequence-based algorithms. Intrinsic disorder may explain why traditional structure determination of HIV-1 Vif has been elusive, making structure-based drug design impossible. To characterize HIV-1 Vif's structural topology and to map the domains involved in oligomerization we used chemical cross-linking, proteolysis, and mass spectrometry. Cross-linking showed evidence of monomer, dimer, and trimer species via denaturing gel analysis and an additional tetramer via western blot analysis. We identified 47 unique linear peptides and 24 (13 intramolecular; 11 intermolecular) noncontiguous, cross-linked peptides, among the noncross-linked monomer, cross-linked monomer, cross-linked dimer, and cross-linked trimer samples. Almost complete peptide coverage of the N-terminus is observed in all samples analyzed, however reduced peptide coverage in the C-terminal region is observed in the dimer and trimer samples. These differences in peptide coverage or "protections" between dimer and trimer indicate specific differences in packing between the two oligomeric forms. Intramolecular cross-links within the monomer suggest that the N-terminus is likely folded into a compact domain, while the C-terminus remains intrinsically disordered. Upon oligomerization, as evidenced by the intermolecular cross-links, the C-terminus of one Vif protein becomes ordered by wrapping back on the N-terminal domain of another. In addition, the majority of the intramolecular cross-links map to regions that have been previously reported to be necessary for viral infectivity. Thus, this data suggests HIV-1 Vif is in a dynamic equilibrium between the various oligomers potentially allowing it to interact with other binding partners.     

Cynomolgus monkey liver aldehyde oxidase: extremely high oxidase activity and an attempt at purification

Aldehyde oxidase (EC 1.2.3.1) in monkey (Macaca fascicularis) liver was characterized. Liver cytosol exhibited extremely high benzaldehyde and phthalazine oxidase activities based on aldehyde oxidase, compared with those of rabbits, rats, mice and guinea pigs. Monkey liver aldehyde oxidase showed broad substrate specificity distinct from that of the enzyme from other mammals. Purified aldehyde oxidase from monkey liver cytosol showed two major bands and two minor bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These bands were also observed in Western blotting analysis using anti-rat aldehyde oxidase. The molecular mass of the enzyme was estimated to be 130-151 kDa by SDS-PAGE, and to be about 285 kDa by HPLC gel filtration. The results suggest that isoforms of aldehyde oxidase exist in monkey livers.     

Intersubunit disulfides of the follitropin receptor

The electrophoretic mobility of radioiodinated follitropin (FSH) alpha and beta subunits as well as the alpha beta dimer changed markedly depending on the concentration of reducing agents such as dithiothreitol. The changes were more dramatic in the beta subunit than in the alpha subunit. 125I-FSH, complexed to the receptor on porcine granulosa cells or in Triton X-100 extracts, was cross-linked with a cleavable (nondisulfide) homobifunctional reagent, solubilized in sodium dodecyl sulfate without reducing agents, and electrophoresed. The cross-linked sample revealed three bands of high molecular mass, in addition to the hormone subunit and dimer bands. The band of lightest mass, 110 kDa, was the major band and the other two of 76 and 62 kDa were barely noticeable. Upon reduction with dithiothreitol, the 110-kDa band decreased while the 76- and 62-kDa bands increased, indicating the existence of disulfides between components of the 110-kDa complex. Formation of the disulfide-linked complexes requires 125I-FSH, specifically bound to the hormone receptor and cross-linking, and can be prevented with an excess of native FSH but not human choriogonadotropin. Complex formation was independent of blocking free sulfhydryl groups with N-ethylmaleimide. When the cross-linked complexes were reduced in the gel matrix and analyzed on fresh gels, the 76- and 62-kDa complexes were generated from the 110-kDa band, indicating the loss of two components. The lost components were estimated to be at 14 and 34 kDa. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22-, 14-, and 34-kDa components to the FSH alpha beta dimer. The results of reduction of the cross-linked complexes demonstrate the existence of disulfide linkage between the three components.     

gp160, the envelope glycoprotein of human immunodeficiency virus type 1, is a dimer of 125-kilodalton subunits stabilized through interactions between their gp41 domains.

The molecular masses, carbohydrate contents, oligomeric status, and overall molecular structure of the env glycoproteins of human immunodeficiency virus type 1--gp120, gp160, and gp41--have been determined by quantitative electron microscopy. Using purified gp160s, a water-soluble form of env purified from a recombinant vaccinia virus expression system, we have measured the masses of several hundred individual molecules by dark-field scanning transmission electron microscopy. When combined with sequence-based information, these mass measurements establish that gp160s is a dimer of subunits with an average monomer mass of 123 kDa, of which approximately 32 kDa is carbohydrate and 91 kDa is protein. Similarly, gp120 was found to be a monomer of 89 kDa and to contain virtually all of env's glycosylation. gp41 is glycosylated only slightly, if at all, and is responsible for the interactions that stabilize the gp160s dimer. A molecular mass map of gp160s derived by image processing depicts an asymmetric dumbbell whose two domains have masses of approximately 173 and approximately 73 kDa, corresponding to a gp120 dimer and a gp41 dimer, respectively. We infer that the average monomer mass of native gp160 is 125 kDa and that in situ, env is either a dimer or a tetramer but is most unlikely to be a trimer.     

Isolation and characterization of a thermolabile beta-2 macroglycoprotein ('thermolabile substance' or 'Hakata antigen') detected by precipitating (auto) antibody in sera of patients with systemic lupus erythematosus.

A novel thermolabile beta-2 macroglycoprotein ('thermolabile substance' (TLS) or 'Hakata antigen' (HA], which was detected by the precipitating (auto) antibodies of patients with systemic lupus erythematosus, was isolated and characterized. The purification procedure entailed the following steps: isoelectric precipitation in the range between pH 5.2-6.1, hydroxyapatite absorption chromatography, 35% saturated ammonium sulfate precipitation, Sephadex G-200 gel filtration, Pevikon block electrophoresis, lentil lectin affinity chromatography and immobilized rabbit anti-human whole serum IgG column chromatography. Utilizing these procedures, 0.1 mg of HA was purified from 3 1 of pooled human serum. The molecular mass of HA was determined as 650 kDa by Sepharose 4B gel filtration. On SDS-PAGE analysis, HA showed a single band at 35 kDa under reduced conditions and numerous ladder bands between 35 kDa to more than 300 kDa under nonreduced conditions. On analytical ultracentrifugation, HA gave a molecular mass of 520 kDa with a single meniscus and a sedimentation constant of 12.0. The amino acid and carbohydrate analysis of reduced and S-pyridylethylated HA revealed that it contained five residues of hydroxyproline and an N-linked type sugar chain.     

SAXS studies of human protein HC (alpha1-microglobulin)

Protein HC is a low molecular weight heterogeneous glycoprotein widely distributed in human body fluids and belonging to the lipocalin superfamily. The monomer contains a single (183 amino acid residues long) peptide chain with 3 cysteine residues (2 of which form a disulfide bridge) and is glycosylated. The molecular mass of the glycosylated protein is about 27 kDa. Native gel electrophoresis results revealed partial oligomerisation of protein HC, which therefore was analysed by gel filtration. Two forms (monomer and dimer) of the protein HC were isolated. The SAXS data were recorded on an X33 camera using synchrotron radiation (lambda=0.15 nm) at X33 beamline at the DORIS storage ring of DESY (Hamburg, Germany). Solution scattering results permitted determination of the structural parameters of both forms of the protein studied. The monomer of protein HC is characterised by a radius of gyration R(G)= 2.20 nm and D(max)=6.3 nm and the dimer by R(G)=2.99 nm and D(max)=9.5 nm.     

A metalloproteinase extracellularly released by Crithidia deanei

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The incorporation of gelatin into SDS-PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 degrees C and pH 6.0 and showed 25% of residual activity at 28 degrees C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.