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The structural organization of the genes encoding Bungarus multicinctus protease inhibitor-like proteins (PILPs), PILP-1, PILP-2 and PILP-3, are reported in this study. Unlike PILP-2 and PILP-3, recombinant PILP-1 exhibited inhibitory activity on trypsin. PILP genes and B chain genes shared identical organization with three exons interrupted by two introns in similar positions. On the contrary, intron 1 of these genes had a similar size, a notable variation with the size of intron 2 was observed. It was found that two regions at the second intron of B1 chain and B2 chain genes were absent in that of PILP genes. Noticeably, intronic insertion in the second intron of B chain genes appeared in the promoter region of PILP-1 gene, but not in that of PILP-2 and PILP-3 genes. Comparative analyses of PILP genes and B chain genes showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. These results suggest that PILP genes and B chain genes originate from a common ancestor, and that accelerated evolution may diversify PILP and B chain genes as that proposed for snake venom phospholipase A(2), neurotoxin and cardiotoxin genes.

Compounds 2a- h and 6 displayed significant GI 50 values of 10(-7)-10(-6) M against various cancer cell lines. Of these compounds, 2-(6-(2-trifluoromethylphenyl))-3(Z)-hexen-1,5-diynyl)aniline (2c) showed the most potent growth inhibition activity. Compound 2c also arrested cancer cells in the G2/M phase and in low concentration reduced a significant percentage of MDA-MB-231/ATCC breast cancer tetraploid cells. In addition to the G2/M block, compound 2c caused microtubule depolymerization and induced apoptosis via activation of the caspase family.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. When K562 cells were treated with CTX III, cytosolic calcium concentration was rapidly and persistently increased. This CTX III-induced cell death was partially reversed by pretreatment with BAPTA/AM (20 microM), a chelator of intracellular Ca2+. Moreover, CTX III-induced apoptotic signals, such as caspase-12 and c-Jun N-terminal kinase (JNK) activation, were induced in a time-dependent manner and inhibited by BAPTA/AM. In contrast, the neutral protease micro-calpain, a key enzyme in endoplasmic reticulum (ER) stress-related apoptosis via caspase-12 activation, was unchanged during apoptosis. Taken together, our findings suggest CTX III-induced apoptosis is triggered by Ca2+ influx, then activated caspase-12 and JNK through micro-calpain-independent cascade, and consequently caused apoptosis.

Modification of His-47 and removal of the N-terminal octapeptide caused a different effect on the structure of Naja naja atra (Taiwan cobra) phospholipase A2 (PLA2). Unlike native enzyme, Ca2+ induced an alteration in the structural flexibility of His-modified PLA2. Moreover, the spatial positions of Trp residues in His-modified PLA2 were not properly rearranged toward lipid-water interface in the presence of Ca2+. CD spectra and fluorescence measurement showed that the dynamic properties of Trp residues and the gross conformation of N-terminally truncated PLA2 were totally different from native enzyme. Although a precipitous drop in the enzymatic activity was observed with modified PLA2, His-modified PLA2 and N-terminally truncated PLA2 retained cytotoxicity on inducing necrotic death of human neuroblastoma SK-N-SH cells. Our data suggest that structural perturbations elicited by the chemical modification cause a dissociation of enzymatic activity and cytotoxicity of PLA2.

beta-Bungarotoxin (beta-Bgt), a presynaptic phospholipase A(2) (PLA(2)) neurotoxin isolated from the venom of Bungarus multicinctus, consists of A chain and B chain. The goal of the present study is to explore the functional contribution of the two subunits to the toxicity of beta-Bgt. beta-Bgt was found to induce apoptotic death of SK-N-SH cells via elevating intracellular Ca(2+) and intracellular ROS production. Moreover, an activation of p38 MAPK was associated with the cytotoxicity of beta-Bgt. SB202190 (p38 MAPK inhibitor), N-acetylcysteine (antioxidant reagent), 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) (Ca(2+) chelator) and the inhibitors of Ca(2+) release from intracellular depots (ruthenium red and 2-aminoethoxydiphenyl borate) effectively attenuated the cytotoxicity of beta-Bgt. In sharp contrast to the inability of A chain, B chain was able to induce cytotoxic effects on SK-N-SH cells as beta-Bgt did. Abolishment of PLA(2) activity did not significantly alter the cytotoxic activity of beta-Bgt. MK801 (an NMDA receptor antagonist), antibodies against NMDA receptor and 4-aminopyridine (a potassium channel blocker) markedly reduced the cytotoxic effects of beta-Bgt, B chain and catalytically inactivated beta-Bgt. Moreover, antibodies against NMDA receptor blocked the binding of rhodamine-labeled beta-Bgt to SK-N-SH cells. Taken together, our data indicate that B chain is a functional subunit responsible for the cytotoxicity of beta-Bgt, and suggest that the cytotoxicity of beta-Bgt is mediated by NMDA receptor and potassium conductance.

To elucidate whether the phospholipase A(2) (PLA(2)) activity of notexin was exclusively associated with the manifestation of its pharmacological activities, the interaction of notexin with phospholipid liposomes was explored by fluorescence and CD measurement underlying the conditions of depriving its PLA(2) activity. Although a higher membrane-damaging activity was noted with Ca(2+)-bound notexin, abolishment of PLA(2) activity by EDTA and Sr(2+) could not diminish the membrane-damaging activity of notexin. Fluorescence-quenching studies and CD measurement indicated that Ca(2+)-bound, Sr(2+)-bound or metal-free notexin did not adopt the same conformation upon binding with phospholipids. Regardless of the presence of Ca(2+), Sr(2+) or EDTA, self-quenching assay with rhodamine-labeled notexin revealed that the toxin pertained to form oligomer when it bound with liposomes. Although Lys-modified notexin retained full PLA(2) activity, a notable decrease in membrane-damaging activity was observed. These results indicate that notexin could directly cause a leakage of membrane via a PLA(2) activity-independent manner, and implicate that another biological event contributes to the activity of notexin in vivo.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, may have a potentiality as a structural template for rational drug design in killing cancer cells. Treatment of K562 cells with 0.3 microM of CTX III resulted in G2/M phase cell cycle arrest that was associated with a marked decline in protein levels of G2/M regulatory proteins including cyclin A, cyclin B1, Cdk2 and Cdc25C. In contrast to no effect on the phosphorylation of ERK, p38 MAPK and Akt, an activation of JNK was noted when K562 cells were exposed to CTX III. CTX III-mediated G2/M phase arrest and apoptosis were reduced by treatment with the JNK-specific inhibitor SP600125, but not by ERK and p38MAPK inhibitors. Further investigation showed that the specific JNK inhibitor, SP600125, reduced the activation of caspase-3, caspase-9, and reversed the decline in the expression of cyclin B1. Taken together, our data show for the first time that JNK, but not ERK, p38MAPK or Akt signaling, plays an important role in CTX III-mediated G2/M arrest and apoptosis in K562 cancer cells.

DC-81, an antitumor antibiotic produced by the Streptomyces species, belongs to pyrrolo[2,1-c] [1,4]benzodiazepine (PBD), which are potent inhibitors of nucleic acid synthesis. We previously reported an efficient synthesis of PBD hybrids linked with indole carboxylates. This is the first demonstration on the mechanism of the anticancer effect of PBD hybrid (IN6CPBD) agent on human melanoma A375 cells. IN6CPBD-treated cells exhibited higher cytotoxicity than DC-81 and displayed several features of apoptosis, including an increase in the sub-G1 population, a significantly increased annexin V binding, a degradation of caspase-3, and poly (ADP-ribose) polymerase (PARP) cleavage. Because degradative changes associated with apoptosis are often preceded by the disruption of mitochondrial function, the assessment of mitochondrial function in IN6CPBD-treated cells is worthy of investigation. Our data revealed that treatment of A375 cells with IN6CPBD resulted in the loss of mitochondrial membrane potential (DeltaPsimt), a decrease in intracellular pH (pHi), a reduction of ATP synthesis, increased reactive oxygen species (ROS) generation, and cytochrome c release. Collectively, our studies indicate that IN6CPBD induces apoptosis in A375 cells through a mitochondrial dysfunction pathway, leading to caspase-3 substrate PARP cleavage and subsequent apoptotic cell death.

The functions of centrosomal protein ninein may be involved in microtubule minus end capping, centriole positioning, protein anchoring and microtubule nucleation, but the true physiological function of various human hNinein isoforms remains to be determined. Here we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. These hNinein isoforms exhibit centrosomal (concentrated) and noncentrosomal (aggregated) localization when GFP-tagged fusion proteins are expressed transiently in mammalian cells. In a kinase assay, we show that the CCII region of hNinein provides a differential phosphorylation site by GSK3beta. In addition, our data indicate that either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits gamma-tubulin to centrosomal or noncentrosomal hNinein-containing sites, implying that the gamma-tubulin localization may be hNinein-dependent. Further, our RNA interference experiment against all hNinein isoforms caused a significant decrease in the gamma-tubulin signal in the centrosome. In domain swapping, we clearly show that the CCIIX-CCIIY region provides docking sites for gamma-tubulin. Moreover, our data also show that nucleation of microtubules from the centrosome is significantly affected by the presence of either the full -length hNinein or CCIIX-CCIIY region overexpression. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit gamma-tubulin at centrosomal and noncentrosomal sites.

Selective reduction on the Cys28-Cys32 disulfide of Ophiophagus hannah neurotoxins, Oh-4 and Oh-5, revealed that isomerization of this disulfide linkage caused the two toxins to have distinct conformation and different retention time on a reversed-phase column. The Cys28-Cys32 disulfide of Oh-4 and Oh-5 was prone to form mixed disulfides with glutathione following pseudo-first-order kinetics. In addition to glutathionylated proteins, Oh-4 could be promoted to convert into Oh-5 by thiol compounds. Isomerization of Oh-5 into Oh-4 was not observed in the presence of thiol compounds. Dethiolation of glutathionylated proteins produced Oh-4 and Oh-5. Oxidation of the partially reduced toxin with reduced Cys28 and Cys32 was exclusively converted into Oh-5 regardless of the absence or presence of GSH/GSSG. Acrylamide quenching studies revealed difference in degree of exposure of the single Trp27 between Oh-4 and Oh-5. Synthesized peptides with substitution of Trp27 or Phe31 with Gly abolished entirely the formation of disulfide-linked dimeric product noted with the peptide of wild-type sequence. These results suggest that disulfide formation and isomerization of Cys28-Cys32 could be regulated by thiolation, and that the bulky aromatic residues Trp27 and Phe31 facilitate favorably the occurrence of disulfide isomerization of Cys28-Cys32.

Three cytotoxin-like proteins from the venom of Ophiophagus hannah were isolated by a combination of ion exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that these proteins all consisted of 63 amino acids and shared approximate 50% and 56% sequence identity with Naja naja atra cardiotoxins and cardiotoxin-like basic proteins (CLBPs), respectively. CD spectra revealed that their secondary structure was dominated with beta-sheet as those noted with cardiotoxins and CLBPs. O. hannah cytotoxin-like protein exhibited a cell-lytic activity on SK-N-SH cells, but its activity was more weak than that noted for N. naja atra cardiotoxin 3. Alternatively, apoptotic cell death was induced by the addition of N. naja atra CLBP. Based on the sequence information with the toxin molecules, the functional residues and regions related to the differential activity with O. hannah cytotoxin-like protein, cardiotoxin and CLBP are discussed.

The mechanism of the action of beta-bungarotoxin (beta-BuTx) in the facilitation of spontaneous transmitter release at neuromuscular synapse was investigated in Xenopus cell culture using whole-cell patch clamp recording. Exposure of the culture to beta-BuTx dose-dependently enhances the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca2+ with BAPTA-AM hampered the facilitation of SSC frequency induced by beta-BuTx. The beta-BuTx-enhanced SSC frequency was reduced when the pharmacological Ca2+ -ATPase inhibitor thapsigargin was used to deplete intracellular Ca2+ store. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP3) but not ryanodine receptors effectively occluded the increase of SSC frequency elicited by beta-BuTx. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI3K) and with phospholipase C (PLC) inhibitor U73122, abolished the beta-BuTx-induced facilitation of synaptic transmission. The beta-BuTx-induced synaptic facilitation was completely abolished while there was presynaptic loading of the motoneuron with GDPbetaS, a non-hydrolyzable GDP analogue and inhibitor of G protein. Taken collectively, these results suggest that beta-BuTx elicits Ca2+ release from the IP3 sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PI3K/PLC signaling cascades and G protein activation, leading to an enhancement of spontaneous transmitter release.

The goal of the present study is to explore whether Ca2+ and Mg2+-binding properties of isomeric Kv channel-interacting proteins (KChIPs) have different effects on their molecular structure and the binding with Kv channel. 8-Anilinonaphthalene- 1-sulfonate fluorescence measurement showed that KChIP4.1 and KChIP2.2 possessed one and two types of Ca2+-binding sites, respectively, and only one type of Mg2+-binding site was noted in the two KChIP proteins. Removal of EF-hand 4 (EF-4) caused a marked drop in their high affinities for Ca2+, but the binding affinity for Mg2+ remained mostly the same. Unlike KChIP4.1, the intact EF-4 was essential for the Kv channel-binding ability of KChIP2.2 in a metal-free buffer. Nevertheless, the interaction of wild-type KChIPs and EF-4-truncated mutants with Kv channel was enhanced by the addition of Mg2+ and Ca2+. In contrast to KChIP4.1, the thermal stability of KChIP2.2 was decreased by the binding of Mg2+ and Ca2+. These results suggest that the conformational change with metal-bound KChIP4.1 is crucial for its interaction with Kv channel but not for KChIP2.2, and that the Mg2+- and Ca2+-binding properties of KChIP2.2 and KChIP4.1 have different effects on their molecular structure.

Ala-screening mutagenesis studies on Arg1, Pro2, Arg3, Phe4 and Thr54 of Naja naja atra (Taiwan cobra) chymotrypsin inhibitor showed that inhibitory potency and gross conformation of the mutants were not significantly different from those of wild-type inhibitor. Nevertheless, the R1A mutant had an appreciable decrease in the structural stability underlying thermal unfolding and urea-induced denaturation. Alternatively, deleting the first three residues at the N-terminus caused a reduction in structural stability as well as inhibitory potency. In sharp contrast to wild-type and other mutated inhibitors, R1A mutant and truncated mutant completely lost their inhibitory activity when the inhibitors were incubated with chymotrypsin for periods of up to 3 h. The loss of activity correlated with chymotryptic cleavage of inhibitors as evidenced by SDA-PAGE. Taken together, these results reflect that the globally structural rigidity of N. naja atra chymotrypsin inhibitor functionally affects the sustainable period in inhibiting chymotrypsin activity, and that the intact N-terminus might contribute to this event.

The interaction of human visinin-like protein 1 (VILIP1) and visinin-like protein 3 (VILIP3) with divalent cations (Mg2+, Ca2+, Sr2+ and Ba2+) was explored using circular dichroism and fluorescence measurement. These results showed that the four cations each induced a different subtle change in the conformation of VILIPs. Moreover, VILIP1 and VILIP3 bound with Ca2+ or Mg2+ in a cooperative manner. Studies on the truncated mutants showed that the intact EF-3 and EF-4 were essential for the binding of VILIP1 with Ca2+ and Mg2+. Pull-down assay revealed that Ca2+ and Mg2+ enhanced the intermolecular interaction of VILIPs, and led to the formation of homo- and hetero-oligomer of VILIPs. Together with previous findings that Ca2+-dependent localization of VILIPs may be involved in the regulation of distinct cascades and deprivation of Ca2+-binding capacity of VILIPs did not completely eliminate their activity, it is likely to reflect that Mg2+-bound VILIPs may play a role in regulating the biological function of VILIPs in response to a concentration fluctuation of Ca2+ in cells.

To assess whether chemical modification of phospholipase A(2) (PLA(2)) enzymes may affect their fine structure and consequently alter their enzymatic activity, the present study was carried out. Both Lys-6 and Lys-65 in the Taiwan cobra (Naja naja atra) PLA(2) were selectively modified with trinitrobenzene sulfonate and pyridoxal-5'-phosphate (PLP), respectively. Incorporation of either trinitrophenylated (TNP) or PLP groups on Lys-6 and Lys-65 caused a drop in PLA(2) activity, but the Ca(2+)-binding ability and global conformation of modified derivatives were not significantly different from that of native enzyme. A distinct enhancement of stability was observed with native PLA(2) when thermal unfolding was conducted in the presence of 20 mM Ca(2+). Conformational transition induced by guanidine hydrochloride was also attenuated by the addition of Ca(2+). Conversely, a marked decrease in the structural stability was noted with modified derivatives, and the enhancing effect of Ca(2+) pronouncedly decreased. Together with the finding that the incorporated TNP and PLP groups did not equally affect enzymatic activity and structural stability of PLA(2), our data suggest that an alteration in the fine structure owing to the incorporated groups should contribute to the observed decrease in PLA(2) activity.

A cDNA encoding chymotrypsin inhibitor was constructed from the cellular RNA isolated from the venom glands of Naja atra (Taiwan cobra). The resultant amino acid sequence showed that the mature protein is comprised of 57 amino acid residues with six cysteine residues. Cloned protein was expressed and isolated from the inclusion bodies of E. coli and refolded into a functional protein in vitro. Deleting the first three residues at its N-terminus caused a moderate increase in the inhibitory constant (K(i)) against chymotrypsin. The genomic DNA encoding the chymotrypsin inhibitor was amplified by PCR. The gene shares virtually an identical structural organization with the beta-bungarotoxin B1 chain (a snake Kunitz/BPTI neurotoxic homolog) gene. Moreover, the overall sequence identity of the N. atra chymotrypsin inhibitor and beta-bungarotoxin B1 chain genes was up to 83%. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.

Two beta-bungarotoxin isotoxins BM12 and BM13 were isolated from Bungarus multicinctus (Taiwan banded krait) venom by sequential chromatography on ion-exchange and reverse phase columns. The two toxins have the same A chain, but different B chains. Different phospholipase A2 activity and different potencies in inhibiting the spontaneous enhancement of spontaneous synaptic current frequency and muscle contraction were observed for BM12 and BM13. Nevertheless, modification of Lys-64 in the A chain of BM12 and BM13 similarly reduced in their phospholipase A2 activity and toxicity. The modified derivatives retained their affinity with Ca2+ and their conformation as deduced by CD. These results suggest that Lys-64 of the A chain is involved in the phospholipase A2 activity and in the neurotoxic effect of beta-bungarotoxin.

Two proteins G2a and G2b with molecular masses of approximately 24 kDa were isolated from Naja naja atra (Taiwan cobra) venom using sequential chromatography on gel filtration, ion-exchanger and reverse phase columns. The results of Edman degradation and mass analysis revealed that G2a is a cysteine-rich protein reported previously, and G2b is a novel polypeptide. CD spectra showed that the gross conformation of G2a and G2b notably differed. G2a exhibited an activity higher than that noted with G2b on inhibiting carbachol-induced muscle contraction. However, the two proteins weakly blocked muscle contraction evoked by K+. The observations that the two proteins exhibit the toxic activity in the concentration of micromolar range suggest that they are inherently weak toxins as other snake venom cysteine-rich proteins.

Genomic DNAs encoding the precursors of eight cardiotoxins and two cardiotoxin-like basic proteins (CLBP) were isolated from the liver of Naja naja atra (Taiwan cobra). The cardiotoxin and CLBP genes have three exons like alpha-neurotoxin precursors. The promoter regions of these genes are highly conserved and contain the consensus transcriptional factor-binding sites for TBP, NF-1, CACCC-binding site, Spl and EFII, suggesting that these genes are regulated using similar transcriptional mechanisms. The introns and flanking regions of these genes share a high degree of nucleotide sequence identity, but except for the signal peptide domain the protein-coding regions are much more diversified than introns. The ratio of nonsynonymous to synonymous substitution is higher than one, reflecting that adaptive selection occurred during the evolution of cardiotoxin and CLBP proteins. Phylogenetic trees separate CLBPs and cardiotoxins into two clusters, suggesting that the CLBP gene and the cardiotoxin gene diverged earlier before the appearance of numerous cardiotoxins and CLBP.

Gamma-bungarotoxin, a snake venom protein isolated from Bungarus multicinctus, contains 68 amino acids, including 10 cysteine residues and a TAVRGDGP sequence at positions 30-37. The solution structure of gamma-bungarotoxin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The structure is similar to that of the short-chain neurotoxins that contain three loops extending from a disulfide-bridged core. The tripeptide Arg-Gly-Asp (RGD) sequence is located at the apex of the flexible loop and is similar to that of other RGD-containing proteins. However, gamma-bungarotoxin only inhibits platelet aggregations with an IC50 of 34 microM. To understand its weak activity in inhibiting platelet aggregation, we mutated the RGD loop sequences of rhodostomin, a potent platelet aggregation inhibitor, from RIPRGDMP to TAVRGDGP, resulting in a 196-fold decrease in activity. In addition, the average Calpha-to-Calpha distance between R33 and G36 of gamma-bungarotoxin is 6.02 A, i.e., shorter than that of other RGD-containing proteins that range from 6.55 to 7.46 A. These results suggested that the amino acid residues flanking the RGD motif might control the width of the RGD loop. This structural difference may be responsible for its decrease in platelet aggregation inhibition compared with other RGD-containing proteins., (Copyright 2004 Wiley-Liss, Inc.)

Notexin, a presynaptic phospholipase A2 (PLA2) neurotoxin isolated from Notechis scutatus scutatus venom, was inactivated by arginine-specific reagents, phenylglyoxal and 1,2-cyclohexanedione. Kinetic analyses of the modification reaction revealed that the inactivation of notexin followed pseudo-first order kinetics and the loss of PLA2 activity was correlated with the incorporation of one molecule of modification reagent per toxin molecule. However, the results of amino acid analysis and sequence determination revealed that two arginine residues at positions 43 and 79 of notexin were modified simultaneously. Modification of the arginine residues was accompanied with a decrease in the ability to inhibit the indirectly evoked contraction of chick biventer cervicis muscle and bind with synaptic membranes. The secondary structure of the toxin molecule did not significantly change after modification with phenylglyoxal as revealed by the CD spectra. The modified derivative retained its affinity for Ca2+, indicating that the modified arginine residues did not participate in Ca2+ -binding. Together with the notion that Arg-43 and Arg-79 of notexin are located in the proximity of its catalytic site and toxic site, respectively, our results suggest that modification of Arg-43 and Arg-79 should differently contribute to the observed decrease in the PLA2 activity and neurotoxic effect of notexin.

Different types of cardiotoxin (I-V and n) were isolated and purified from the venom of the Taiwan cobra (Naja naja atra). The effects of these cardiotoxins were studied on membrane-bound acetylcholinesterase, which was isolated from a sheep's brain cortex. The results showed that cardiotoxins I-III, V, and n activated the enzyme by modification of substrate inhibition, but cardiotoxin IV's reaction was different. The inhibition and activation of acetylcholinesterase were linked to the functions of the hydrophobicity index, presence of a cationic cluster, and the accessible arginine residue. Our results indicate that Cardiotoxins have neither a cationic cluster nor an arginine residue in their surface area of loop I; therefore, in contrast to fasciculin, cardiotoxins are attached by loop II to the peripheral site of the enzyme. As a result, fasciculin seems to stabilize nonfunctional conformation, but cardiotoxins seem to stabilize the functional conformation of the enzyme. Based on our experimental and theoretical findings, similar secondary and tertiary structures of cardiotoxins and fasciculin seem to have an opposite function once they interact with acetylcholinesterase.

To investigate the metal-binding properties of KChIP1, the interaction of KChIP1 and mutated KChIP1 with divalent cations (Mg(2+), Ca(2+), Sr(2+), and Ba(2+)) was explored by 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence. It showed that KChIP1 possessed two types of Ca(2+)-binding sites, high-affinity and low-affinity Ca(2+)-binding sites. However, only low-affinity-binding site for Mg(2+), Sr(2+), and Ba(2+) was observed. The metal-binding properties of KChIP1 are not appreciably affected after removal of the N-terminal portion and EF-hand 1. Deleting the EF-hand 4 of KChIP1 abolishes its high-affinity Ca(2+)-binding site, but retains the intact low-affinity-binding site for metal ions. A decrease in the nonpolarity of ANS-binding site occurs with all mutants. However, the binding of ANS with KChIP1 is no longer observed after removal of EF-hands 3 and 4. Intermolecular interaction assessed by chemical cross-linking suggested that KChIP1 had a propensity to form dimer in the absence of metal ions, and a KChIP1 tetramer was pronouncedly produced in the presence of metal ions. Noticeably, the oligomerization state depends on the integrity of EF-hand 4. Taken together, our data suggest that EF-hand 4 is of structural importance as well as functional importance for fulfilling the physiological function of KChIP1.

Two novel neurotoxins BM10-1 and BM10-2 were isolated from Bungarus multicinctus (Taiwan banded krait) venom using the combinations of chromatography on a SP-Sephadex C-25 column and a reverse phase HPLC column. BM10-1 contained 66 amino acid residues including 10 Cys residues, while BM10-2 consisted of 65 amino acid residues with 8 Cys residues. The secondary structure of both BM10-1 and BM10-2 was dominated with beta-sheet, but their gross conformation differed as evidenced by CD spectra and acrylamide quenching studies. BM10-1 inhibited carbachol-induced muscle contraction in a reversible manner and the dose for achieving 50% inhibition was approximately fourfold that of alpha-bungarotoxin. BM10-2 exhibited an irreversible but weak inhibition on carbachol-induced muscle contraction. Sequence alignment of neurotoxins with BM10-1 and BM10-2 suggested that the manner in the manifestation of their activity could be partly elucidated by the residues at toxin second loop. The genomic DNAs encoding BM10-1 and BM10-1-like protein (BM10-1L) were amplified by PCR. The two genes shared virtually identical structural organization and high degree of sequence identity with B. multicinctus neurotoxin genes. Compared to intron sequences of these genes, the protein-coding regions were highly variable. The difference between BM10-1 gene and BM10-1L gene notably arose from the third exon. These results suggest the evolution of B. multicinctus neurotoxins via the path of gene duplication.

The repertoire of antibodies producing by immunizing rabbits with cobrotoxin and dimeric glutaraldehyde-modified cobrotoxin (dGA-cobrotoxin) was analyzed by studying the immunoreactivity of the two antibody preparations toward cobrotoxin, GA-cobrotoxin and recombinant cobrotoxin. The results of enzyme-linked immunoassay revealed that the two antibody preparations exhibited a higher reactivity against their cognate antigen. Moreover, different behavior was observed for the reactivity of the two antibody preparations against GA-cobrotoxin and recombinant cobrotoxin. Notably, distortion of disulfide linkages at the C-terminus resulted in a reduced decrease in the antigenic activity of recombinant cobrotoxin toward anti-cobrotoxin antibodies compared to anti-dGA-cobrotoxin antibodies. Affinity purification of the antibodies against the C-terminus of cobrotoxin revealed that its amount represented 77% and 35.5% of the total anti-dGA-cobrotoxin antibodies and the total anti-cobrotoxin antibodies, respectively. These findings suggest that the antibody preparation elicited by dGA-cobrotoxin enriches the content of antibodies recognizes the C-terminal region of native cobrotoxin.

Four novel small nucleolar RNAs (snoRNAs), h5sn1, h5sn2, h5sn3, and h5sn4, were successfully amplified from human total RNAs using RT-PCR. They exhibited the structural hallmarks of box H/ACA snoRNAs and formed sequence complementarity to 5S rRNA. The nucleotide sequences of the snoRNAs from different donors were highly conserved as evidenced by single-stranded conformational polymorphism and direct nucleotide sequence analysis. Although their host genes had no protein-coding potential, the expression of the snoRNAs was differentially displayed in different tissues. Noticeably, h5sn2 was highly expressed in normal brain, but its expression drastically decreased in meningioma. This opens the fascinating possibility of the relationship between the processing of snoRNAs and carcinogenesis.

Bungarus multicinctus (Taiwan banded krait) beta-bungarotoxins consist of two dissimilar polypeptide chains, A and B. The A chain is structurally homologous to phospholipase A(2) (PLA(2)) enzymes. The structural organization of the genes encoding A1, A2 and A8 chains are reported in this study. Their nucleotide sequences shared up to 97.5% identity. Alignment of the determined A chain genes with their cDNAs revealed that A1 chain gene organized with four exons and three introns, while A2 chain gene comprised three exons and two introns. When A2 chain is expressed, the region corresponding to the first exon of A1 chain gene is skipped instead of the inclusion of intronic sequence adjacent to the second exon. The resulting A2 chain mRNA encoded a 25 residue signal peptide, which is different from A1 chain mRNA with a 27 residue signal peptide. Nevertheless, expression of the A chain genes was partly regulated by a common mechanism as evidenced by sequence conservation of their promoter region and consensus transcriptional factor binding-sites inside this region. 5'-RACE analyses revealed that A chain mRNAs with 27 residue signal peptide represented the predominant species in the preparation of B. multicinctus venom gland mRNAs. Comparative analyses on PLA(2) genes and cDNAs suggest that this is the first report on the skipping of exon which changes the signal peptide sequence of snake venom proteins., (Copyright 2002 Elsevier Science Ltd.)

Two novel proteins, BM8 and BM14, were isolated from Bungarus multicinctus (Taiwan banded krait) venom using the combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase HPLC column. Both proteins contained 82 amino acid residues including 10 cysteine residues, but there were two amino acid substitutions at positions 37 and 38 (Glu37-Ala38 in BM8; Lys37-Lys38 in BM14). CD spectra and acrylamide quenching studies revealed that the gross conformation of BM8 and BM14 differed. In contrast to BM8, BM14 inhibited the binding of [3H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine (mAchR) receptor subtype. Trinitrophenylation of Lys residues abolished the mAchR-binding activity of BM14, indicating that the Lys substitutions at positions 37 and 38 played a crucial role in the activity of BM14. The genomic DNA encoding the precursor of BM14 was amplified by PCR. The gene shared virtually identical structural organization with alpha-neurotoxin and cardiotoxin genes. The intron sequences of these genes shared a sequence identity up to 84%, but the protein-coding regions were highly variable. These results suggest that BM8, BM14, neurotoxins and cardiotoxins may have originated from a common ancestor, and the evolution of snake venom proteins shows a tendency to diversify their functions.

A neurotoxin, Oh9-1, from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that Oh9-1 consists of 57 amino acids and eight cysteine residues. This protein was mainly constituted with beta-sheet as evidenced by CD spectrum. Oh9-1 inhibited carbachol-induced muscle contraction in an irreversible manner and the dose for achieving 50% inhibition was approximately fourfold that of alpha-bungarotoxin. Since the residues in alpha-neurotoxins closely involve in the binding with acetylcholine receptors are not highly conserved in this toxin molecule, Oh9-1 represents a novel type of neurotoxin structurally distinct from alpha-neurotoxins.

Two genomic DNAs were isolated from the liver of Bungarus multicinctus (Taiwan banded krait) encoded kappa1-bungarotoxin and kappa3-bungarotoxin precursors, respectively. They shared virtually identical overall organization with three exons separated by two introns and a high degree of nucleotide-sequence identity with alpha-neurotoxin genes, including similar intron insertions. This suggests that kappa-neurotoxins and alpha-neurotoxins might have originated from a common ancestor. The consensus transcriptional factor binding sites within the promoter regions of these genes indicate that their transcriptions are, at least in part, regulated under the same mechanism. Comparative analyses on kappa-bungarotoxin and alpha-neurotoxin genes revealed that the protein-coding regions of exons were much more diversified than introns except for the signal peptide domain. Phylogenetic analyses on the exon and intron regions of kappa-bungarotoxin and alpha-neurotoxin genes showed that the evolution of exon regions were not in consensus with that of intron regions. The ratio of nonsynonymous to synonymous substitution is higher than 1, reflecting the occurrence of an adaptive selection during the evolution of kappa-bungarotoxins. In contrast to a conserved size of the second intron, segmental insertions and/or deletions within the first intron accelerate the evolutionary divergence of kappa- and alpha-neurotoxin genes.

gamma-Bungarotoxin was isolated from Bungarus multicinctus (Taiwan banded krait) venom using a combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase high-performance liquid chromatography column. Circular dichroism (CD) measurement revealed that its secondary structure was dominant with beta-sheet structure as is that of snake venom alpha-neurotoxins and cardiotoxins. gamma-Bungarotoxin exhibits activity on inhibiting the binding of [3H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine receptor subtype, and competes weakly with radioiodinated alpha-bungarotoxin for binding to the Torpedo nicotinic acetylcholine receptor. Moreover, the toxin inhibits collagen-induced platelet aggregation, with an IC50 of approximately 200 nM. The genomic DNA encoding the gamma-bungarotoxin precursor is amplified by polymerase chain reaction (PCR). The gene is organized with three exons separated by two introns, and shares virtually identical overall organization with those reported for alpha-neurotoxin and cardiotoxin genes, including similar intron insertions. The intron sequences of these genes share sequence identity up to 85%, but the exon sequences are highly variable. These observations suggest that gamma-bungarotoxin, alpha-neurotoxins, and cardiotoxins originate from a common ancestor, and the evolution of these genes shows a tendency to diversify the functions of snake venom proteins.

Six cardiotoxins (CTXs) and one cardiotoxin-like basic protein (CLBP) from Naja naja atra (Taiwan cobra) venom were separated by a SP-Sephadex C-25 column. CTXn and CTXI were well separated by eluting with ammonium acetate buffer, and the separation of CLBP from CTXIV and CTXV mixtures was achieved using sodium phosphate buffer. These findings suggest a differential interaction of CTXs with the chromatographic matrix using different buffer systems. Chemical modification studies on cationic residues of CTXI suggested that there was no single lysine or arginine residue exclusively responsible for its biological activity. Moreover, it was found that the cytotoxicity and hemolytic sites of CTXI could be dissociated by chemical modifications. It suggests the potentiality for preparing toxin derivatives in which a specific activity is retained.