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A T Tu

Publications and source records attributed to A T Tu.

At least 55 records · Page 3Linked to original sources
PubMed1988

Characterization of nicked myotoxin a and its effect on the sarcoplasmic reticulum calcium pump.

Myotoxin a, a muscle-necrotizing polypeptide isolated from Crotalus viridis viridis (prairie rattlesnake) venom, was nicked at Met-28 by cyanogen bromide. Amino acid analysis indicated that the methionine content was reduced to zero from the original 1 mol. Judging from circular dichroism, the nicked myotoxin a had a conformation similar to that of original myotoxin. Raman spectra indicated that the conformations of the three disulfide bonds are not affected in nicked myotoxin a. Like the original toxin, nicked myotoxin a was myotoxic and inhibited calcium ion loading activity, although the inhibitory action was slightly lower than that of the original myotoxin a. Both modified and unmodified myotoxin a showed myonecrotic activity as determined by examining histological slides. The modified toxin also inhibited the formation of decavanadate-induced two-dimensional crystalline arrays of the sarcoplasmic reticulum Ca2+-ATPase just as the original myotoxin a does.

Animals↗
PubMed1988

Isolation and primary structure of the major toxin from sea snake, Acalyptophis peronii, venom.

The major neurotoxin from the venom of Acalyptophis peronii captured in the Gulf of Thailand was isolated. Although there are two toxic fractions in the venom, the most toxic and abundant fraction was selected for purification and chemical characterization. The LD50 of the major toxin is 0.125 micrograms/g mice, indicating an extremely toxic nature. The toxin consists of 60 amino acid residues with methionine as the amino-terminal and asparagine as the carboxy-terminal end. It contains nine half-cystine residues. There is 1 mol each of tryptophan, tyrosine, methionine, valine, aspartic acid, leucine, and alanine, and there is no phenylalanine. The molecular weight calculated from the amino acid sequence determination was 6600. The toxin replaces alpha-bungarotoxin in binding with the acetylcholine receptor, indicating that the A. peronii major neurotoxin competes with alpha-bungarotoxin for the same binding site of the acetylcholine receptor.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of horridum toxin with arginine ester hydrolase activity from Heloderma horridum (beaded lizard) venom.

A hemorrhagic toxin with lethal and arginine ester hydrolytic activities was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel, and Q-Sepharose column chromatography. The hemorrhagic toxin was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis and immunodiffusion. Its molecular weight is approximately 31,000 with an isoelectric point of 3.9. Hemorrhagic, lethal, and benzoyl-L-arginine ethyl ester hydrolytic activities of this preparation were inhibited by diisopropyl fluorophosphate (DFP), N-bromosuccinimide, and beta-mercaptoethanol, suggesting that serine, tryptophan, and disulfide bonds are involved in these activities. Also there was an increase in creatine kinase activity in mice serum which is an indicator that the toxin is involved in muscle damage. This protein was stable to heat and pH ranges between 2 and 11. The Michaelis constant (Km), for benzoyl-L-arginine ethyl ester, and inhibition constant (Ki), for DFP, were found to be 6.9 X 10(-3) and 1.93 X 10(-4) M, respectively.

Amino Acid Sequence↗
PubMed1988

Amino-acid sequence of the minor neurotoxin from Acalyptophis peronii venom.

Sea snakes, Acalyptophis peronii, were captured in the Gulf of Thailand and their venom was isolated. A. peronii venom contains two neurotoxins called major and minor toxin. The complete amino-acid sequence of the minor toxin was identified and compared to that of the major toxin. The only difference between the major and the minor toxins is in the 43rd residue. The major toxin at this position contains glutamine, while the minor toxin contains glutamic acid. The LD50 of the minor toxin is 0.170 microgram/g in mice when injected intravenously. The toxicity is slightly lower than that of the major toxins, which has an LD50 of 0.125 microgram/g.

Amino Acid Sequence↗
PubMed1987

Crystallization of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum. Inhibition by myotoxin a.

Decavanadate produces extensive ordered arrays of Ca2+-ATPase molecules on sarcoplasmic reticulum (SR) vesicle surfaces [(1984) J. Bioenerg. Biomembranes 16, 491-505] and the basic unit of these crystalline structures seems to be a dimer of Ca2+-ATPase [(1983) J. Ultrastruct. Res. 24, 454-464; (1984) J. Mol. Biol. 174, 193-204]. Myotoxin a, isolated from the venom of the prairie rattlesnake Crotalus viridis viridis, is a muscle-degenerating polypeptide and its primary site of interaction is the SR membrane, where it uncouples CA2+-translocation from CA2+-dependent ATP hydrolysis [(1986) Arch. Biochem. Biophys. 246, 90-97]. The effect of myotoxin a on decavanadate-induced two-dimensional Ca2+-ATPase crystals of SR membranes has been investigated. The toxin inhibits the formation of two-dimensional SR-membrane crystals and disrupts previously formed crystals in a time- and concentration-dependent manner, which parallels the uncoupling of ATP hydrolysis from Ca2+ translocation. Two-dimensional crystalline arrays of the SR membrane have a typical diffraction pattern which, after myotoxin a treatment, displays a progressive loss of order. Decavanadate is an uncompetitive inhibitor of the Ca2+-ATPase enzyme-myotoxin a complex. The present results suggest that a Ca2+-ATPase dimer is required for coupling Ca2+ translocation to Ca2+-dependent ATP hydrolysis.

Animals↗
PubMed1987

Biochemical characterization of hemorrhagic toxins with fibrinogenase activity isolated from Crotalus ruber ruber venom.

Three hemorrhagic toxins with proteolytic activity were isolated from the venom of Crotalus ruber ruber (red rattlesnake). Molecular weights of HT-1, HT-2, and HT-3 were 60,000, 25,000, and 25,500, respectively. Although HT-3 was a basic protein, HT-1 and HT-2 were slightly acidic proteins. Total amino acid residues were 482,207, and 221 for HT-1, HT-2, and HT-3, respectively. Protease activity of all the toxins was inhibited in the presence of EDTA or o-phenanthroline, suggesting that the toxins are metalloproteins. Analyses for various metals by inductively coupled plasma-atomic emission spectrometry indicated that sodium, potassium, zinc, and calcium atoms were present in significant quantities. With all three toxins, there was roughly 1 mol of zinc to 1 mol of protein; the results for calcium were not consistent. All three hemorrhagic toxins degraded the A alpha chain of fibrinogen, while HT-1 also degraded the B beta chain. Although fibrinogen was degraded by the three toxins, no clots were observed, indicating that the proteolytic specificities of the three toxins were different from those of thrombin. The hemorrhagic toxins increased creatine kinase activity in mice serum, indicating muscle damage, which was substantiated by histological examination.

Amino Acids↗
PubMed1987

A calorimetric examination of the effect of myotoxin a on the thermotropic phase behavior of model lipid membranes.

The effect of myotoxin a on the thermotropic phase behavior of aqueous dispersions of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) was examined using differential scanning calorimetry (DSC). Myotoxin a significantly altered the normal phase behavior of DMPC in a concentration dependent fashion. This effect is perturbed by Ca2+ and is sensitive to ionic strength and pH. High concentrations of toxin eliminate the characteristic pretransition associated with the polar head group of DMPC. They also increase the temperature of the main gel-to-liquid crystal transition from 23 degrees C to 32-35 degrees C. At low concentrations of toxin, the first visible effect is upon the pretransition which is split into two components that diminish with time. The main transition is less affected at low toxin concentrations, although the magnitude of the transition is reduced while it is simultaneously shifted to higher temperatures. The main transition is also split into multiple components. The toxin also had pH specific effects on the phase behavior of DMPS. Above physiological pH (8.5) the normal transition of DMPS at 36-38 degrees C was split in the presence of myotoxin a and new components appeared centered at 31 degrees C and 35 degrees C. These observations are consistent with reports that the skeletal muscle membrane system is the major site of the myonecrotic effect of myotoxin a.

Calorimetry↗
PubMed1987

Biotoxicology of sea snake venoms.

Sea snakes are the most abundant venomous reptiles, found throughout the Indian and Pacific Oceans. Divided into two subfamilies, Laticaudinae and Hydrophiinae, all sea snakes are poisonous. Venoms are highly toxic, as indicated by low LD50 values in test animals. Toxic compounds include presynaptic and postsynaptic neurotoxins. Similarities in phylogeny are reflected in immunodiffusion patterns, immunoelectrophoresis, cross-neutralization by antivenin against heterologous venoms, and amino acid composition. The clinical syndrome following a bite is largely neurotoxic and myotoxic, with rare hepatotoxicity and nephrotoxicity. Proper emergency field therapy and timely administration of antivenin can be lifesaving. Hemodialysis may be useful when antivenin is not available.

Animals↗
PubMed1986

Isolation and characterization of an anticoagulant proteinase, cerastase F-4, from Cerastes cerastes (Egyptian sand viper) venom.

An anticoagulant protease, Cerastase F-4, was isolated from the venom of Cerastes cerastes (Egyptian sand viper) by a combination of gel filtration, ion-exchange chromatography, and HPLC. Homogeneity of the purified anticoagulant was established by discontinuous polyacrylamide disc gel electrophoresis and by isotachophoresis. The anticoagulant enzyme is a single polypeptide chain without subunits having a molecular weight of 22,500. It consists of 28% aspartic acid residues and only 7% are basic amino acids. This agrees well with the fact that the anticoagulant is an acidic protein with an isoelectric point of 5.2. The anticoagulant is a proteolytic enzyme which hydrolyzes casein, fibrinogen and fibrin. The enzyme's optimum activity occurs around 55 degrees C. The anticoagulant showed no phospholipase A activity, low lethal activity, low hemorrhagic and capillary permeability activity, and no myotoxic activity.

Amino Acids↗
PubMed1986

Mechanism of the anticoagulant, Cerastase F-4, isolated from Cerastes cerastes (Egyptian sand viper) venom.

An anticoagulant enzyme, Cerastase F-4, from the venom of Cerastes cerastes was purified to homogeneity and was characterized (1). In the present report the mode of its fibrinogenolytic and fibrinolytic actions, and its effects on some other blood coagulation factors are described. Cerastes F-4 was shown to readily hydrolyze the alpha A chain of fibrinogen followed by the hydrolysis of the beta B chain. The gamma-chain was relatively resistant to hydrolysis. It also degrades the three chains of fibrin at different rates. The degradation products of the two substrates shown on SDS-polyacrylamide gel were quite different from those produced by plasmin, indicating different sites of cleavage by the enzyme. Using specific chromogenic substrates, Cerastase F-4 seems not to show thrombin-like, plasmin-like, kallikrein-like, antithrombin, or antiplasmin actions. Also, it does not activate prothrombin or plasminogen but degrades both of them slowly. It is concluded that the anticoagulation property of the purified enzyme, Cerastase F-4, is due to its destruction of fibrinogen.

Blood Coagulation↗
PubMed1986

Interaction of myotoxin a with the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum.

Myotoxin a is a muscle-damaging toxin isolated from the venom of Crotalus viridis viridis. Its interaction with the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles purified from rabbit skeletal muscle was investigated. Myotoxin a inhibited Ca2+ loading and stimulated Ca2+-dependent ATPase without affecting unidirectional Ca2+ efflux. Its action was dose, time, and temperature dependent. Myotoxin a partially blocked the binding of specific anti-(rabbit SR Ca2+-ATPase) antibodies. It is concluded that myotoxin a attaches to the SR Ca2+-ATPase and uncouples Ca2+ uptake from Ca2+-dependent ATP hydrolysis. Myotoxin a also prevented the formation of decavanadate-induced two-dimensional crystalline arrays of the SR Ca2+-ATPase.

Animals↗
PubMed1986

Reevaluation of hemorrhagic toxin, HR-I, from Agkistrodon halys blomhoffii venom: proof of proteolytic enzyme.

HR-I is a hemorrhagic toxin originally isolated from Agkistrodon halys blomhoffii (Mamushi) venom by Oshima et al. (1972). It was reported by the original investigators that it was nonproteolytic when casein was used as the substrate. HR-I was isolated again and proteolytic activity was tested using different substrates and assay methods. It is shown that HR-I is indeed a proteolytic enzyme hydrolyzing a number of peptide bonds. This present investigation suggests that more than one method should be used for proteolytic enzyme assay of hemorrhagic toxins. Toxicological and biochemical properties of HR-I were further investigated and are reported in this paper.

Amino Acids↗
PubMed1985

Interaction of myotoxin a with artificial membranes: Raman spectroscopic investigation.

Myotoxin a from the venom of Crotalus viridis viridis (prairie rattlesnake) is a small protein which is responsible for myonecrosis. It is a basic protein with 42 amino acid residues of known sequence. Three disulfide bonds give it a highly compact structure. Microscopic examination of the toxin's effects reveals that the most pronounced and earliest visible damage occurs intracellularly, in the sarcoplasmic reticulum membrane system of skeletal muscle. A better understanding of its mechanism of action is therefore of particular interest. The interaction of myotoxin a with artificial membranes (multibilamellar phospholipid dispersions) was investigated by using dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS). Two regions of the Raman spectrum were examined for information: the C-H stretching region between 2800 and 3000 cm-1 and the C-C stretching region between 1000 and 1300 cm-1. The effects of myotoxin a on the thermotropic phase behavior of the artificial membranes were determined. This was done by monitoring three structurally sensitive Raman intensity ratios, I2932/2880, I2880/2850, and I1088/1126. It was found that myotoxin alpha destabilized the ordered structure of the gel phase of phospholipid bilayers. This effect was seen with both DMPC and DMPS. The pretransition of DMPC was perturbed by myotoxin a, while the main gel to liquid-crystal phase transition temperature was decreased. The effect of myotoxin a on the phase behavior of DMPS was found to be pH dependent with the least effect observed at low pH values. These results suggest the involvement of negatively charged phosphate groups of phospholipids in the interaction of myotoxin a with artificial membranes.

Binding Sites↗
PubMed1985

Specificity of hemorrhagic proteinase from Crotalus atrox (western diamondback rattlesnake) venom.

Hemorrhagic proteinase, HTb, isolated from Crotalus atrox (western diamondback rattlesnake) venom was studied for its specificity. HTb showed fibrinogenase activity, hydrolyzing the A alpha chain of fibrinogen first, followed by the cleavage of the B beta chain. HTb is different from thrombin and did not produce a fibrin clot. The degradation products of fibrinogen were found to be different, indicating that the cleavage sites in the A alpha and B beta chains are different from those of thrombin. N-Benzoyl-Phe-Val-Arg-p-nitroanilide was not hydrolyzed by HTb, although this substrate was hydrolyzed by thrombin and reptilase.

Animals↗
PubMed1985

Proteolytic specificity of hemorrhagic toxin b from Crotalus atrox (western diamondback rattlesnake) venom.

In our effort to identify the proteolytic specificity of various hemorrhagic toxins isolated from western diamondback rattlesnake venom, hemorrhagic toxin b was isolated in homogeneous form by previously published methods. Hemorrhagic toxin b hydrolyzed glucagon, producing six fragments. The proteolytic sites were identified as Thr(5)-Phe(6), Thr(10)-Ser(11), Asp(15)-Ser(16), Asp(21)-Phe(22) and Try(25)-Leu(26). When oxidized insulin B chain was used, proteolysis occurred at four sites: Asn(3)-Gln(4), His(10)-Leu(11), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The proteolytic specificity of hemorrhagic toxin b is quite different from those of the nonvenom proteases such as thermomycolin, aspergillopeptidase c, alkaline protease from Aspergillus flavus, elastase, subtilisin and papain.

Animals↗
PubMed1985

The effect of tryptophan modification on the structure and function of a sea snake neurotoxin.

A neurotoxin has been isolated from the venom of the sea snake Lapemis hardwickii. This neurotoxin contains only one tryptophan residue which was chemically modified with two different reagents, 2-hydroxy-5-nitrobenzyl bromide and N-bromosuccinimide. Following modification, the neurotoxin almost completely lost its acetylcholine receptor-binding activity. To examine the effect tryptophan modification had on the conformation of the neurotoxin, circular dichroic and Raman spectra were taken of the modified neurotoxin and compared with those of the native toxin. The overall conformation of the modified neurotoxin was very similar to the native conformation. The Raman data also indicated some side chain conformations in the modified toxin were very similar to those of the native toxin. These data suggest that the tryptophan in sea snake venom short chain neurotoxins may have a direct role in the acetylcholine receptor binding process as well as a role in stabilizing the neurotoxin's active site in the proper conformation for optimum binding.

2-Hydroxy-5-nitrobenzyl Bromide↗
PubMed1985

Isotachophoretic analysis of bloodstains: differentiation of human, menstrual, bovine, and ovine bloods.

Isotachophoresis, a technique to separate components by constant current electrophoresis, was used to differentiate between bloodstains of male, female, menstrual, bovine, and ovine bloods on cotton cloth and filter paper. Bloodstain analysis by isotachophoresis of stains from male and female subjects showed identical cationic patterns, but gave different profiles in the anionic system. Plasma had one extra peak in the anionic system when compared to the profile of serum. This extra peak is due to the presence of fibrinogen in plasma. Some hemoglobin peaks overlapped with serum protein peaks, but these could be identified by comparisons at lower concentrations. Menstrual blood had a much different pattern than normal human blood as was expected since many more compounds are found in menstrual blood than in normally circulating blood. Human, bovine, and ovine bloodstains showed different profiles both in the cationic and anionic systems. These results indicate that isotachophoresis can be used for the rapid and simple analysis of bloodstains to differentiate reliably human male, female, and menstrual blood and also to distinguish human bloodstains from those of cattle or sheep.

Animals↗