[show abstract][hide abstract] ABSTRACT: alpha-Crystallin, a major protein of all vertebrate lenses, consists of two subunits, alphaA and alphaB, which form polymeric aggregates with an average molecular mass of about 800kDa. In this study, we have employed various biophysical methods to study aggregate sizes and conformational properties of purified alphaA, alphaB subunits, and cloned recombinant alphaB subunit. From far- and near-UV CD spectra, native alpha-, alphaA-, alphaB-, and recombinant alphaB-crystallins from porcine lenses all show similar beta-sheet conformation to that from bovine and human lenses as reported previously. By means of gel-filtration chromatography and dynamic light scattering, we have found that the molecular sizes of all four crystallin aggregates are polydispersedly distributed in the following order of aggregate sizes, i.e., native alpha>alphaA>alphaB approximately recombinant alphaB. To investigate the structural and functional relationships, we have also compared the chaperone activities of all four alpha-crystallin aggregates at different temperatures. From the results of chaperone-activity assays, ANS (8-anilinonaphthalene-1-sulfonic acid) binding and thermal stability studies, there appeared to be at least two factors playing major roles in the chaperone-like activity of these lens proteins: one is the hydrophobicity of the exposed protein surface and the other is the structural stability associated with each protein. We showed that alphaA-crystallin is a better chaperone to protect gamma-crystallin against UV irradiation than alphaB-crystallin, in contrast to the observation that alphaB is generally a better chaperoning protein than alphaA for enzyme protective assays at physiological temperatures.
Biochemical and Biophysical Research Communications 07/2002; 295(4):854-61. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bungarus fasciatus fraction IX (BF9), a chymotrypsin inhibitor, consists of 65 amino acid residues with three disulfide bridges. It was isolated from the snake venom of B. fasciatus by ion-exchange chromatography and belongs to the bovine pancreatic trypsin inhibitor (BPTI)-like superfamily. It showed a dissociation constant of 5.8 x 10(-8) m with alpha-chymotrypsin as measured by a BIAcore binding assay system. The isothermal titration calorimetry revealed a 1:1 binding stoichiometry between this inhibitor and chymotrypsin and apparently no binding with trypsin. We further used CD and NMR to determine the solution structure of this venom-derived chymotrypsin inhibitor. The three-dimensional NMR solution structures of BF9 were determined on the basis of 582 restraints by simulated annealing and energy minimization calculations. The final set of 10 NMR structures was well defined, with average root mean square deviations of 0.47 A for the backbone atoms in the secondary structure regions and 0.86 A for residues The side chains of Phe(23), Tyr(24), Tyr(25), Phe(35), and Phe(47) exhibited many long-range nuclear Overhauser effects and were the principal components of the hydrophobic core in BF9. To gain insight into the structure-function relationships among proteins in the BPTI-like superfamily, we compared the three-dimensional structure of BF9 with three BPTI-like proteins that possess distinct biological functions. These proteins possessed similar secondary structure elements, but the loop regions and beta-turn were different from one another. Based on residues at the functional site of each protein, we suggest that the flexibility, rigidity, and variations of the amino acid residues in both the loop and beta-turn regions are related to their biological functions.
Journal of Biological Chemistry 12/2001; 276(48):45079-87. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Two venom proteases with fibrinogenolytic activity were isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus), one major crotalid snake species in Taiwan. The purified enzymes showed a strong beta-fibrinogenolytic activity, cleaving the beta-chain of fibrinogen molecules specifically. They also showed strong kallikrein-like activity in vitro, releasing bradykinin from kininogen. The purified enzymes did not coagulate human plasma, yet decreasing fibrinogen levels in plasma and prolonging bleeding without formation of fibrin clots, indicating that both proteases have specificities different from thrombin and the thrombin-like proteases of snake venom reported previously. They also exhibit amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, which is a specific synthetic substrate for kallikrein-like proteases. Their stability at high temperatures was examined and found to be more stable when compared with ancrod and thrombin. Intravenous injection of either protease was shown to lower blood pressure in experimental rats. Most noteworthy is the observation that the proteases can cleave angiotensin I and release bradykinin from plasma kininogen in vitro, which is a strong vasodilator and probably responsible for the in vivo hypotensive effect of these venom proteases.
Biochemical and Biophysical Research Communications 04/2001; 281(4):1012-8. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: In order to circumvent the difficulty encountered in the expression and purification of the recombinant products in E. coli system, we have developed a novel and facile method of removing the polyhistidine tag from target proteins after heterologous gene expression. The expression of a serine protease (Tm-5) from Taiwan habu (Trimeresurus mucrosquamatus) is taken as an exemplar to illustrate the basic rationales and protocols involved. In place of an enterokinase recognition site, a polyhistidine tag linked to an autocatalyzed site based on cleavage specificity of the serine protease flanking on the 5'-end of Tm-5 clone sequence was engineered before protein expression in E. coli system. Renaturation of the fusion protein after expression revealed that the recombinant protease had refolded successfully from the inclusion bodies. Upon autocleavage of the expressed protease, the polyhistidine tag with additional amino acid residues appended to the N-terminus of the coding sequence is found to be removed accordingly. The protein expressed and purified by this new strategy possesses a molecular weight of approximately 28,000 in accord with the expected value for this venom protease. Further characterization of the recombinant protein employing a variety of techniques which include immunoblot analysis, RP-HPLC, ESI-MS, and N-terminal amino acid sequencing all shows indistinguishable properties to those of the isolated native protease. Most noteworthy is that the recombinant Tm-5 protease also exhibits amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, a unique and strict substrate for native Tm proteases reported previously.
Biochemical and Biophysical Research Communications 10/2000; 275(3):924-30. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Penicillium notatum is a well-known indoor aeroallergen and is frequently included in skin test panels for allergic diagnosis. On two-dimensional immunoblotting using patients' sera containing IgE and monoclonal antibody D7B8 specific for Pen c 1 of P. citrinum, two allergens with a molecular mass of 33 kDa but different isoelectric points were identified. A novel cDNA coding for Pen n 13 was cloned and sequenced. The nucleotide sequence codes for a protein 397 amino acids including a putative signal peptide of 25 amino acids and a propeptide of 90 amino acids. The allergen is an alkaline serine protease that shares more than 39% identical residues with other kinds of mold allergens. The coding cDNA of Pen n 13 was cloned into vector pQE-30 and expressed in E. coli M15 as a His-tag fusion protein and purified to homogeneity. The fusion protein reacted with monoclonal antibodies of Pen c 1 and with IgE from Penicillium-allergic patients. Furthermore, it also cross-reacted strongly with IgE specific for the natural Pen c 1, indicating that similar IgE binding epitopes may exist in the allergens of P. notatum and P. citrinum. Antigenicity index plots indicated that there are several similar epitope regions of high antigenic indices in Pen c 1 and Pen n 13, corroborating that mold allergens belonging to the alkaline serine protease family possess similar protein structure and strong antigenic cross-reactivity.
Biochemical and Biophysical Research Communications 04/2000; 269(1):14-20. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Aspergillus species of fungi have been known to be one of the most prevalent aeroallergens. One important A. flavus allergen (Asp fl 1) was identified by means of immunoblotting with a serum pool of allergic patients on a two-dimensional electrophoretic gel. The cDNA coding for Asp fl 1 was cloned and sequenced. The clone encodes a full-length protein of 403 amino acid precursors of 42 kDa. After cleavage of a putative signal peptide of 21 amino acids and a prepeptide of 100 amino acids, a mature protein of 282 amino acids was obtained with a molecular mass of 33 kDa and a pI of 6.3. A degree of identity was found in a range of 27 to 84% among related allergens derived from bacteria allergen subtilisin, mold allergen Pen c 1, and virulence factor of A. fumigatus. Recombinant Asp fl 1 (rAsp fl 1) was cloned into vector pQE-30 and expressed in E. coli M15 as a histidine-tag fusion protein and purified to homogeneity. The IgE binding capacity of rAsp fl 1 was tested by immunoblotting using a serum pool of Aspergillus-allergic patients. Recombinant allergen cross-reacted strongly with IgE specific for natural Asp fl 1 and Pen c 1, indicating that common IgE epitopes may exist between allergens of A. flavus and P. citrinum.
Biochemical and Biophysical Research Communications 09/1999; 261(3):669-75. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: We present a systematic structure comparison of three major classes of postsynaptic snake toxins, which include short and long chain alpha-type neurotoxins plus one angusticeps-type toxin of black mamba snake family. Two novel alpha-type neurotoxins isolated from Taiwan cobra (Naja naja atra) possessing distinct primary sequences and different postsynaptic neurotoxicities were taken as exemplars for short and long chain neurotoxins and compared with the major lethal short-chain neurotoxin in the same venom, i.e., cobrotoxin, based on the derived three-dimensional structure of this toxin in solution by NMR spectroscopy. A structure comparison among these two alpha-neurotoxins and angusticeps-type toxin (denoted as FS2) was carried out by the secondary-structure prediction together with computer homology-modeling based on multiple sequence alignment of their primary sequences and established NMR structures of cobrotoxin and FS2. It is of interest to find that upon pairwise superpositions of these modeled three-dimensional polypeptide chains, distinct differences in the overall peptide flexibility and interior microenvironment between these toxins can be detected along the three constituting polypeptide loops, which may reflect some intrinsic differences in the surface hydrophobicity of several hydrophobic peptide segments present on the surface loops of these toxin molecules as revealed by hydropathy profiles. Construction of a phylogenetic tree for these structurally related and functionally distinct toxins corroborates that all long and short toxins present in diverse snake families are evolutionarily related to each other, supposedly derived from an ancestral polypeptide by gene duplication and subsequent mutational substitutions leading to divergence of multiple three-loop toxin peptides.
Biochemical and Biophysical Research Communications 05/1999; 257(2):500-10. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: The soluble S-crystallin constitutes the major lens protein in cephalopods. The primary amino acid sequence of S-crystallin shows an overall 41% identity with the digestive gland sigma-class glutathione transferase (GST) of cephalopod. However, the lens S-crystallin fails to bind to the S-hexylglutathione affinity column and shows very little GST activity in the nucleophilic aromatic substitution reaction between GSH and 1-chloro-2,4-dinitrobenzene. When compared with other classes of GST, the S-crystallin has an 11-amino acid residues insertion between the conserved alpha4 and alpha5 helices. Based on the crystal structure of squid sigma-class GST, a tertiary structure model for the octopus lens S-crystallin is constructed. The modeled S-crystallin structure has an overall topology similar to the squid sigma-class GST, albeit with longer alpha4 and alpha5 helical chains, corresponding to the long insertion. This insertion, however, makes the active center region of S-crystallin to be in a more closed conformation than the sigma-class GST. The active center region of S-crystallin is even more shielded and buried after dimerization, which may explain for the failure of S-crystallin to bind to the immobilized-glutathione in affinity chromatography. In the active site region, the electrostatic potential surface calculated from the modeled structure is quite different from that of squid GST. The positively charged environment, which contributes to stabilize the negatively charged Meisenheimer complex, is altered in S-crystallin probably because of mutation of Asn99 in GST to Asp101 in S-crystallin. Furthermore, the important Phe106 in authentic GST is changed to His108 in S-crystallin. Combining the topological differences as revealed by computer graphics and sequence variation at these structurally relevant residues provide strong structural evidences to account for the much decreased GST activity of S-crystallin as compared with the authentic GST of the digestive gland.
[show abstract][hide abstract] ABSTRACT: gammaS-Crystallin from catfish eye lenses, formerly designated betas-crystallin in mammalian lenses, is structurally characterized in this study by cDNA cloning and sequencing. To facilitate sequence characterization of gammaS-crystallin with structural properties lying between beta- and gamma-crystallins, a cDNA mixture was constructed from the poly(A)+ mRNA isolated from catfish eye lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain nucleotide segments encoding multiple gammaS-crystallin isoforms. Sequencing several positive clones revealed that at least two distinct isoforms exist in the gammaS-crystallin class of this teleostean fish, similar to the authentic gamma-crystallin family characterized previously in species of the piscine class. Comparison of protein sequences encoded by two representative catfish gammaS1 and gammaS2 cDNAs with the published sequences of beta-, gamma-, and gammaS-crystallins from shark, carp, bullfrog, bovine, and human lenses indicates that there is about 20-50% sequence homology between catfish gammaS-crystallins and various members of the related beta/gamma-crystallin superfamily from different evolutionary classes, with a higher sequence similarity being found between catfish gammaS- and mammalian gamma-crystallins than between catfish gammaS- and bovine or carp gammaS-crystallins. Phylogenetic trees constructed on the basis of the nucleotide and protein sequence divergence among various beta-, gamma-, and gammaS-crystallins corroborate the closer relatedness of catfish gammaS- to authentic gamma-crystallin than to bovine and carp gammaS-crystallins. The results suggest that evolution of catfish gammaS-crystallins follows a different path from that of bovine and carp gammaS-crystallins and may represent a more ancient offshoot from the ancestral gamma/gammaS coding gene than carp and bovine gammaS-crystallins.
Biochemical and Biophysical Research Communications 12/1998; 252(2):412-9. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Extracellular and secretory phospholipase A2 (PLA2), a class of phospholipid digesting enzyme, is widely distributed in animal venoms of reptiles and insects. Two cDNAs encoding PLA2 isoenzymes from Taiwan Cobra (Naja naja atra) were cloned into pQE-30 plasmid vector and expressed in Escherichia coli. The recombinant products were subjected to refolding using sulfonation under reduction/oxidation conditions with glutathione and enterokinase removal of His-tag, resulting in the active recombinant PLA2 with the same molecular masses of native enzymes as determined by mass spectrometry. The recombinant PLA2 was also shown by circular dichroism to possess a secondary structure similar to native PLA2. The enzymatic activity of the major isoenzyme (PLA2-1) is higher than the other minor isoenzyme (PLA2-2), which shows two amino acid difference from PLA2-1. Site-directed mutagenesis was used to probe the structure/function relationship of two highly conserved residues among all reported PLA2, i.e., His-47 and Asp-93. Replacement of His-47 residue by either Ala or Arg resulted in the complete loss of activity. Similarly, the mutant Asp-93 --> Asn (D93N) also retained little activity. These results suggest that both His-47 and Asp-93 are essential for the catalytic activity of PLA2. Computer graphic study, based on homology modelling, highlights the differences between native PLA2 isoenzymes and their site-directed mutants, which may account for the differences in the observed biological activity.
Biochemical and Biophysical Research Communications 09/1998; 250(1):154-60. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Three small peptide components were isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus), which show specific activity to inhibit the strong proteolytic activity of multiple metalloproteinases present in the crude venom. Using multiple chromatographies coupled with successive ultrafiltrations, three inhibitors, i.e. pyroglutamate-lysine-tryptophan (pyroGlu-Lys-Trp), pyroglutamate-asparagine-tryptophan (pyroGlu-Asn-Trp) and pyroglutamate-glutamine-tryptophan (pyroGlu-Gln-Trp) were obtained in good yields and high homogeneity. The yields of these peptide fractions were estimated to be about 0.65 mg, 0.55 mg and 0.42 mg from 250 mg total lyophilized crude venom, which corresponded to the approximate concentrations of 8.4 mM, 7.3 mM and 5.4 mM respectively in venom secretion. Detailed and unambiguous structural determination was established by amino acid analyses, mass spectrometry and microsequencing of purified peptides. Further functional characterization of these three tripeptides showed that they could weakly inhibit three metalloproteinases previously isolated from the same venom. The inhibitory activities were similar among these tripeptides and their IC50 (concentration for 50% inhibition) were estimated in a range of 0.20-0.95 mM, which is much more effective than citrate, another venom protease inhibitor of low molecular-weight component. Since these tripeptides are the endogenous peptide inhibitors present in the lumen of venom glands, it is conceivable that they may act as a self-defensive mechanism against the auto-digestive deleterious effect of the strong metalloproteinases in vivo, particularly several zinc-dependent metalloproteinases present in crotalid and viperid venoms.
Biochemical and Biophysical Research Communications 08/1998; 248(3):562-8. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: The endogenous argininosuccinate lyase activity of duck delta2-crystallin was specifically inactivated by the histidine-specific reagent, diethyl pyrocarbonate. The protein was protected by l-citrulline or l-arginine from the diethyl pyrocarbonate inactivation. To characterize further the chemical mechanism of the delta2-crystallin-catalysed reaction, deuterium-labelled argininosuccinate was enzymically synthesized from fumarate and l-arginine with delta2-crystallin in 2H2O. The argininosuccinate synthesized contained about 19% of the anhydride form; however, the deuterium was clearly demonstrated to be incorporated enantioselectively. Only the pro-HR atom at C-9 of the succinate moiety was labelled in the [2H]argininosuccinate-9-d synthesized, which indicates an anti-elimination mechanism for the endogenous argininosuccinate lyase activity of delta2-crystallin. The enzymic activity of duck lens delta2-crystallin in the pH range 5.5-8.5 was investigated using both protium- and deuterium-labelled argininosuccinate as the substrate. From the logkcat versus pH plot, two molecular pKa values of 6.18+/-0.02 and 8.75+/-0.03 were detected in the delta2-crystallin-argininosuccinate binary complex. The former must be dehydronated and the latter hydronated to achieve an optimum reaction rate. The logkcat/Km versus pH plot suggested two molecular pKa values of 5.96+/-0.09 and 8.29+/-0.10 for the free delta2-crystallin to be involved in the substrate binding. Small kinetic isotope effects of 1.17+/-0.02 and 1.05+/-0.09 were found for kcat and kcat/Km respectively. Combining results from labelling and kinetic analysis indicates that the endogenous argininosuccinate lyase activity of duck delta2-crystallin is compatible with a stepwise E1cB mechanism, the rate-limiting step probably at the C-N bond-cleavage step.
[show abstract][hide abstract] ABSTRACT: Two novel postsynaptic neurotoxins (alpha-neurotoxins) isolated and purified from the Taiwan cobra venom (Naja naja atra) possess distinct primary sequences and different neurotoxicities as compared with the most abundant and lethal component in the venom, i.e., cobrotoxin characterized before from the same venom. The complete sequences of two neurotoxin analogues were determined by N-terminal Edman degradation and comparison of amino acid compositions of proteolytic toxin fragments with other homologous toxins of known sequences. The short-chain neurotoxin consists of 61 amino acid residues with eight conserved cysteine residues and is found to show 78% sequence identity with cobrotoxin. The other toxin, consisting of 65 residues with ten cysteines, belongs to the family of long-chain neurotoxins. It is the first long-chain alpha-neurotoxin reported from the Taiwan cobra. The lethal toxicities of these two novel neurotoxins were much lower than cobrotoxin, albeit with close structural homology among the three toxins in terms of their primary sequences and tertiary structure predicted by homology modeling. Multiple sequence alignment and comparison coupled with construction of a phylogenetic tree for various alpha-neurotoxins of Naja and closely related genuses have established that all nicotinic alpha-neurotoxins present in the snake family of Elapidae are closely related to each other, presumably derived from an ancestral polypeptide by gene duplication and subsequent multiple mutational substitutions.
Journal of Protein Chemistry 02/1998; 17(2):107-14.
[show abstract][hide abstract] ABSTRACT: gamma S-Crystallin from shark eye lenses, formerly termed beta s crystallin in mammalian lenses, is structurally characterized in this study by cDNA cloning and sequencing. To facilitate sequence characterization of gamma S-crystallin possessing intermediate structural properties between beta- and gamma-crystallins, cDNA mixture was constructed from the poly(A)+ mRNA isolated from shark eye lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain nucleotide segments encoding multiple shark gamma S-crystallins. Sequencing several positive clones revealed that a multiplicity of isoforms exists in the gamma S-crystallin class of this cartilaginous fish, similar to authentic gamma-crystallin family characterized from the same shark species. Comparison of protein sequences encoded by two representative shark gamma S1 and gamma S2 cDNAs with those published sequences of beta-, gamma-, and gamma S crystallins from bovine, human, bullfrog and carp lenses indicated that there is about 35-64% sequence homology between shark gamma S crystallins and structurally related crystallins from different evolutionary classes, with a higher sequence similarity between shark gamma S and mammalian gamma-crystallins than that of shark gamma S and carp gamma S or bovine gamma S crystallins. A phylogenetic tree constructed on the basis of the sequence divergence among various beta-, gamma-, and gamma S crystallins corroborates the closer relatedness of shark gamma S to authentic gamma-crystallin than to mammalian and teleostean gamma S crystallins. It further strengthens the supposition that ancestral precursors of gamma S-crystallins were present in the shark lens long before the appearance of present-day teleostean and mammalian gamma S-crystallins.
Biochemical and Biophysical Research Communications 12/1997; 240(1):51-6. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: gamma-Crystallin is a common lens protein of most vertebrate eye lenses and the major protein component in lenses of fishes and in many mammalian species during embryonic and neonatal stages. To facilitate the structural characterization of gamma-crystallin possessing extensive charge heterogeneity, a cDNA mixture was constructed from the poly(A)+ mRNA isolated from shark eye lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain cDNAs encoding multiple shark gamma-crystallins. Sequencing analysis of multiple positive clones containing PCR-amplified inserts revealed the presence of a multiplicity of isoforms in the gamma-crystallin class of this cartilaginous fish. It was of interest to find that two shark cDNA sequences coexist, one encoding gamma-crystallin (gamma M1) of high methionine content (15.5%) and the other encoding one (gamma M2) of low methionine content (5.1%), each corresponding to the major teleostean and mammalian gamma-crystallins, respectively. Comparison of protein sequences encoded by these two shark cDNAs with published sequences of gamma-crystallins from mouse, bovine, human, frog, and carp lenses indicated that there is about 61-80% sequence homology between different species of the piscine class, whereas only 47-66% is found between mammals and shark. A phylogenetic tree constructed on the basis of sequence divergence among various gamma-crystallin cDNAs revealed the close relatedness between shark gamma M2-crystallin and mammalian gamma-crystallins and that between shark gamma M1 and teleostean gamma-crystallins. The results pointed to the fact that ancestral precursors of gamma-crystallins were present in the sharp lens long before the appearance of modern-day mammalian and teleostean gamma-crystallins.
Journal of Protein Chemistry 06/1997; 16(4):299-307.
[show abstract][hide abstract] ABSTRACT: A new destruxin [destruxin E2 chlorohydrin] was isolated from the culture medium of Metarrhizium anisopliae and its structure was determined by NMR spectroscopy and mass spectrometry. As compared with other destruxins, the new destruxin showed a lower suppressive activity on the production of hepatitis B virus surface antigen in human hepatoma Hep3B cells. NMR study coupled with molecular modeling by computer graphics has revealed that the hydrophobicity nature of the convex surface characteristic of all destruxin molecules plays an important role in their biological activity.
Biochemical and Biophysical Research Communications 01/1997; 229(1):65-72. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: The tertiary structure of waglerin I has been determined by NMR and dynamic simulated annealing [Chuang et al., Biochim. Biophys. Acta 1292, 145-155 (1996)]. It is believed that the peptide basicity of waglerin may play an important role for its activity due to its high content of basic amino acids. In order to investigate the active site of the toxin, seven analogues of waglerin, [Ala3]-waglerin, [Ala7]-waglerin, [Ala10]-waglerin, [Ala14]-waglerin, [Ala18]-waglerin, [Ala20]-waglerin and [Ala22]-waglerin have been synthesized chemically by single replacement of basic amino acid residues one by one with Ala. By correlation of structures for each analogue with LD50 toxicity bioassays, it is found that the [Ala10]-waglerin exhibits no toxicity and the active site of the native toxin seems to reside in the proximity of the disulfide loop, which is spatially close to His10. Furthermore, the closer is the disulfide loop to the basic amino acid in waglerin, the more influential is the basic amino acid on the toxicity of waglerin. Based on the tertiary structure of waglerin, the structures of all synthetic analogues were derived based on computer-simulated modelling. By the pair-wise structural comparison, the disulfide loop in [Ala10]-waglerin analogue is found to be twisted as compared to the native form, in agreement with the lack of toxicity for this synthetic analogue.
Biochemical and Biophysical Research Communications 11/1996; 227(1):59-63. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: One novel venom factor was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using two consecutive anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of the purified protein indicated that it lacks the proteolytic activity toward fibrinogen molecules, suggesting that this protein factor does not belong to the familes of metalloproteinases and thrombin-like serine proteases commonly found in the crude venoms of various crotalid snakes. The purified protein exists as a native dimeric protein of 26 kDa, consisting of two closely similar subunits of 16 and 13 kDa, held together by disulfide linkage. N-Terminal sequence analysis revealed that both chains are homologous to each other at the N-terminal fragment and also similar to the factors IX/X-binding protein isolated from Trimeresurus flavoviridis and botrocetin from Bothrops jararaca. This study points to the existence of one new two-chain venom factor without fibrinogenase activity from Taiwan habu, which, in contrast to botrocetin, promotes platelet agglutination even in the absence of von Willebrand factor. Unlike factors IX/X-binding proteins, it did not show affinity to coagulation factors IX and X in the presence of Ca2+ ion. It also shows no inhibition on thrombin, in contrast with bothrojaracin, a thrombin inhibitor isolated from Bothrops jararaca venom. We have therefore named this novel venom factor trimecetin to distinguish it from some structurally related venom factors present in various crotalid and viperid snakes.
Journal of Protein Chemistry 11/1996; 15(7):667-74.
[show abstract][hide abstract] ABSTRACT: beta-Crystallins composed of the most heterogeneous group of subunit chains among the three major crystallin families of vertebrates, i.e. alpha-, beta- and gamma-crystallins, are less well understood at the structural and functional levels than the other two. They comprise a multigene family with at least three basic (betaB1-3) and four acidic (betaA1-4) subunit polypeptides. In order to facilitate the determination of the primary sequences of all these ubiquitous crystallin subunits present in all vertebrate species, cDNA mixture was synthesized from the poly(A)+ mRNA isolated from bullfrog eye lenses. We report here a protocol of Rapid Amplification of cDNA Ends (RACE) was used to amplify cDNAs encoding beta-crystallin acidic subunit polypeptides by polymerase chain reaction (PCR). Four complete full-length reading frames with two each of 597 and 648 base pairs, which cover four deduced protein sequences of 198 (betaA1-1 and betaA1-2) and 215 (betaA3-1 and betaA3-2) amino acids including the universal initiating methionine, were revealed by nucleotide sequencing. They show about 96-98% sequence similarity among themselves and 76-80%, 80-83% to the homologous betaA1/A3 crystallins of bovine and human species respectively, revealing the close structural relationship among acidic subunits of all beta-crystallins even from remotely related species. In this study a phylogenetic comparison based on amino-acid sequences of various betaA1/A3 crystallins plus the major basic beta-crystallin (betaBp) and gamma-crystallin from different vertebrate species is made using a combination of distance matrix and approximate parsimony methods, which correctly groups these betaA crystallin chains together as one family distinct from basic beta-crystallins and gamma-crystallin and further corroborates the supposition that beta- and gamma-crystallins form a superfamily with a common ancestry.
Biochemical and Biophysical Research Communications 05/1996; 221(2):219-28. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: An echistatin analogue, designated as des(46-49)-[Ala8,37]-echistatin gamma, was synthesized chemically by solid-phase peptide synthesis. The analogue was made by replacing Cys8 and Cys37 residues with two alanines and the deletion of C-terminal peptide 46-49 of echistatin gamma, resulting in an artificial polypeptide of 45 amino acids with three disulfide bonds. In the platelet aggregation assay, the analogue exhibits almost the same activity as echistatin gamma, indicating that the linear sequence of des(46-49)-[Ala8,37]-echistatin gamma contains all of the primary-structure information that is required for proper folding of this synthetic polypeptide. The tertiary structure of the analogue, as determined from high-resolution nuclear magnetic resonance (NMR) coupled with dynamic simulated annealing, is very similar to that of echistatin alpha1 which differs from echistatin gamma by 8 residues. In particular the two important sites of the Arg-Gly-Asp (RGD) loop and the C-terminal Lys45, both of which show some degree of disorder, are maintained in similar spatial orientation and proximity as those in echistatin alpha 1 even without the constraint provided by the disulfide bond of the (Cys8-Cys37) pair. These results provide new insights in further defining distinct structural features of echistatin gamma, which are involved in supporting the active polypeptide conformation to achieve biological activity in the absence of one pair of disulfide bonds.
Biochemical and Biophysical Research Communications 04/1996; 220(2):246-54. · 2.41 Impact Factor