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PubMed1993

On the stability of nucleic acid structures in solution: enthalpy-entropy compensations, internal rotations and reversibility.

The thermodynamics of self-association (stacking) of free bases and nucleotides, intramolecular stacking in dinucleotides, nearest-neighbour base pair stacking interactions in duplex DNA and RNA, and the formation of hairpin loops illustrate enthalpy/entropy compensations. Large stacking exothermicities are associated with large negative entropy changes that ensure that delta G is small, permitting readily reversible associations in solution. We rationalise enthalpy/entropy compensations with reference to residual motions and torsional vibrations which make a larger entropic contribution to binding when - delta H approximately kT (thermal energy at room temperature), than when - delta H >> kT. We present a factorisation of experimental free energies for helix formation in terms of approximate contributions from the restriction of rotations, hydrophobic interactions, electrostatic interactions due to base stacking, and contributions from hydrogen bonding, and estimate the adverse free energy cost per rotor (mainly entropy) of ordering the phosphate backbone as between 1.9 and 5.4 kJ mol-1 [averaged over 12 rotors per base pair for A-U on A-U stacking (lower limit), and G-C on C-G stacking (upper limit)]. The largest cost is associated with the most exothermic stacking interactions, while the range of values is consistent with earlier conclusions from data on the fusion of hydrocarbon chains (lower value), and with entropy changes in covalent isomerisations of small molecules involving severe restrictions (upper value).

Base Composition↗

PubMed1983

Biochemistry of the polyamines.

The naturally-occurring polyamines exist in the free form, as N-acetyl derivatives and bound to protein. Their biosynthesis is subject to sensitive control, particularly of ornithine decarboxylase. This enzyme may be multifunctional and a key regulatory protein. Studies, principally with selective inhibitors, have elucidated the roles of polyamines in cell proliferation. Oxidized polyamines, in contrast, can be potent mitotic inhibitors. These effects are reviewed in terms of their chemistry and biochemistry. Their principal distinctions are that they can be made or degraded intracellularly, they can associate electrostatically with macromolecules by means of their spaced cationic groups, and these can be readily converted to covalent bonds.

Animals↗

PubMed1991

Initial binding of 2'-deoxynucleoside 5'-triphosphates to human immunodeficiency virus type 1 reverse transcriptase.

Human immunodeficiency virus type 1 reverse transcriptase (EC 2.7.7.49), a heterodimer consisting of two polypeptide chains of molecular weights 66,000 and 51,000, fluoresces due to the presence of 36 tryptophan residues with an emission peak centered at 338 nm. The association of 2'-deoxynucleoside 5'-triphosphates with the enzyme results in a decrease in the intensity of the tryptophan emission spectrum, which can be used to calculate apparent dissociation constants. The Kd values determined for binding of the four natural 2'-deoxynucleoside 5'-triphosphates to the free enzyme range from 36.7 +/- 1.8 microM for dTTP to 47.3 +/- 3.9 microM for dATP. The 5'-triphosphate of zidovudine has a Kd of 54.1 +/- 1.3 microM. The enzyme shows no preference for purine or pyrimidine nucleotides. Hill coefficients and the results of dual ligand titration experiments demonstrate that the free enzyme possesses a single dNTP binding site for which the four natural substrates and the 5'-triphosphate of zidovudine compete. The presence of homopolymeric template-primers does not result in selective binding of the complementary 2'-deoxynucleoside 5'-triphosphate, indicating that Watson-Crick base pairing is not involved in the initial binding reaction. The major force driving the association of the ligands with the binding site is hydrophobic. Approximately 14% of the binding energy is derived from electrostatic interactions. Although Mg2+ is required for catalytic activity, it is not absolutely required for initial binding.

Binding, Competitive↗

PubMed1994

Reverse transphosphorylation by ribonuclease A needs an intact p2-binding site. Point mutations at Lys-7 and Arg-10 alter the catalytic properties of the enzyme.

Bovine pancreatic ribonuclease A interacts with RNA along multiple binding subsites that essentially recognize the negatively charged phosphates of the substrate. This work gives additional strong support to the existence of the postulated phosphate-binding subsite p2 (Parés, X., Llorens, R., Arús, C., and Cuchillo, C. M. (1980) Eur. J. Biochem. 105, 571-579) and confirms the central role of Lys-7 and Arg-10 in establishing an electrostatic interaction with a phosphate group of the substrate. The effects of charge elimination by Lys-7-->Gln (K7Q) and/or Arg-10-->Gln (R10Q) substitutions in catalytic and ligand-binding properties of ribonuclease A have been studied. The values of Km for cytidine 2',3'-cyclic phosphate and cytidylyl-3',5'-adenosine are not altered but are significantly increased for poly(C). In all cases, kcat values are lower. Synthetic activity, i.e. the reversion of the transphosphorylation reaction, is reduced for K7Q and R10Q mutants and is practically abolished in the double mutant. Finally, the extent of the reaction of the mutants with 6-chloropurine-9-beta-D-ribofuranosyl 5'-monophosphate indicates that the phosphate ionic interaction in p2 is weakened. Thus, p2 modification alters both the catalytic efficiency and the extent of the processes in which an interaction of the phosphate group of the substrate or ligand with the p2-binding subsite is involved.

Amino Acid Sequence↗

PubMed1997

Experimental quantitation of cation effect on nucleic acid stability.

Cation effect on DNA/DNA, RNA/RNA, and RNA/DNA stabilities was investigated quantitatively. Plots of predicted delta G degree 37 values vs. measured delta G degree 37 ones showed the same relationship for DNA/DNA, RNA/RNA, and RNA/DNA. The number of sodium ion concerned in a double helix formation was estimated, and the result indicated that the number of bound sodium ion may determine the duplex stability regardless of DNA/DNA, RNA/RNA, and RNA/DNA. Since these duplex stability can be predicted with the nearest-neighbor model, it is considered that the number of bound sodium ions is affected by the nearest-neighbor bases. The importance of the screening effect of the electrostatic repulsion by bound cations was also indicated for determining the duplex stability as well as the hydrogen bonding and stacking interactions.

Base Sequence↗

PubMed1988

Interactions of porphyrins and transfer RNA.

The interactions of the free base porphyrin, tetra-(4N-methylpyridyl)porphyrin and its copper(II), manganese(III) and zinc(II) complexes with brewer's yeast type V phenylalaninyl tRNA were evaluated by UV-visible spectroscopy, circular dichroism and melting temperature studies over a range of magnesium ion concentrations and ionic strengths. Scatchard analysis of absorption spectra of the porphyrins in the presence of tRNA showed the free base, copper and zinc porphyrins to have binding constants of 7.3 X 10(7), 1.7 X 10(6) and 2.3 X 10(8), respectively; the manganese(III) complex did not demonstrate changes in its electronic spectra that enable the calculation of a binding constant. The results of the spectroscopic studies indicate a mode of binding for the free base, copper(II) and zinc(II) complexes that is neither intercalative nor simply outside electrostatic. The magnitude of the binding constants and the UV-visible results support intercalation, but the analyses of the thermal denaturation studies and the circular dichroism evaluations suggest that the porphyrins are associating at a single site in a fold of the tertiary structure of the tRNA close to several crucial hydrogen bonds, perhaps in the vicinity of the P10 loop. That the manganese(III) complex does not bind in this site points to constraints on the axial thickness of a molecule that may be accommodated in this locus.

Cations, Divalent↗

PubMed1992

Characterization of the nucleic acid-binding activities of the isolated amino-terminal head domain of the intermediate filament protein vimentin reveals its close relationship to the DNA-binding regions of some prokaryotic single-stranded DNA-binding proteins.

In order to demonstrate that the nucleic acid-binding activities of vimentin are dictated by its Arg-rich N-terminal head domain, this was cut off at position Lys96 with lysine-specific endoproteinase and analysed for its capacity to associate with a variety of synthetic and naturally occurring nucleic acids. The isolated polypeptide (vim NT) showed a preference for single-stranded (ss) polynucleotides, particularly for ssDNAs of high G-content. A comparison of the sequence and predicted secondary structure of vim NT with that of two prokaryotic ssDNA-binding proteins, G5P and G32P of bacteriophages fd and T4, respectively, revealed that the nucleic acid-binding region of all three polypeptides is almost entirely in the beta-conformation and characterized by a very similar distribution of aromatic amino acid residues. A partial sequence of vim NT can be folded into the same beta-loop structure as the DNA-binding wing of G5P of bacteriophage fd and related viruses. As in the case of G5P, nitration of the Tyr residues with tetranitromethane was blocked by single-stranded nucleic acids. This and spectroscopic data indicate intercalation of the Tyr aromatic ring systems between the bases of the nucleic acids and thus the contribution of a stacking component to the binding reaction. The binding was accompanied by significant changes in the ultraviolet absorption spectra of both vim NT and single-stranded nucleic acids. Upon mixing of vim NT with nucleic acids, massive precipitation of the reactants occurred, followed by the quick rearrangement of the aggregates with the formation of specific and soluble association products. Even at very high ionic strengths, at which no electrostatic reaction should be expected, a distinct fraction of vim NT incorporated naturally occurring ssRNAs and ssDNAs into fast sedimenting complexes, suggesting co-operative interaction of the polypeptide with the nucleic acids. In electron microscopy, the complexes obtained from 28 S rRNA appeared as networks of extended nucleic acid strands densely covered with vim NT, in contrast to the compact random coils of uncomplexed RNA. The networks produced from fd DNA were heterogeneous in appearance and their nucleoprotein strands in rare cases were very similar to the rod-like structures of G5P-fd DNA complexes.

Amino Acid Sequence↗

PubMed1994

2.1 A structure of Serratia endonuclease suggests a mechanism for binding to double-stranded DNA.

The crystal structure of Serratia endonuclease has been solved to 2.1 A by multiple isomorphous replacement. This magnesium-dependent enzyme is equally active against single- and double-stranded DNA, as well as RNA, without any apparent base preference. The Serratia endonuclease fold is distinct from that of other nucleases that have been solved by X-ray diffraction. The refined structure consists of a central layer containing six antiparallel beta-strands which is flanked on one side by a helical domain and on the opposite side by one dominant helix and a very long coiled loop. Electrostatic calculations reveal a strongly polarized molecular surface and suggest that a cleft between this long helix and loop, near His 89, may contain the active site of the enzyme.

Amino Acid Sequence↗

PubMed1990

Selective staining of nucleic acids by osmium-ammine complex in thin sections from lowicryl-embedded samples.

Osmium-ammine (OA)/SO2 selectively contrasted RNA- and DNA-containing structures in thin sections from Lowicryl-embedded samples. No cell structures were stained after Epon embedding. RNAse and DNAse digestion experiments demonstrated that only RNA and DNA were stained in Lowicryl thin sections. Protease digestion did not modify the staining reaction. The very fine end-reaction produced a very high resolution of the stained structures. The staining reaction was not due to the presence of SO2 but to the low pH of the solution (ranging from 1.5-2.2). OA in glycine buffer, pH 1.5, selectively contrasted nucleic acids. Electrostatic bonds between nucleic acids and OA complex were probably involved in the staining reaction. Increasing the pH value of the staining medium resulted in loss of OA specificity for nucleic acids. The high electrolyte concentration of the staining medium hindered the staining reaction.

Acrylic Resins↗

PubMed2000

Amino acid substitution within the S2 and S4 transmembrane segments in Shaker potassium channel modulates channel gating.

To investigate of the gating properties in the voltage-activated potassium channel, we have mutated a variety of S2 and S4 residues in the Shaker potassium protein. Results showed that the R365C and R368C, but not the E283C, R362C, R365S, R368S or the ShB-IR, were sensitive to micromolar concentrations of Cd(2+) ions. This indicates that R365 and R368 play a crucial role in the channel gating due to a conformational modulation of the channel structure. Doubly mutated channels of the E283C/R365E and E283C/R368E caused a transient increase in current amplitude, which reached a peak within a few seconds and then decreased toward initial levels, despite the continual presence of Cd(2+). Taken together, our results suggest that E283, R365, and R368 form a network of strong, local, and electrostatic interactions that relate closely to the mechanism of the channel gating.

Amino Acid Substitution↗

PubMed2001

Membrane activity of the southern cowpea mosaic virus coat protein: the role of basic amino acids, helix-forming potential, and lipid composition.

Southern cowpea mosaic virus (SCPMV) is a spherical RNA virus with T = 3 icosahedral symmetry. The particle is composed of 180 subunits of the coat protein (CP) and one copy of the positive-sense viral RNA. The CP has two domains, the random (R) domain formed by the N-terminal 64 aa and the shell (S) domain (aa 65--260). The R domain is highly charged, with 11 of the N-terminal 30 residues being basic. It is localized to the interior of the native particle where it may interact with the viral RNA, but under certain pH and salt conditions the topology of the particle changes to externalize the R domain. Since the CPs of several spherical RNA viruses have been shown to interact with host membranes during infection, we have begun investigating the membrane interactions of the SCPMV CP using the artificial liposome membranes. Both the native CP and the R domain overexpressed in Escherichia coli were observed to interact with liposomes. The interaction between the R domain and liposomes required either anionic phospholipids or non-bilayer-forming lipids and involved electrostatic interactions since it was shown to be both pH and ionic strength dependent. The analysis of four different deletion and six different site-directed substitution mutations partially mapped the region responsible for this interaction to residues 1--30. Analysis of this region of the R domain by circular dichroism indicated that it assumes an alpha-helical structure when exposed to liposomes composed of anionic lipids. Mutations, which extend the helical nature of this region, promoted an increased interaction. The possible role of the CP/lipid interaction in the SCPMV infection is discussed.

Amino Acid Sequence↗

PubMed1985

Mechanism of codon recognition by transfer RNA studied with oligonucleotides larger than triplets.

The binding of yeast tRNAPhe to UUCA, UUCC, UUCCC, UUCUUCU, U4, U5, U6 and U7 was analysed by fluorescence temperature jump and equilibrium sedimentation measurements. In all cases the two observed relaxation processes can be assigned to alpha) an intramolecular conformation change of the anticodon loop and beta) preferential binding of the oligonucleotides to one of the anticodon conformations. The anticodon loop transition is associated with inner sphere complexation of Mg2+ and proceeds with rate constants of about 10(3) s-1. The rate constants of oligonucleotide binding are between 4 and 10 X 10(6) M-1s-1 and reflect an increase of the association rate with the number of binding sites compensated to some degree by electrostatic repulsion in the preequilibrium complex. Neither temperature jump nor equilibrium sedimentation experiments provided evidence for UUCA or UUCC induced tRNA dimerisation, although UUC binding leads to strong tRNA dimerisation under equivalent conditions. The results obtained for the longer oligonucleotides are similar. In the case of UUCUUCU with its two potential binding sites for tRNAPhe there was no evidence for the formation of 'ternary' complexes. Apparently tRNAPhe binds preferentially to the second UUC of this 'messenger' and forms additional contacts with residues on either side of the codon. Some evidence for the formation of ternary complexes is obtained for U6 and U7, although the extent of this reaction remains very small. Our results demonstrate that the mode of tRNA binding to a codon is strongly influenced by residues next to the codon. The formation of cooperative contacts between tRNA molecules at adjacent codons apparently requires support by a catalyst adjusting an appropriate conformation of messenger and tRNA molecules.

Anticodon↗

PubMed1999

Solution structure and thermodynamics of a divalent metal ion binding site in an RNA pseudoknot.

Identification and characterization of a metal ion binding site in an RNA pseudoknot was accomplished using cobalt (III) hexammine, Co(NH3)63+, as a probe for magnesium (II) hexahydrate, Mg(H2O)62+, in nuclear magnetic resonance (NMR) structural studies. The pseudoknot causes efficient -1 ribosomal frameshifting in mouse mammary tumor virus. Divalent metal ions, such as Mg2+, are critical for RNA structure and function; Mg2+preferentially stabilizes the pseudoknot relative to its constituent hairpins. The use of Co(NH3)63+as a substitute for Mg2+was investigated by ultraviolet absorbance melting curves, NMR titrations of the imino protons, and analysis of NMR spectra in the presence of Mg2+or Co (NH3)63+. The structure of the pseudoknot-Co(NH3)63+complex reveals an ion-binding pocket formed by a short, two-nucleotide loop and the major groove of a stem. Co(NH3)63+stabilizes the sharp loop-to-stem turn and reduces the electrostatic repulsion of the phosphates in three proximal strands. Hydrogen bonds are identified between the Co(NH3)63+protons and non-bridging phosphate oxygen atoms, 2' hydroxyl groups, and nitrogen and oxygen acceptors on the bases. The binding site is significantly different from that previously characterized in the major groove surface of tandem G.U base-pairs, but is similar to those observed in crystal structures of a fragment of the 5 S rRNA and the P5c helix of the Tetrahymena thermophila group I intron. Changes in chemical shifts occurred at the same pseudoknot protons on addition of Mg2+as on addition of Co(NH3)63+, indicating that both ions bind at the same site. Ion binding dissociation constants of approximately 0.6 mM and 5 mM (in 200 mM Na+and a temperature of 15 degrees C) were obtained for Co(NH3)63+and Mg2+, respectively, from the change in chemical shift as a function of metal ion concentration. An extensive array of non-sequence-specific hydrogen bond acceptors coupled with conserved structural elements within the binding pocket suggest a general mode of divalent metal ion stabilization of this type of frameshifter pseudoknot. These results provide new thermodynamic and structural insights into the role divalent metal ions play in stabilizing RNA tertiary structural motifs such as pseudoknots.

Binding Sites↗

PubMed2001

Streptococcus pneumonia YlxR at 1.35 A shows a putative new fold.

The structure of the YlxR protein of unknown function from Streptococcus pneumonia was determined to 1.35 A. YlxR is expressed from the nusA/infB operon in bacteria and belongs to a small protein family (COG2740) that shares a conserved sequence motif GRGA(Y/W). The family shows no significant amino-acid sequence similarity with other proteins. Three-wavelength diffraction MAD data were collected to 1.7 A from orthorhombic crystals using synchrotron radiation and the structure was determined using a semi-automated approach. The YlxR structure resembles a two-layer alpha/beta sandwich with the overall shape of a cylinder and shows no structural homology to proteins of known structure. Structural analysis revealed that the YlxR structure represents a new protein fold that belongs to the alpha-beta plait superfamily. The distribution of the electrostatic surface potential shows a large positively charged patch on one side of the protein, a feature often found in nucleic acid-binding proteins. Three sulfate ions bind to this positively charged surface. Analysis of potential binding sites uncovered several substantial clefts, with the largest spanning 3/4 of the protein. A similar distribution of binding sites and a large sharply bent cleft are observed in RNA-binding proteins that are unrelated in sequence and structure. It is proposed that YlxR is an RNA-binding protein.

Amino Acid Sequence↗

PubMed1978

Ultracentrifugation studies of yeast valyl-tRNA synthetase and of its interaction with tRNAVal.

Yeast valyl-tRNA synthetase and its complexes with yeast tRNAVal were investigated by means of analytical ultracentrifugation. A molecular weight of 125 700 +/- 1500 and a sedimentation coefficient (SO 20, w) of 6.3 +/- 0.3 were found for the native enzyme. When the enzyme (3--60 muM) was mixed with its cognate tRNA, several types of complex were observed, depending on the relative amounts of the two macromolecules. In the presence of equimolecular amounts of tRNA and enzyme, a complex formed by the association of one of each molecule was observed with a sedimentation coefficient of about 7.3 S. However, for tRNA/enzyme stoichiometries lower than one, beside the 1 : 1 complex, a complex of higher molecular weight was observed, with a sedimentation coefficient of about 10.0 S which fits with the association of two valyl-tRNA synthetase molecules with one tRNA molecule. This 2 : 1 complex was predominant from tRNA/enzyme stoichiometries lower than 0.3. It dissociated into the 1 : 1 complex upon addition of monovalent salts or MgCl2, suggesting the electrostatic nature of the interaction in this association. All these association and dissociation phenomena were detected over a large range of pH (6.0--7.5) and in various buffers.

Amino Acyl-tRNA Synthetases↗

PubMed2001

tRNA-guanine transglycosylase from Escherichia coli: molecular mechanism and role of aspartate 89.

The enzyme tRNA-guanine transglycosylase (TGT, EC 2.4.2.29) catalyzes a posttranscriptional transglycosylation reaction involved in the incorporation of the modified base queuine [Q, 7-(4,5-cis-dihydroxy-2-cyclopenten-1-ylaminomethyl)-7-deazaguanine] into tRNA. Previously, the crystal structure of the TGT from Zymomonas mobilis was solved in complex with preQ(1) (the substrate for the eubacterial TGT) [Romier et al. (1996) EMBO J. 15, 2850-2857]. An aspartate residue at position 102 (position 89 in the Escherichia coli TGT) was proposed to play a nucleophilic role in an associative catalytic mechanism. Although this is an attractive and precedented mechanism, a dissociative mechanism is equally plausible. In a dissociative mechanism, aspartate 89 would provide electrostatic stabilization of an oxocarbenium ion intermediate that is formed by dissociation of guanine. To clarify the nature of the catalytic mechanism of TGT, we have generated and characterized four mutations of aspartate 89 in the E. coli TGT (alanine, asparagine, cysteine, and glutamate). All four mutant TGTs were able to noncovalently bind tRNA, but only the glutamate mutant was able to form a stable complex with the RNA substrate under denaturing conditions that was comparable to wild type. Furthermore, the glutamate mutant was the only mutant TGT that demonstrated significant activity. Kinetic parameters were determined for this enzyme and shown to be comparable to wild type, revealing that the enzyme is considerably tolerant of the positioning of the carboxylate. Under conditions of high enzyme concentrations and long time courses, the alanine, asparagine, and cysteine mutants showed very low levels (ca. 10(3)-fold lower than wild type) of activity that were linear with respect to enzyme concentration and dependent upon pH in a fashion similar to that of the wild type. However, the observed initial velocities were too low to accurately determine k(cat) and K(m) values. We hypothesize that the activity observed for these mutants is most likely derived from host strain TGT (wt) contamination. These results are most consistent with aspartate 89 acting as a nucleophile in an associative catalytic mechanism.

Aspartic Acid↗

PubMed2005

Crystal structure of the first KH domain of human poly(C)-binding protein-2 in complex with a C-rich strand of human telomeric DNA at 1.7 A.

Recognition of poly(C) DNA and RNA sequences in mammalian cells is achieved by a subfamily of the KH (hnRNP K homology) domain-containing proteins known as poly(C)-binding proteins (PCBPs). To reveal the molecular basis of poly(C) sequence recognition, we have determined the crystal structure, at 1.7-A resolution, of PCBP2 KH1 in complex with a 7-nucleotide DNA sequence (5'-AACCCTA-3') corresponding to one repeat of the human C-rich strand telomeric DNA. The protein-DNA interaction is mediated by the combination of several stabilizing forces including hydrogen bonding, electrostatic interactions, van der Waals contacts, and shape complementarities. Specific recognition of the three cytosine residues is realized by a dense network of hydrogen bonds involving the side chains of two conserved lysines and one glutamic acid. The co-crystal structure also reveals a protein-protein dimerization interface of PCBP2 KH1 located on the opposite side of the protein from the DNA binding groove. Numerous stabilizing protein-protein interactions, including hydrophobic contacts, stacking of aromatic side chains, and a large number of hydrogen bonds, indicate that the protein-protein interaction interface is most likely genuine. Interaction of PCBP2 KH1 with the C-rich strand of human telomeric DNA suggests that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.

Amino Acid Sequence↗

PubMed2000

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.

Amino Acid Sequence↗