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ABSTRACT: Pangasianodon hypophthalmus serum was fractionated by affinity chromatography on 12 different Sepharose-carbohydrate columns and proteins eluted by the corresponding sugar. Binding to the affinity matrices is dependent on Ca(2+) ions. Upon gel filtration using Superose-12, essentially one fraction was obtained, eluting as a protein with a molecular mass of about 900 kDa. SDS-PAGE in reducing conditions revealed the presence of large (72 kDa) subunits (H-chains) and one up to three small (24, 26 and/or 28-29 kDa) subunits (L-chains). The isolated proteins were shown to be IgM since they bind monoclonal anti-P. hypophthalmus IgM antibodies. Rabbit polyclonal anti-galactose-binding IgM only cross-react with some sugar-binding IgM. The H-chains of the anti-carbohydrate IgM are glycosylated. Circular dichroism studies revealed that the IgMs have an "all-β" type of structure, and that Ca(2+) ions, though essential for carbohydrate-binding activity, are not required for the structural integrity of the molecules. In non-reducing SDS-PAGE, only monomers and halfmers were obtained, showing that there are no disulfide bonds linking the monomers, and that a disulfide bond connecting both H-chains within one monomer is only present in 45% of the molecules. Both the monomers and the halfmers display molecular mass heterogeneity which is indicative for redox forms at the level of the intradomain disulfide bonds. The native carbohydrate-binding IgMs agglutinate erythrocytes from different animals, as well as fish pathogenic bacteria. Similar proteins could not be isolated from another catfish, Clarias gariepinus.
Developmental and comparative immunology 09/2011; 36(2):418-32. · 3.29 Impact Factor
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ABSTRACT: One hundred and fifteen semen samples were collected from 115 different boars from two farms in Cuba. The boars belonged to five different breeds. Evaluation of the semen sample characteristics (volume, pH, colour, smell, motility of sperm cells) revealed that they meet international standards. The samples were also tested for the presence of agglutinated sperm cells and for bacterial contamination. Seventy five percent of the ejaculates were contaminated with at least one type of bacteria and E. coli was by far the major contaminant, being present in 79% of the contaminated semen samples (n=68). Other contaminating bacteria belonged to the genera Proteus (n=31), Serratia (n=31), Enterobacter (n=24), Klebsiella (n=12), Staphylococcus (n=10), Streptococcus (n=8) and Pseudomonas (n=7). Only in one sample anaerobic bacteria were detected. Pearson's analysis of the data revealed that there is a positive correlation between the presence of E. coli and sperm agglutination, and a negative correlation between sperm agglutination and litter size. One-way ANOVA and post hoc Tukey analysis of 378 litters showed that the litter size is significantly reduced when semen is used that is contaminated with spermagglutinating E. coli above a threshold value of 3.5x10(3)CFU/ml.
Animal reproduction science 07/2010; 120(1-4):95-104. · 1.56 Impact Factor
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ABSTRACT: From 1 kg of defatted Pterocarpus angolensis (mukwa tree) seed meal, 21.6 grams of an alpha,D-mannose/glucose-specific lectin can be purified on mannose-Sepharose. Relative affinities for several (oligo)saccharides and glycoproteins were studied by haemagglutination-inhibition. Gel filtration shows that the lectin exists as a dimer above pH 5 and as a monomer below pH 3.5. This is confirmed by studies on the release of lectin subunits that were adsorbed from solution to lectin monomers immobilized onto Eupergit-c. From the gel filtration patterns it is calculated that a residue with pK(a) of about 4.4 is involved in dimer dissociation. Titration of glutamic acids (E60, E209) is postulated to be involved. CD spectroscopy shows that the secondary structure of the lectin is unchanged between pH 1 and 12.5, and that the tertiary structure remains unchanged between pH 5 and 12. In the acid pH region, reversible spectral changes occur that may be due to the titration of one or more amino acids with a pK(a) value of 3.9-4.2, probably aspartic acid. These residues are implicated in sugar-binding but not in dimerization of the lectin. Only at pH 12.5, irreversible denaturation occurs. Mukwa lectin displays full carbohydrate-binding capacity between pH 4 and 12, as is concluded from ELLA (Enzyme Linked Lectin Assay) using ovalbumin and fetuin, and from binding of the same glycoproteins to immobilized lectin monomers. The lectin is rapidly and fully reversibly demetallized at pH 2.5 with 5 mM EDTA. The demetallized lectin is completely devoid of sugar-binding activity. Mukwa lectin is a very thermostable molecule (at least till 85 degrees C). However, addition of non-ionic detergents substantially lowers its thermostability.
Protein and Peptide Letters 10/2009; 16(9):1120-34. · 1.94 Impact Factor
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ABSTRACT: World-wide, enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC)-induced diarrhea are economically important for porcine producers. Our aim was to investigate the prevalence of toxin and fimbrial genes among E. coli isolated from diarrheic piglets from randomly selected piggeries in Zimbabwe. We used multiplex PCR for screening STa, STb, LT, and Stx-2e toxins. Subsequently F4, F5, F6, F18 and F41 fimbriae genes were screened in toxin positive isolates. Toxin positive strains lacking tested fimbriae genes were characterized using transmission electron microscopy, agglutination and agglutination inhibition tests. Approximately 32% of the 1,984 isolates tested positive for STa, STb, LT or Stx-2e genes. Of these, approximately 81% had F4, F5, F6, F18 or F41 fimbriae genes. The remaining toxin positive strains lacked tested fimbriae genes and appeared to either express F1-like fimbriae, or lacked fimbriae. The data constitute an important framework for implementation of prevention measures, such as using relevant fimbriae-based vaccines against ETEC induced diarrhea or VTEC-induced edema.
Tropical Animal Health and Production 05/2009; 41(7):1539-47. · 1.12 Impact Factor
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ABSTRACT: A new procedure has been developed for the isolation of fumarase (EC 4.2.1.2). It is described for the purification of pig heart and liver enzyme. Pyromellitic acid has been covalently coupled to Sepharose-4B with diaminopropanol as spacer arm. When a dialysed 0.55 saturated ammonium sulphate precipitate is applied to the column, in Tris-acetate buffer, pH 7.3, fumarase remains quantitatively bound. It is eluted by competition, together with a few other proteins, by the natural product l-malate. Malate is removed from the eluate by dialysis. After this highly efficient purification step the enzyme is very easily crystallized. The final yield is 67% for both pig heart and liver preparations. The specific activity of fumarase purified from both tissues is found to be the same. Polyacrylamide gel electrophoresis in dodecylsulphate shows one single band corresponding with a subunit molecular weight of 48500. A single band is also obtained by electrophoresis in acid urea. This new procedure based on biospecific affinity chromatography allows a fast and easy preparation of gram quantities of fumarase.
European Journal of Biochemistry. 06/2008; 78(2):437 - 444.
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ABSTRACT: The crystal structure of Pterocarpus angolensis lectin is determined in its ligand-free state, in complex with the fucosylated biantennary complex type decasaccharide NA2F, and in complex with a series of smaller oligosaccharide constituents of NA2F. These results together with thermodynamic binding data indicate that the complete oligosaccharide binding site of the lectin consists of five subsites allowing the specific recognition of the pentasaccharide GlcNAc beta(1-2)Man alpha(1-3)[GlcNAc beta(1-2)Man alpha(1-6)]Man. The mannose on the 1-6 arm occupies the monosaccharide binding site while the GlcNAc residue on this arm occupies a subsite that is almost identical to that of concanavalin A (con A). The core mannose and the GlcNAc beta(1-2)Man moiety on the 1-3 arm on the other hand occupy a series of subsites distinct from those of con A.
FEBS Journal 07/2006; 273(11):2407-20. · 3.79 Impact Factor
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ABSTRACT: The Man/Glc-specific legume lectin from the seeds of the African bloodwood tree (Pterocarpus angolensis) was crystallized in the presence of the disaccharide ligand Man(alpha1-3)ManMe. Small crystals initially appeared from a preliminary screen, but proved difficult to reproduce. The initial crystals were used to prepare microseeds, leading to a reproducible crystallization protocol. All attempts to obtain crystals directly of the ligand-free protein or of other carbohydrate complexes failed. However, the Man(alpha1-3)ManMe co-crystals withstand soaking with ten other carbohydrates known to bind to the lectin. Soaking for 15 min in 100 mM carbohydrate typically resulted in complete replacement of Man(alpha1-3)ManMe by the desired carbohydrate despite the involvement of lattice contacts at the binding site. Transferring the crystals for two weeks in carbohydrate-free artificial mother liquor resulted in the complete removal of the sugar from one of the two monomers in the asymmetric unit. Additional treatment of these crystals with 100 mM EDTA for two weeks resulted in removal of the structural calcium and manganese ions, which is accompanied by significant structural rearrangements of the loops that constitute the carbohydrate-binding site.
Acta Crystallographica Section D Biological Crystallography 07/2005; 61(Pt 6):685-9. · 12.62 Impact Factor
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ABSTRACT: The crystal structure of a Man/Glc-specific lectin from the seeds of the bloodwood tree (Pterocarpus angolensis), a leguminous plant from central Africa, has been determined in complex with mannose and five manno-oligosaccharides. The lectin contains a classical mannose-specificity loop, but its metal-binding loop resembles that of lectins of unrelated specificity from Ulex europaeus and Maackia amurensis. As a consequence, the interactions with mannose in the primary binding site are conserved, but details of carbohydrate-binding outside the primary binding site differ from those seen in the equivalent carbohydrate complexes of concanavalin A. These observations explain the differences in their respective fine specificity profiles for oligomannoses. While Man(alpha1-3)Man and Man(alpha1-3)[Man(alpha1-6)]Man bind to PAL in low-energy conformations identical with that of ConA, Man(alpha1-6)Man is required to adopt a different conformation. Man(alpha1-2)Man can bind only in a single binding mode, in sharp contrast to ConA, which creates a higher affinity for this disaccharide by allowing two binding modes.
Journal of Molecular Biology 02/2004; 335(5):1227-40. · 4.00 Impact Factor
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ABSTRACT: The crystal structure of the Man/Glc-specific seed lectin from Pterocarpus angolensis was determined in complex with methyl-α-d-glucose, sucrose, and turanose. The carbohydrate binding site contains a classic Man/Glc type specificity loop. Its metal
binding loop on the other hand is of the long type, different from what is observed in other Man/Glc-specific legume lectins.
Glucose binding in the primary binding site is reminiscent of the glucose complexes of concanavalin A and lentil lectin. Sucrose
is found to be bound in a conformation similar as seen in the binding site of lentil lectin. A direct hydrogen bond between
Ser-137(OG) to Fru(O2) in Pterocarpus angolensis lectin replaces a water-mediated interaction in the equivalent complex of lentil lectin. In the turanose complex, the binding
site of the first molecule in the asymmetric unit contains the αGlc1–3βFruf form of furanose while the second molecule contains the αGlc1–3βFrup form in its binding site.
Journal of Biological Chemistry 05/2003; 278(18):16297-16303. · 4.77 Impact Factor
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ABSTRACT: Two different approaches provided evidence for a physical interaction between the carbamate kinase-like carbamoyl-phosphate synthetase (CKase) and ornithine carbamoyltransferase (OTCase) from the hyperthermophilic archaeon Pyrococcus furiosus. Affinity electrophoresis indicated that CKase and OTCase associate into a multienzyme cluster. Further evidence for a biologically significant interaction between CKase and OTCase was obtained by co-immunoprecipitation combined with formaldehyde cross-linking experiments. These experiments support the hypothesis that CKase and OTCase form an efficient channeling cluster for carbamoyl phosphate, an extremely thermolabile and potentially toxic metabolic intermediate. Therefore, by physically interacting with each other, CKase and OTCase prevent the thermodenaturation of carbamoyl phosphate in the aqueous cytoplasmic environment.
Journal of Biological Chemistry 06/2002; 277(21):18517-22. · 4.77 Impact Factor
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ABSTRACT: Two different approaches provided evidence for a physical interaction between the carbamate kinase-like carbamoyl-phosphate
synthetase (CKase) and ornithine carbamoyltransferase (OTCase) from the hyperthermophilic archaeonPyrococcus furiosus. Affinity electrophoresis indicated that CKase and OTCase associate into a multienzyme cluster. Further evidence for a biologically
significant interaction between CKase and OTCase was obtained by co-immunoprecipitation combined with formaldehyde cross-linking
experiments. These experiments support the hypothesis that CKase and OTCase form an efficient channeling cluster for carbamoyl
phosphate, an extremely thermolabile and potentially toxic metabolic intermediate. Therefore, by physically interacting with
each other, CKase and OTCase prevent the thermodenaturation of carbamoyl phosphate in the aqueous cytoplasmic environment.
Journal of Biological Chemistry 05/2002; 277(21):18517-18522. · 4.77 Impact Factor
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ABSTRACT: Samples of seeds from eight legume species and Triticum vulgaris grains were extracted with buffer and lectin activity in the extracts was determined in haemagglutination experiments using normal or Pronase-treated rabbit erythrocytes. The effect of the addition of spray dried porcine and bovine plasma powder, whole egg powder, galactosides, whey powder and specific inhibitors (eg mannose, galactose, N-acetyl galactosamine, fetuin) on haemagglutination activity (HA) was determined. Plasma powders were potent inhibitors of HA in extracts of Pisum sativum, Vicia faba, Vicia sativa, Lens culinaris and Phaseolus vulgaris. HA in extracts of Lupinus sp and Phaseolus vulgaris was efficiently decreased by whole egg powder, while the lectin of Glycine max could only be inhibited by addition of galactosides, whole and defatted milk powder and whey powder. Inhibitors (plasma and whole egg powder and fetuin) were subjected to SDS-PAGE and Western blotting and blots were incubated with biotinylated lectins, except for Lupinus lectin. Results of the HA experiments were confirmed: lectins which were not influenced by inhibitory compounds in HA experiments also showed no binding with proteins of the blotted inhibitor. There were strong indications that lectins were not bound to the albumin fraction of the plasma powders. Results are discussed in view of future in vivo experiments. © 1998 SCI.
Journal of the Science of Food and Agriculture 03/1999; 77(3):319 - 326. · 1.44 Impact Factor
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ABSTRACT: By means of covalently immobilized fumarase and mitochondrial or cytoplasmic malate dehydrogenase we were able to detect physical interactions between different enzymes of the citric acid cycle (fumarase with malate dehydrogenase, malate dehydrogenase with citrate synthase and fumarase with citrate synthase) and between the enzymes of both mitochondria) and cytoplasmic halves of the aspartate-malate shuttle (aspartate aminotransierase and malate dehydrogenase). The interactions between fumarase and malate dehydrogenase were also investigated by immobilizing one enzyme indirectly through antibodies bound to Sepharose-protein A. Our results are consistent with a model in which maximally four molecules of malate dehydrogenase are bound to one fumarase molecule. This complex is able to bind either citrate synthase or aspartate aminotransferase. We propose that these enzymes bind alternatively, in order to allow the cell to perform citric acid cycle or shuttle reactions, according to its needs. The physiological meaning and implications on the regulation of metabolism of the existence of a large citric acid cycle/malate-aspartate shuttle multienzyme complex are discussed.
European Journal of Biochemistry. 06/1981; 117(3):527 - 535.
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ABSTRACT: A purification scheme is described for the glyoxylate cycle enzyme isocitrate lyase from maize scutella. Purification involves an acetone precipitation and a heat denaturation step, followed by ammonium sulfate precipitation and chromatography on DEAE-cellulose and on blue-Sepharose. The latter step results in the removal of the remaining malate dehydrogenase activity, and of a high molecular mass (62 kDa) but inactive degradation product of isocitrate lyase. Catalase can be completely removed by performing the DEAE-cellulose chromatography in the presence of Triton X-100. Pure isocitrate lyase can be stored without appreciable loss of activity at −70 °C in 5 mm triethanolamine buffer containing 6 mm MgCl2, 7 mm 2-mercaptoethanol, and 50% () glycerol, pH 7.6. Maize isocitrate lyase is a tetrameric protein with a subunit molecular mass of 64 kDa. Purity of the enzyme preparation was demonstrated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, in acid (pH 3.2) urea and by isoelectric focusing (pI = 5.1). Maize isocitrate lyase is devoid of covalently linked sugar residues. From circular dichroism measurements we estimate that its structure comprises 30% α-helical and 15% β-pleated sheet segments. The enzyme requires Mg2+ ions for activity, and only Mn2+ apparently is able to replace this cation to a certain extent. The kinetics of the isocitrate lyase-catalyzed cleavage reaction were investigated, and the amino acid composition of the maize enzyme was determined. Finally the occurrence of an association between maize isocitrate lyase and catalase was observed. Such a multienzyme complex may be postulated to play a protective role in vivo.
Archives of Biochemistry and Biophysics.
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ABSTRACT: From the serum of Pangasianodon hypophthalmus, two proteins were isolated by affinity chromatography on Sepharose and phosphorylcholine-Sepharose. Their binding on the affinity matrices critically depends on the presence of Ca2+ ions. N-terminal sequencing and sequencing of internal tryptic peptides identified the proteins as pentraxins and from their binding properties they are identified as SAP (serum amyloid P component) and CRP (C-reactive protein). Per ml serum, 36 microg SAP and 56 microg CRP was purified. Upon gel filtration, both the SAP and CRP elute as trimers of respectively 24 kDa and 28 kDa subunits. Both proteins are devoid of inter-chain disulfide bonds. Both SAP and CRP are glycosylated and agglutinate rabbit erythrocytes and pathogenic bacteria Edwardsiella ictaluri and Aeromonas hydrophila, but not Micrococcus lysodeikticus or Escherichia coli. Haemagglutination of SAP and CRP is inhibited by galactose (MIC = 1 mM) and by phosphorylcholine (MIC = 1-2 mM), respectively. Circular dichroism studies revealed that antiparallel beta-pleated sheets are dominating the secondary structure. Upon removing the Ca(2+) ions by EDTA, slight structural changes are observed by CD spectroscopy in the near-UV region. Immunodiffusion shows that P. hypophthalmus SAP and CRP do not cross-react.
Fish & Shellfish Immunology 28(5-6):743-53. · 3.32 Impact Factor
