Homology between the seed cytolysin enterolobin and bacterial aerolysins

Enterolobin, a 55-kDa cytolytic, inflammatory, and insecticidal protein isolated from seeds of the Brazilian treeEnterolobium contortisiliquum (Leguminosae-Mimosoideae) has been further purified and partially sequenced by using both manual and automated methods. A computational search of enterolobin partial amino acid sequence against the PIR database revealed possible sequence similarities with aerolysins, cytolytic proteins fromAeromonas species. An alignment of enterolobin partial sequence to the amino acid sequences ofA. hydrophila andA. sobria aerolysins showed several similar regions with many residue identites. The seed protein enterolobin and the bacterial aerolysins may be homologous proteins despite the distant phylogenetic relationship.     

A phylogenetic analysis of species in the Bufo crucifer group (Anura: Bufonidae), based on indolealkylamines and proteins from skin secretions

This research tested the utility of two classes of skin secretion compounds to the phylogeny of the Bufo crucifer group. Skin secretions from specimens of nine populations of B. crucifer group were obtained and submitted to qualitative analysis. We observed a clear difference in the composition of the skin secretion molecules obtained from the species of Bufo studied. Fifty-nine molecules, 16 indolealkylamines and 43 proteins, were used as characters, and 39 of these were parsimonious informative. The tree topology of the skin secretion combined data showed areas of congruence and conflict when compared to an mtDNA phylogeny of the B. crucifer group. We used the Templeton test to evaluate the heterogeneity between the skin secretion and mtDNA data. Although not recommended, we performed a combined analysis with the two partitions. The skin secretion characters from the species of Bufo studied have phylogenetic signal. These data are indicative, at least as a preliminary study, of the phylogenetic relationships among the B. crucifer group taxa.     

Gill microsomal (Na+,K+)-ATPase from the blue crab Callinectes danae: Interactions at cationic sites

Euryhaline crustaceans tolerate exposure to a wide range of dilute media, using compensatory, ion regulatory mechanisms. However, data on molecular interactions occurring at cationic sites on the crustacean gill (Na⁺,K⁺)-ATPase, a key enzyme in this hyperosmoregulatory process, are unavailable. We report that Na⁺ binding at the activating site leads to cooperative, heterotropic interactions that are insensitive to K⁺. The binding of K⁺ ions to their high affinity sites displaces Na⁺ ions from their sites. The increase in Na⁺ ion concentrations increases heterotropic interactions with the K⁺ ions, with no changes in K_0.5 for K⁺ ion activation at the extracellular sites. Differently from mammalian (Na⁺,K⁺)-ATPases, that from C. danae exhibits additional NH₄⁺ ion binding sites that synergistically activate the enzyme at saturating concentrations of Na⁺ and K⁺ ions. NH₄⁺ binding is cooperative, and heterotropic NH₄⁺ ion interactions are insensitive to Na⁺ ions, but Na⁺ ions displace NH₄⁺ ions from their sites. NH₄⁺ ions also displace Na⁺ ions from their sites. Mg²⁺ ions modulate enzyme stimulation by NH₄⁺ ions, displacing NH₄⁺ ion from its sites. These interactions may modulate NH₄⁺ ion excretion and Na⁺ ion uptake by the gill epithelium in euryhaline crustaceans that confront hyposmotic media.     

Structural and functional characterization of BnSP-7, a Lys49 myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom

BnSP-7, a Lys49 myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom, was structurally and functionally characterized. Several biological activities were assayed and compared with those of the chemically modified toxin involving specific amino acid residues. The cDNA produced from the total RNA by RT-PCR contained approximately 400 bp which codified its 121 amino acid residues with a calculated pI and molecular weight of 8.9 and 13,727, respectively. Its amino acid sequence showed strong similarities with several Lys49 phospholipase A(2) homologues from other Bothrops sp. venoms. By affinity chromatography and gel diffusion, it was demonstrated that heparin formed a complex with BnSP-7, held at least in part by electrostatic interactions. BnSP-7 displayed bactericidal activity and promoted the blockage of the neuromuscular contraction of the chick biventer cervicis muscle. In addition to its in vivo myotoxic and edema-inducing activity, it disrupted artificial membranes. Both BnSP-7 and the crude venom released creatine kinase from the mouse gastrocnemius muscle and induced the development of a dose-dependent edema. His, Tyr, and Lys residues of the toxin were chemically modified by 4-bromophenacyl bromide (BPB), 2-nitrobenzenesulfonyl fluoride (NBSF), and acetic anhydride (AA), respectively. Cleavage of its N-terminal octapeptide was achieved with cyanogen bromide (CNBr). The bactericidal action of BnSP-7 on Escherichia coli was almost completely abolished by acetylation or cleavage of the N-terminal octapeptide. The neuromuscular effect induced by BnSP-7 was completely inhibited by heparin, BPB, acetylation, and CNBr treatment. The creatine kinase releasing and edema-inducing effects were partially inhibited by heparin or modification by BPB and almost completely abolished by acetylation or cleavage of the N-terminal octapeptide. The rupture of liposomes by BnSP-7 and crude venom was dose and temperature dependent. Incubation of BnSP-7 with EDTA did not change this effect, suggesting a Ca(2+)-independent membrane lytic activity. BnSP-7 cross-reacted with antibodies raised against B. moojeni (MjTX-II), B. jararacussu (BthTX-I), and B. asper (Basp-II) myotoxins as well as against the C-terminal peptide (residues 115-129) from Basp-II.     

Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein

The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, a SBP isoform (GmSBP2/S64) was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as inclusion bodies. The renatured protein was studied by circular dichroism (CD), intrinsic fluorescence, and binding of the hydrophobic probes ANS and Bis-ANS. The estimated content of secondary structure of the renatured protein was consistent with that obtained by theoretical modeling with a large predominance of beta-strand structure (42%) over the alpha-helix (9.9%). The fluorescence emission maximum of 303 nm for SBP2 indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. We also measured the equilibrium dissociation constant (K(d)) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity (K(d)=2.79+/-0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, these results indicate that the folded structure of the renatured protein was similar to the native SBP protein. As a member of the bicupin family of proteins, which includes highly stable seed storage proteins, SBP2 was fairly stable at high temperatures. Likewise, it remained folded to a similar extent in the presence or absence of 7.6M urea or 6.7 M GdmHCl. The high stability of the renatured protein may be a reminiscent property of SBP from its evolutionary relatedness to the seed storage proteins.