Biomedical subjects

S H Chiou

Publications and source records attributed to S H Chiou.

At least 73 records · Page 4Linked to original sources

PubMed1994

cDNA and protein sequences coding for the precursor of phospholipase A2 from Taiwan cobra, Naja naja atra.

The cDNA sequence encoding phospholipase A2 (PLA2) was determined by analysis of polymerase-chain-reaction (PCR) product amplified from total cDNA mixture which had been constructed from the poly(A)+RNA of venom glands obtained from Taiwan cobras. Two oligonucleotide segments corresponding to the 5'- and 3'-noncoding regions of sea-snake PLA2 gene were used as primers for PCR-amplified reaction. Plasmids of transformed E. coli strain JM109 containing amplified PLA2 cDNA were purified and prepared for nucleotide sequencing by dideoxynucleotide chain-termination method. Sequencing more than five clones containing about 0.5 kb DNA inserts revealed two isoforms with complete reading frames of 468 base pairs each covering a precursor for phospholipase A2 with a deduced mature protein sequence of 119 amino acids and a 27 amino-acid signal peptide. These two enzymes of Group I PLA2 differ in six nucleotide residues at the gene level and three amino acids along the whole polypeptide chain, each consisting of 14 cysteine residues similar to all reported PLA2 of different snake venoms. The signal peptides and hydropathy profiles of Group I PLA2 reported here are distinctly different from those of Group II PLA2 in viperid snakes.

Amino Acid Sequence↗

PubMed1994

Taxon-specific lens crystallins with endogenous enzymic activities: some perspectives on synthetic applications.

A systematic and general approach has been carried out to provide basic structural and mechanistic information on epsilon- and delta-crystallin with lactate dehydrogenase and argininosuccinate lyase activities respectively from representative species of birds. A detailed kinetic analysis of these two crystallins revealed considerable differences in the enzymic activities among different species of birds. Especially noteworthy is the abundant presence of high activity in duck and goose epsilon- and delta-crystallins in contrast with the total lack of epsilon-crystallin and diminished activities of delta-crystallins in chicken and pigeon lenses. Sequence comparison of inactive pigeon delta-crystallin and active duck crystallin revealed several sequence variations and replacements which may account for the loss of argininosuccinate lyase activity in the lenses of certain birds. Bio-organic synthesis based on epsilon- and delta-crystallins isolated from duck lenses provides an efficient and practical tool for asymmetric or enantioselective synthesis of some pharmaceutically important chiral intermediates which may open new avenues and potentials in crystallin research.

Amino Acid Sequence↗

PubMed1993

Inactivation of the endogenous argininosuccinate lyase activity of duck delta-crystallin by modification of an essential histidine residue with diethyl pyrocarbonate.

The argininosuccinate lyase activity of duck delta-crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and delta-crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of delta-crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the delta-crystallin-L-arginine and delta-crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified delta-crystallin had minimum, if any, conformational changes as compared with the native delta-crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of delta-crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.

Animals↗

PubMed1993

Facile cloning and sequence analysis of goose delta-crystallin gene based on polymerase chain reaction.

To facilitate the cloning of delta-crystallin gene, the product of which is a major lens protein present in the avian and reptilian lenses, polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of goose lenses. The PCR product was then subcloned into pUC19 vector and transformed in E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by dideoxynucleotide chain-termination method. Sequencing several clones containing 1.4 kb DNA inserts encoding delta-crystallin constructed a complete and unambiguous full-length reading frame of 1401 base pairs covering a deduced protein sequence of 465 amino acids excluding the universal translation-initiating methionine. The goose delta-crystallin shows 88, 94, 88 and 69% sequence identity to pigeon delta, duck delta 2, chicken delta 1 crystallins and human argininosuccinate lyase respectively. It is also shown that, similar to duck delta 2 and in contrast to pigeon delta crystallin, goose delta-crystallin appears to possess high argininosuccinate lyase activity despite the fact that a highly homologous structure is shared among these homologous proteins. Structural analysis and comparison of these closely related delta-crystallin homologues with or without enzymatic activity should be of value in unraveling the intriguing evolutionary process leading to the development and evolution of active enzymatic crystallins in the lenses of certain avian species.

Amino Acid Sequence↗

PubMed1993

Cobra venom cardiotoxin (cytotoxin) isoforms and neurotoxin: comparative potency of protein kinase C inhibition and cancer cell cytotoxicity and modes of enzyme inhibition.

Effects of cobra cardiotoxin (cytotoxin) and its isoforms, and neurotoxin, on protein kinase C (PKC) and cancer cells were investigated. A positive correlation existed between hydrophobicities and activities of the toxins to inhibit PKC activity (assayed using phosphatidylserine vesicle, arachidonate monomer, and Triton/phosphatidylserine mixed micelle systems), phorbol ester binding to PKC, proliferation of several cancer cell lines, and phorbol ester-induced HL60 cell differentiation. Their relative hydrophobicities and activities, in a decreasing order, were cardiotoxin-1 approximately cardiotoxin-3 > cardiotoxin (a mixture of isoforms) > cardiotoxin-4 >> neurotoxin (inactive). Under the mixed micelle assay system (containing 0.3% Triton X-100, 8 mol % phosphatidylserine, 2 mol % diolein, and 200 microM CaCl2), cardiotoxin inhibited PKC competitively with respect to phosphatidylserine (apparent Ki of about 0.06 mol % or 2.5 microM), and in a mixed-type manner with respect to both diolein (apparent Ki of about 0.04 mol % or 1.7 microM) and Ca2+ (apparent Ki of about 2.9 microM). On the basis of findings that IC50 (approximately 0.3 microM) of cardiotoxin for inhibition of HL60 cell proliferation and differentiation was lower than its IC50 (9 microM) for PKC inhibition in vitro in the phosphatidylserine vesicle system and that PKC inhibition was the only known molecular mechanism of cardiotoxin, it was suggested that cardiotoxin might be highly membrane interacting and that the observed cellular effects of cardiotoxin might be mediated, in part, via PKC inhibition.

Amino Acid Sequence↗

PubMed1993

Sequence characterization of a novel alpha-neurotoxin from the king cobra (Ophiophagus hannah) venom.

Several postsynaptic neurotoxins (alpha-neurotoxins) with distinct pharmacological and biochemical properties were isolated and purified from the King cobra venom (Ophiophagus hannah) by employing sequentially preparative-scale cation-exchange chromatography on SP-Sephadex C-25 coupled with gel filtration and reversed-phase HPLC. The complete sequence of one neurotoxin was determined by N-terminal Edman degradation with the automatic pulsed-liquid phase sequencer on some peptide fragments generated from the endopeptidases, i.e. trypsin, S. aureus V8 protease and lysyl endopeptidase. This novel neurotoxin is a basic polypeptide of pI 9.05, consisting of 72 amino-acid residues with 10 cysteine residues. It is found to share about 60% sequence homology with Toxins a and b isolated from the same venom and the well established alpha-bungarotoxin, a major postsynaptic toxic ligand for acetylcholine receptor isolated from Bungarus multicinctus. The characterized alpha-neurotoxin molecules were also shown to bind specifically with nicotinic acetylcholine receptors of the electric eel, Torpedo californica.

Amino Acid Sequence↗

PubMed1993

A comparative study on commercial samples of phellodendri cortex.

A total of 31 commercial samples of Phellodendri cortex (Rutaceous plant) which originated from PHELLODENDRON AMURENSE Ruprecht, P. CHINENSE Schneid, P. WILSONII Hayata et Kanehira, and P. AMURENSE Rupr. var. SACHALINENSE Fr. Schm., respectively, were collected from the Taiwan and Japan herbal markets. The contents of five quaternary alkaloids (berberine, palmatine, jatrorrhizine, phellodendrine and magnoflorine) in these samples were determined by capillary electrophoresis. It was found that P. WILSONII (4.10 +/- 0.78%) and P. AMURENSE var. SACHALINENSE (4.18 +/- 1.03%) were superior to P. AMURENSE (1.55 +/- 0.72%) and P. CHINENSE (1.54 +/- 0.60%). Berberine was the major alkaloid in almost all samples. In comprised about 80% of the total alkaloids in the first two of the four herbs named above, but only about 40% in the latter two. From the data on the chemical analysis of the herb's constituents, as well as the herb's texture and color, we can postulate the origin and quality of a herb drug.

Journal Article↗

PubMed1993

Sequence characterization of cardiotoxins from Taiwan cobra: isolation of a new isoform.

Cardiotoxins (or called cytotoxins) are a group of very basic polypeptides present in some snake venoms, which are especially abundant in Formosan cobras. Several cardiotoxins with distinct pharmacological and biochemical properties were isolated and purified from the Taiwan cobra venom (Naja naja atra) by employing sequentially preparative-scale cation-exchange chromatography on TSK CM-650 coupled with the improved separation of toxin components on reversed-phase HPLC based on their hydrophobicity. The complete sequences of six cardiotoxin analogues, including one novel isoform never reported previously in this venom, were determined by N-terminal Edman degradation with the automatic pulsed-liquid phase sequencer and comparison of amino-acid compositions of purified toxin components. This novel cardiotoxin, a basic polypeptide of pI 10.13, comprises about 5% of total crude venom. It consists of 60 amino-acid residues with four disulfide bonds and shares about 80-92% sequence identity with several cytotoxins isolated from varied sources of different snakes. A systematic structure comparison of six major cardiotoxin isoforms was made by the secondary-structure predictions and hydropathy profiles based on their primary sequences. It is of interest to find that some subtle differences among these natural cardiotoxin analogues may lie in the intrinsic differences of the surface hydrophobicity of these isotoxins.

Amino Acid Sequence↗

PubMed1993

Characterization of three fibrinogenolytic proteases isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus).

Snake venoms, particularly those belonging to Crotalidae and Viperidae families, are known to possess fibrinogenolytic proteases which strongly affect the blood coagulation system. Three fibrinogenolytic enzymes, named as TM-1, TM-2 and TM-3, were purified from the venom of Taiwan habu using anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. These enzyme fractions were shown to be homogeneous as judged by SDS-gel electrophoresis. Further characterization of these purified fractions with fibrinogenase activity indicated that they are single-chain proteases possessing basic isoelectric points and molecular masses of approximately 27 kDa. These enzymes cleaved alpha-chain of fibrinogen first and then beta-chain, with relatively low activity on gamma-chain. Fibrinogenolytic activities of these enzymes were not significantly affected at extreme pH values, activity loss occurred only at temperatures higher than 65 degrees C. Enzyme activities were strongly inhibited by some metal chelators such as EDTA or 1,10-phenanthroline, suggesting that these fibrinogenases belong to metalloproteases. The closely similar amino-acid compositions coupled with identical cleaved products of similar molecular masses observed on the autolysis of TM-1 and TM-2 suggest that these two proteases are similar in primary structures and probably mutually related.

Amino Acids↗

PubMed1992

Biochemical characterization of crystallins from pigeon lenses: structural and sequence analysis of pigeon delta-crystallin.

Crystallins from pigeon eye lenses were isolated and purified by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid composition and sequence analysis. Alpha- and beta-crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta-crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of the gel filtration column. Pigeon delta-crystallin, similar to duck and reptilian delta-crystallins, exists as a tetrameric structure of about 200 kDa in the native form and is composed of one major subunit of 50 kDa with heterogeneous isoelectric points spreading in a range of 4.7 to 6.8. In contrast to those obtained from duck, goose and caiman, delta-crystallin isolated from the pigeon lens possessed very little argininosuccinate lyase activity. However, pigeon delta-crystallin can still cross-react with the antibody against enzymically active duck delta-crystallin as revealed by the sensitive immunoblotting technique. It was also shown that the delta-crystallin content of the total pigeon soluble proteins decreased with the age of the animal. Structural analysis of purified delta-crystallin fraction was made with respect to its amino-acid composition and protein primary sequence. N-terminal sequence analysis indicated the presence of blocked amino-termini in all crystallin fractions of pigeon lenses. Therefore, a sequence analysis of PCR (polymerase chain reaction) amplified delta-crystallin cDNA was employed to deduce the protein sequence of this crystallin. Structural comparison of delta-crystallin sequences from pigeon, chicken and duck lenses casts some doubts on the recent claim that His-89-->Gln mutation in the chicken delta-crystallin may account for the loss of argininosuccinate lyase activity in this avian species, as compared to high enzymic activity in the duck crystallin (Barbosa et al. (1991) J. Biol. Chem. 266, 5286-5290).

Amino Acid Sequence↗

PubMed1992

Sequence analysis of pigeon delta-crystallin gene and its deduced primary structure. Comparison of avian delta-crystallins with and without endogenous argininosuccinate lyase activity.

delta-Crystallin is a major lens protein present in the avian and reptilian lenses. To facilitate the cloning of the delta-crystallin gene, cDNA was constructed from the poly(A)+ RNA of pigeon lenses, amplified by the polymerase chain reaction (PCR). The PCR product was then subcloned into pUC19 vector and transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing two clones, containing 1.4 kb DNA inserts coding for delta-crystallin allowed the construction of a complete, full-length reading frame of 1,417 bp covering a deduced protein sequence of 466 amino acids, including the universal translation-initiating methionine. The pigeon delta-crystallin shows 88, 83 and 69% sequence identity to duck delta 2, chicken delta 1 crystallins and human argininosuccinate lyase respectively. It is also shown that, in contrast to duck delta 2 crystallin which has a high argininosuccinate lyase activity, pigeon delta-crystallin appears to contain very low activity of this enzyme, despite the fact that they share a highly homologous structure. A structural comparison of delta-crystallins with or without enzymatic activity suggested several amino acid replacements which may account for the loss of argininosuccinate lyase activity in the lenses of certain avian species.

Amino Acid Sequence↗

PubMed1992

Characterization of a protease with alpha- and beta-fibrinogenase activity from the Western diamondback rattlesnake, Crotalus atrox.

A metalloprotease from the rattlesnake Crotalus atrox venom was isolated and purified from multiple-step chromatographies including anion-exchange chromatography, gel permeation and reversed-phase HPLC. The fraction was shown to be homogeneous as judged by SDS-gel electrophoresis. It also showed a high proteolytic activity against alpha- and beta-chains of fibrinogen molecules. Further characterization of the purified fraction with fibrinogenase activity indicated that it is a single-chain protease with a molecular mass of about 24 kDa and an acidic isoelectric point. It is relatively heat stable up to about 65 degrees C, inhibited by EDTA, beta-mercaptoethanol, but not by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, soybean trypsin inhibitor and aprotinin. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to some metalloproteases characterized before from the closely related rattlesnake venoms. N-Terminal sequence analysis of the enzyme corroborated some similarity between this enzyme and the reported sequences of these enzymes characterized from the Crotalidae snake family. This study indicated the presence of a novel fibrinogenase (termed Catroxase) with N-terminal sequence different from the metalloprotease with hemorrhagic activity isolated from the same Western diamondback rattlesnake.

Amino Acid Sequence↗

PubMed1992

Kinetic analysis of duck epsilon-crystallin with L-lactate dehydrogenase activity: determination of kinetic constants and comparison of substrate specificity.

A systematic analysis of the kinetic properties of duck lens epsilon-crystallin with lactate dehydrogenase [LDH, (E.C. 1.1.1.27)] activity was carried out by employing some 19 different alpha-keto acids as substrates for this NADH-dependent LDH-catalyzed reaction. The steady-state Michaelis and catalytic constants (Km, kcat) were determined for a broad range of organic compounds. The results provide important insights regarding the binding and affinity of substrates to active sites of this enzyme crystallin and indicate a great potential for the application of the stable epsilon-crystallin as a catalyst to the synthesis of some important chiral alpha-hydroxyacids in a convenient and efficient way. It is also demonstrated for the first time that in addition to the enzymatic activity of lactate dehydrogenase, duck epsilon-crystallin also possesses the enzymatic activity of malate dehydrogenase.

Animals↗

PubMed1992

Facile synthesis of chiral 2-hydroxy acids catalyzed by a stable duck epsilon-crystallin with endogenous L-lactate dehydrogenase activity.

Duck epsilon-crystallin, an abundant structural protein in lenses of some avian species, was shown to possess a genuine and stable L-lactate dehydrogenase (L-LDH; EC 1.1.1.27) activity suitable for the application to enzyme technology as a catalyst for the synthesis of chiral alpha-hydroxy acids. Two pharmaceutically important intermediates, 2-hydroxy acids (S)-2-hydroxybutanoic acid (S)-2-hydroxypentanoic acid have been synthesized in high yields and optical purity utilizing an in situ NADH regeneration system of duck epsilon-crystallin coupled with formate/formate dehydrogenase. This enzyme system is also shown to offer some advantages over the conventional L-LDH sources from several mammalian species.

Animals↗

PubMed1992

Biochemical characterization and kinetic analysis of duck delta-crystallin with endogenous argininosuccinate lyase activity.

Delta-Crystallin, the most abundant crystallin in the avian species, was isolated and purified from duck lenses. It was shown to possess endogenous argininosuccinate lyase activity catalysing the reversible cleavage of argininosuccinate to give fumarate and arginine with an equilibrium constant of 1.8 +/- 0.23 mM. In contrast, chicken lens delta-crystallin showed only 0.4-0.8% of the enzyme activity of duck delta-crystallin under identical assay conditions. Biochemical comparison of delta-crystallins from these two species revealed distinct differences in their structural and kinetic properties. An activity-staining method was developed for the easy detection of endogenous enzyme activity of delta-crystallin from crude lens extracts of different avian species. Two-dimensional gel electrophoresis of lens homogenates indicated that, in the chicken lens, delta-crystallin is composed mainly of a subunit with a pI of 5.9 and a subunit mass of 50 kDa, whereas that of duck lens possesses various 50 kDa subunits in a pI range of 5.9-6.8. Activity staining corroborated the fact that all charge isoenzymes of duck delta-crystallin possess enzyme activity whereas that of chicken delta-crystallin is devoid of activity. For duck delta-crystallin, variation of the enzyme activity with argininosuccinate concentration in the forward reaction followed saturation kinetics with an apparent Michaelis constant for the substrate of 17 +/- 5 microM. In the reverse reaction, initial-velocity studies showed intersecting patterns. Inhibitions of the forward reaction by products (fumarate and arginine) were both non-competitive with respect to argininosuccinate. Citrulline, an analogue of arginine, inhibited the enzyme activity in both directions and was competitive with respect to arginine but non-competitive with respect to fumarate or argininosuccinate. Succinate, which inhibited the bovine argininosuccinate lyase, did not affect the delta-crystallin enzyme activity in a concentration range between 1 and 300 mM. These results suggest a random Uni Bi kinetic mechanism for the argininosuccinate lyase activity of duck delta-crystallin with the formation of various abortive delta-crystallin-argininosuccinate-arginine, delta-crystallin-argininosuccinate-fumarate and delta-crystallin-argininosuccinate-citrulline ternary complexes.

Animals↗

PubMed1992

Specific peptide-bond cleavage by microwave irradiation in weak acid solution.

A rapid and selective peptide-bond cleavage in weak acid, induced by microwave irradiation, has been developed. The specific cleavage sites of peptide bonds located only at the carboxyl- and amino-terminal ends of aspartyl residues along the peptide chain. A systematic study including the time course for the cleavage of various aspartyl-containing peptides, the effect of the acidity of the reaction solution on the completeness of peptide-bond cleavage, and the relationship between the power of microwave irradiation and the reaction time of cleavage are studied.

Amino Acid Sequence↗

PubMed1992

Staining of argininosuccinate lyase activity in polyacrylamide gel.

A procedure for the direct staining of argininosuccinate lyase activity in polyacrylamide gel is described. The method was based on coupling one of the enzymatic products fumarate with fumarase and malic enzyme catalyzed reactions. Fumarate was first converted to L-malate by fumarase. Malic enzyme then catalyzed the oxidative decarboxylation of L-malate to give CO2 and pyruvate with concomitant reduction of NADP+ to NADPH. Finally the reducing power of NADPH was coupled to phenazine methosulfate and in turn to nitroblue tetrazolium yielding a deeply colored insoluble formazan which may be quantitized or semiquantitized by densitometer.

Animals↗

PubMed1992

Studies on the role of the V3 loop in human immunodeficiency virus type 1 envelope glycoprotein function.

Mutations within the principal neutralizing determinant (the V3 loop) of the HIV-1 surface envelope glycoprotein gp120 block or greatly reduce the ability of the HIV-1 envelope glycoprotein to induce cell fusion in CD4+ HeLa T4 cells while keeping its CD4 binding ability. However, when either cysteine or both cysteines forming the V3 disulfide bridge were mutated, the resultant glycoprotein could not mediate cell fusion, undergo proteolytic processing, or bind CD4. To investigate the role that the V3 loop plays in gp160 processing and CD4 binding, we deleted the entire V3 loop region of the HIV-1 env gene. The resultant glycoprotein could not mediate cell fusion in the HeLa T4 cell line and no proteolytic processing of gp160 or CD4 binding could be detected. To test whether any domain of the V3 loop is involved in attaining the proper envelope glycoprotein conformation required for proteolytic processing and CD4 binding, we introduced a series of deletions into the coding region of the V3 loop. Most of the residues within the V3 loop could be removed while retaining gp160 processing and CD4 binding. Our results indicate that the cysteines that form the V3 loop or the disulfide bond itself are important for proper envelope glycoprotein folding and processing. Because many of the mutants constructed in this study do not contain the type-specific neutralizing determinant of HIV-1, they may be potential reagents to bind group-specific neutralizing antibodies or to elicit a group-specific neutralizing response against HIV-1.

Amino Acid Sequence↗