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Biomedical subjects

K T Wang

Publications and source records attributed to K T Wang.

At least 19 recordsLinked to original sources
PubMed1996

Structural characterization of the metal binding site in the cysteine-rich region of HIV-1 Tat protein.

Human immunodeficiency virus type-1 Tat protein transactivates the gene expression of retrovirus. The unique cysteine-rich region of Tat protein is biologically essential. This study characterizes the structural features of a synthetic Tat21-38 peptide covering the cysteine-rich region with Zn binding property. UV titrations confirm Tat peptide binds with two Zn2+ cations maximally per monomer, as previously reported(1). Only monomer is observed from the electrospray mass spectrum. Interestingly, a modified Ellman reaction (2) can differentiate a metal chelated thiolate of cysteine residue from a free one. Three disulfide bonds are formed in apo-Tat21-38 peptide. One Tat21-38 molecule utilize four Cys residues in coordination with the first incorporated Zn2+ cation. Five out of seven Cys residues are coordinating with two Zn cations of the complex Zn-Tat21-38 (2:1). A model of the coordination arrangement of Zn binding sites at different states is proposed.

Amino Acid Sequence↗
PubMed1996

Protein engineering of venom toxins by synthetic approach and NMR dynamic simulation: status of basic amino acid residues in waglerin I.

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.

Amino Acid Sequence↗
PubMed1996

Structural analysis of a biologically active echistatin analogue des(46-49)-[Ala8,37]-echistatin gamma with three disulfide bonds by 2D-NMR and computer graphics.

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.

Amino Acid Sequence↗
PubMed1996

Conformational study of two linear hexapeptides by two-dimensional NMR and computer-simulated modeling: implication for peptide cyclization in solution.

Two linear peptides, D-leucyl-L-prolyl-L-isoleucyl-L-valyl-L-alanyl-beta-alanine (I) and D-leucyl-L-prolyl-L-isoleucyl-L-valyl-N-methyl-L-alanyl-beta-alanine (II), whose sequences were designed from protodestruxin and desmethyldestruxin B by replacing D-leucic acid with D-leucine, two cyclic hexadepsipeptides with insecticidal and immunodepressant activities, have been found to be cyclized in unusually high yields (>85%). In order to gain insight into the conformation and the relative flexibility of different constituent residues in these linear peptides, we have applied various techniques of 2D-NMR spectroscopy coupled with dynamic simulated annealing by computer modeling to establish the solution conformations of these two linear peptides. Based on the derived structures, it is found that the distances between N- and C-terminal residues of both peptides are short enough to facilitate the cyclization, thus collaborating the observation of favorable cyclization yields for both linear peptides.

Amino Acid Sequence↗
PubMed1996

Conformation of Vespa basalis mastoparan-B in trifluoroethanol-containing aqueous solution.

Mastoparan-B, a tetradecapeptide isolated from the venom of the hornet Vespa basalis, belongs to the mastoparan analogs of vespid venom with the lysine residues common for all mastoparan family toxins at positions 4, 11 and 12. Here we use 1H-NMR spectroscopy and hybrid distance geometry-simulated annealing calculation to investigate its three-dimensional structure in trifluoroethanol-containing aqueous solution. The calculated structure shows that residues 3-14 adopt an amphiphilic alpha-helical structure in which the residues with hydrophilic side chains (i.e. Lys-4, Ser-5, Ser-8, Lys-11, Lys-12) are located on one side and the residues with hydrophobic side chains (i.e. Leu-3, Ile-6, Trp-9, Ala-10, Val-13, Leu-14) located on the other side of the molecule. The overall structural features a very similar to the conformation of mastoparan-X reconstituted in vesicle [Wakamatsu et al. (1992) Biochemistry 31, 5654-5660] in spite of the substitutions made for eight residues with distinctly different hydrophobicity. These substitutions lead to a larger hydrophobic moment for the alpha-helical segment and further mobilized N-terminal. This study will help reveal the conformational significance of mastoparan toxins with respect to their potency and activity in G protein regulation.

Amino Acid Sequence↗
PubMed1996

Determination of three-dimensional solution structure of waglerin I, a toxin from Trimeresurus wagleri, using 2D-NMR and molecular dynamics simulation.

The solution conformation of a synthetic snake venom toxin waglerin I, has been determined by using proton nuclear magnetic resonance spectroscopy. By a combination of various two-dimensional NMR techniques, the 1H-NMR spectrum of waglerin I was completely assigned. A set of 247 interproton distance restraints was derived from nuclear Overhauser enhancement (NOE) measurements. These NOE constraints, in addition to the 2 dihedral angle restraints (from coupling constant measurements) and 7 omega torsion angel restraints for prolines, formed the basis of three-dimensional structure determined by molecular dynamics techniques. The 19 structures that were obtained satisfy the experimental restraints, and display small deviation from idealized covalent geometry. Analysis of converged structures indicates that the toxin has no special secondary structure. In the solution structure of waglerin I, the central ring region is well defined but the N- and C-termini possesses more disorder.

Amino Acid Sequence↗
PubMed1996

Structural requirements for the edema-inducing and hemolytic activities of mastoparan B isolated from the hornet (Vespa basalis) venom.

Mastoparan B (MP-B) is a cationic tetradecapeptide isolated from the black-bellied hornet (Vespa basalis) venom. It has a primary structure (LKLKSIVSWAKKVL-CONH2) distinct from other vespine mastoparans. The peptide caused a dose-dependent swelling in rat hind paw and showed a potent hemolytic activity in guinea pig red blood cells. Studies on the structure activity relationship of the peptide showed that replacing lysine at position 2 (Lys2) by asparagine (Asn) in the MP-B sequence caused about 40% decrease in its edema-inducing activity at 50 micrograms/paw and 90% decrease in hemolytic activity at 30 microM of the peptide, while the same substitution at Lys4 did not cause a significant change in either activity. Replacing either Lys11 or Lys12 by leucine (Leu) caused little or no decrease in the edema-inducing and hemolytic activities. Decreases in both activities were observed when both Lys11 and Lys12 were replaced by Leu. On the other hand, replacing tryptophan at position 9 (Trp9) by tyrosine or phenylalanine in MP-B sequence almost abolished its hemolytic activity, while the edema-inducing activity was only partially inhibited. Circular dichroism spectra of the peptides measured in 20% trifluoro-ethanol revealed that substitution of Lys and Trp did not cause a significant change in the conformation of MP-B. it appears that Lys2 is crucial for both hemolytic and edema-inducing activities of MP-B, while Trp9 is of special importance to the hemolytic activity of MP-B. Lys11 and Lys12 in MP-B probably play a lesser role in both activities.

Amino Acid Sequence↗
PubMed1996

[The effect of blood AST, ALT and lactate after short and middle distance exercise training].

Twenty-four nursing college students aged 16 and 17 years were selected as research subjects and divided into two groups. Group A comprised 12 individuals who were trained for short distance running (5km/day) over a four-week period, while group B was trained for middle distance running (7km/day) during the same period. Blood AST (aspartate aminotransferase), ALT (alanine aminotransferase) and lactate were performed at rest (before training) and after exercise every week. After both short and middle distance exercise training, the lactate values after 1-3 week(s) training period were persistently higher than those before training and the differences between then are significant. However, the lactate value after 4 weeks training period is lower than that after the third week training. There are significant differences between the AST values after 1-4 week(s) training period and those before exercise in short distance exercise training. There are no significant differences between the AST values after training and those before exercise in middle distance exercise training. The ALT values after 1-4 week(s) training period were lower than those before exercise in short and middle distance exercise training. In conclusion, after 4 weeks training, the lactate and AST values can't reduce to those before training in middle distance exercise training, and the lactate value in short distance exercise training is the same as former. Further investigation is needed.

Adolescent↗
PubMed1995

Conformation of polymyxin B analogs in DMSO from NMR spectra and molecular modeling.

The tertiary structures of two polymyxin analogues: [formula: see text] and [formula: see text] in DMSO, from solid-phase peptide synthesis and aerobic oxidation were determined from two-dimensional NMR spectra and distance geometry calculations followed by restrained molecular dynamics simulation. The backbone of peptide I had a rectangular shape stabilized by at least two hydrogen bonds and the hydrophilic side chains of five lysine residues, and the hydrophobic side chains of Phe and Leu resided at both sides to form an amphiphilic molecule. This amphiphilic structure of I is likely to interact with lipid A mainly via a hydrophobic interaction. Compared with I, peptide II, which lacks three N-terminal amino-acid residues, exhibits neither amphiphilic property nor binding ability with lipid A.

Amino Acid Sequence↗
PubMed1995

Sequence comparison and computer modelling of cardiotoxins and cobrotoxin isolated from Taiwan cobra.

Six cardiotoxins and one neurotoxin isolated and purified from the Taiwan cobra venom (Naja naja atra) possess distinct pharmacological and biochemical properties despite the existence of a grossly similar tertiary structure among these toxins, i. e., a core consisting of a series of short loops and four disulfide bridges. A systematic structure comparison of these major toxin isoforms was made by the secondary-structure predictions together with computer model-building based on the primary sequences and the established X-ray and NMR structures of one published cardiotoxin isoform and cobrotoxin. It is of interest to find that some defined and subtle differences can be detected upon the superposition of these three-dimensional polypeptide chains, which may reflect the intrinsic differences in the surface hydrophobicity of cardiotoxins and cobrotoxin as revealed by hydropathy profiles of these toxins in one of three major loops. The differences seem to correlate with different inhibitory activities exhibited by cardiotoxins in contrast to the lack of activity by cobrotoxin on protein kinase C (PKC).

Amino Acid Sequence↗
PubMed1995

Physicochemical properties of alkaline serine proteases in alcohol.

The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1 micrograms/ml and that of alcalase was 48.1 micrograms/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters, L-Ala-L-Ala-(D- or L-)Pro-L-Phe-OMe and L-Ala-L-Ala-(D- or L-)Ala-L-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.

Amino Acid Sequence↗
PubMed1995

Immunogenicity of mastoparan B, a cationic tetradecapeptide isolated from the hornet (Vespa basalis) venom, and its structural requirements.

Mastoparan B (MP-B) is a cationic tetradecapeptide (LKLKSIVSWAKKVL-CONH2, mol. wt 1611) isolated from the black-bellied hornet (Vespa basalis) venom. The small peptide itself was capable of inducing antibodies without prior conjugation to a protein carrier in rabbits and mice. The mouse antibody was found to be of IgG1 isotype with kappa-type light chain. The peptide antigen was able to form insoluble complexes with the specific antibody, suggesting that MP-B possessed more than one epitope in its molecule. The finding that MP-B was able to bind with both mouse and rabbit antibodies in sandwich ELISA supports this contention. Synthetic MP-B analogues in which lysine at position 2, 4, 11 or 12 was replaced by neutral amino acids such as asparagine or leucine showed a significant decrease in their antibody-binding activities. Substitution of lysine at position 4 (Lys4) caused the most marked inhibition in its binding activity. However, replacing tryptophan at position 9 by tyrosine caused a relatively small reduction in its binding activity. Replacing both Lys2,4 by asparagine or removing Lys-containing segments at amino or carboxyl terminus in MP-B sequence caused a remarkable decrease in the antibody-binding and immunogenic activities of the peptide. The Lys residues located at amino and carboxyl terminal segments of MP-B, especially Lys4, appear to play a critical role in the binding interaction and the immunogenicity of the peptide.

Amino Acid Sequence↗
PubMed1995

Inhibition of protein kinase C by snake venom toxins: comparison of enzyme inhibition, lethality and hemolysis among different cardiotoxin isoforms.

Cardiotoxins, neurotoxins and phospholipase A2 (PLA2) are three major classes of toxic components present in the Taiwan cobra, Naja naja atra of the Elapidae family. Cardiotoxins (or called cytotoxins), a group of major polypeptides of around 60 amino-acid residues present abundantly in the elapid family of snakes, comprise about 45-55% of the crude venom of Taiwan cobra. In contrast to another prominent group of structurally similar neurotoxins with well-established acetylcholine receptors and modes of action, cardiotoxins showed no defined cellular targets and very diverse pharmacological functions. A systematic structure/function comparison of these toxins was made by their relative inhibitory effects on protein kinase C (PKC) isolated from mouse brains. Lethality and hemolysis of various cardiotoxin isoforms were also compared in order to shed some insight on the biological targets and mechanisms of these surface-active amphiphilic polypeptides. A structure comparison of these cardiotoxins based on computer model-building revealed that some defined and subtle differences can be detected upon the superposition of these three-dimensional polypeptide chains, which may reflect the intrinsic differences in the hydrophobic peptide segments present on the surface loops of toxin molecules. The differences seem to correlate with different inhibitory activities exhibited by cardiotoxins in contrast to the lack of activity by cobrotoxin and PLA2 on PKC.

Animals↗
PubMed1995

[Impact of exercise on athlete's cardiac muscle evaluated by Troponin T test].

Twenty-four nursing college students ages 16 to 17 Year were selected as research subjects and divided into two group. Group A comprised 12 individuals who were trained for short distance running (5KM/day) over a five week period, while Group B was trained for middle distance running (7KM/day) during the same period. Breathing frequency, blood pressure, pulse rate and body surface skin temperature were measured at rest (before training) and after exercise every week. Troponin T (TnT). Creatin kinase (CK), Creatine kinase -MB (CK-MB), and Lactate Dehydrogenase (LDH) tests were performed using blood samples and were used as indicators of the impact on heart muscle during exercise. There were no significant differences in breathing frequency, blood pressure, pulse rate or body surface skin temperature between groups A and B before or after training. After one week, the CK levels in both group were higher after exercising than before the training program had begun, and there were significant differences (P < 0.05) between the two groups. Thenceforth, the CK levels in group A decreased to the normal values and stayed the same throughout the fifth week. The CK levels in group B decreased to the normal value after training for three weeks, and there were significant differences between the values before exercising and those for the forth and fifth weeks of training. Although the levels of CK-MB, LDH, and Troponin-T increased after training, all were within the normal range, and no significant difference was observed before or after five weeks of training.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Cardiovascular effects of mastoparan B and its structural requirements.

Mastoparan B is a cationic, amphiphilic tetradecaeptide (LKLKSIVSWAKKVL-CONH2) isolated from the venom of the hornet Vespa basalis. Intravenous injection of the peptide into rats caused a profound depression of blood pressure and cardiac function, which was inhibited by cyproheptadine, reserpine and multiple doses of compound 48/80, but not by diphenhydramine and cromolyn. Mastoparan from Paravespula lewisii showed little cardiovascular inhibitory activity. A synthetic mastoparan B analog in which lysine at position 2 was replaced by asparagine showed a marked decrease in the cardiovascular depressor effects, while replacing lysine at position 4, 11 or 12 with leucine did not cause a significant reduction in these effects. Replacing lysine at position 12 with leucine even caused a more sustained depressor effect. However, the analog in which lysines at positions 11 and 12 were replaced by leucine lost its cardiovascular inhibitory activity. Replacing tryptophan at position 9 with phenylalanine in mastoparan B did not affect its activity. It is concluded that mastoparan B is involved in the cardiovascular disturbances induced by the hornet venom. Lysine at position 2 is a critical residue for the cardiovascular effects of mastoparan B. A peptide molecule with two lysine residues, one located close to the amino terminus and the other near the carboxyl end of the peptide, appears to be the optimal structure for eliciting the cardiovascular depressor effects.

Amino Acid Sequence↗
PubMed1994

D-Aminoacylase from Alcaligenes faecalis possesses novel activities on D-methionine.

D-Aminoacylase isolated from Alcaligenes faecalis DA1 has a great potential for future application in D-amino acids production. This paper reports for the first time that D-aminoacylase can reverse the catalysis direction on D-Met and deacylate N-Ac-D-Met-OMe and N-Ac-D-Met-Gly. The results provide important insights regarding the binding and affinity of substrates to the active site of this enzyme. Based on a systematic study of kinetic properties and relative reactivities for a broad range of substrates, a model to elucidate the reaction mechanism is proposed.

Acylation↗
PubMed1994

Chemical synthesis of gamma-echistatin analogues: elucidation of status of C-terminal (45-49).

Three analogues of gamma-echistatin, des(45-49)-gamma-echistatin, des(46-49)-gamma-echistatin and des(47-49)-gamma-echistatin, were synthesized by solid-phase methodology and their biological activities were measured and compared. The results reveal that without the C-terminal (45-49) of gamma-echistatin, the folding of the protein to the final active structure is not interfered with and Lys-45 influences the inhibition of platelet aggregation.

Amino Acid Sequence↗