Purification and characterization of a lethal toxin from the venom of Heloderma horridum horridum

A lethal toxin was isolated from the venom of Heloderma h. horridum by gel filtration and ion-exchange chromatography. Molecular weight of the purified toxin was determined to be 28 kDa under reducing and nonreducing conditions. Biological activity, assayed by i.v. routes of injection, shows an LD50 for this preparation of 0.135 micrograms/g. Additionally, the toxin possesses an inhibitory effect on direct electrical stimulation of the isolated mouse hemi-diaphragm. However, neither hemorrhagic nor hemolytic activities were detected. Phospholipase A2 activity, proteolytic activity and arginine esterolytic activity were absent. The amino acid composition of the lethal toxin and the NH2-terminal sequence up to residue number 33 were determined. Neither show similarities to other components from H. h. horridum venom.     

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

1. An arginine ester hydrolase was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel and Q-Sepharose column chromatography, resulting in 5.4 mg of purified enzyme from 320.0 mg of crude venom. 2. The enzyme was shown to be homogeneous by both SDS and non-SDS disc electrophoresis on polyacrylamide gel at pH 8.3. 3. The enzyme possesses arginine ester hydrolase and transglutaminase-like activities, but did not exhibit clotting activity. 4. Molecular weight was determined to be ca 29 kDa, with an isoelectric point of 4.4. 5. The enzyme was stable to heat treatment (95 degrees C, 10 min) and to pH changes over the range 2-11. 6. The arginine ester hydrolase was inactivated by diisopropylfluorophosphate (DFP), beta-mercaptoethanol and N-bromosuccinimide, suggesting that serine, disulfide bonds and tryptophan are involved in enzymatic activity. 7. Amino terminal sequences were determined and appear to be similar to porcine pancreatic kallikrein.     

Separation of Crotalus atrox (western diamondback rattlesnake) alpha-proteinase from serine proteinase and hemorrhagic factor activities

A method for obtaining Crotalus atrox alpha-proteinase (EC 3.4.24.1) in a pure form has been developed. Fractionation of the crude venom on DEAE-Sepharose, followed by gel filtration on Bio-Gel P-150 and chromatography on CM-Sepharose, yielded an alpha-proteinase preparation which showed a single band on disc and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had an activity on casein approximately twice that previously reported. The enzyme is a nonglycosylated single-chain polypeptide with a molecular weight of 26,738 and a pI of 8.15. Proteolytic activity on casein, alpha 1-antichymotrypsin, and Cl-inhibitor was abolished by treatment of alpha-proteinase with 1 mM EDTA, but full activity was retained in the presence of 1 mM phenylmethylsulfonyl fluoride. Caseinolytic activity was increased by 33 and 55% in the presence of 10 mM Mg2+ and Ca2+, respectively. Pure alpha-proteinase is devoid of esterolytic activity on H-D-Pro-Phe-Arg-p-nitroanilide (S-2302), benzoyl-L-arginine ethyl ester, and benzoyl-L-tyrosine ethyl ester. The final preparation has no hemorrhagic factor activity.     

Isolation and characterization of hemorrhagic toxin from the venom of Trimeresurus elegans

1. Hemorrhagic toxin from the venom of Trimeresurus elegans was purified in a homogeneous form using gel filtration and ion exchange chromatographies. 2. Hemorrhagic toxin possessed hemorrhagic and TAME (tosyl-L-arginine methyl ester) hydrolytic activities. These activities were inhibited when hemorrhagic toxin was incubated with benzamidine or N-bromosuccinimide. 3. Its mol. wt was 28,500 and the isoelectric point was 7.4. 4. This toxin contains ca 1.5 mol Ca per mol of protein.     

Beta-fibrinogenase from the venom of Agkistrodon p. piscivorus

1. Beta-fibrinogenase was isolated from the venom of Agkistrodon p. piscivorus by column chromatography on Sephadex G-100, DEAE-Sephacel and by chromatofocusing, with a yield of 2.5 mg of purified enzyme from 1 g of crude venom. 2. The enzyme was homogeneous by SDS and non-SDS disc electrophoresis on polyacrylamide gel at pH 8.3. 3. Beta-fibrinogenase is a glycoprotein possessing both TAME hydrolase and kinin-releasing activities. 4. A mol. wt of approximately 33,500 and an isoelectric point 4.5 was determined. 5. The enzyme is stable to heat treatment and to a pH range of 2-10. 6. Beta-fibrinogenase activity is inactivated by DFP, suggesting that serine is involved in the enzymatic activity. 7. The Michaelis constant (Km) of this enzyme for TAME and inhibition constant (Ki) for DFP were found to be 7.04 X 10(-3) and 4.13 X 10(-3) M, respectively.     

Biochemical characterization of hemorrhagic toxin from crotalus viridis viridis (prairie rattlesnake) venom

Hemorrhage, necrosis and edema are some of the effects often observed following snake bites. This paper reports studies on the isolation and biological properties of hemorrhagic toxin from Crotalus viridis viridis (Prairie rattlesnake) venom. A hemorrhagic toxin was isolated from C. v. viridis venom by Sephadex G-50, DEAE-Sephacel and Q-Sepharose column chromatographies.The hemorrhagic toxin from C. v. viridis venom was shown to be homogenous as demonstrated by a single band on polyacrylamide gel electrophoresis and immunodiffusion. Its molecular weight was approximately 54,000 dallons, and it contained 471 amino acid residues. The toxin possessed hemorrhagic activity with a minimum hemorrhagic dose (MHD) of 0.11 μ g, and hydrolytic activity on dimethylcasein, casein, azocasein, azoalbumin, azocoll and hide powder azure. Hemorrhagic and casein hydrolytic activities were inhibited by EDTA, o-phenanthroline or dithiothreitol. The toxin contained 1 mole of zinc per mole of protein and zinc is essential for both hemorrhagic and proteolytic activities. Hemorrhagic toxin possessed hydrolytic activity on the B-chain of insulin, which cleaves His(5)-Leu(6), His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25) bonds. This toxin also hydrolyzed Aα and Bβ chains of fibrinogen. Intramuscular injections of hemorrhagic toxin caused an increase of creatine phosphokinase activity in mice serum from 50.3 mU/ml to 1133 mU/ml. A toxin isolated from C. v. viridis venom was shown to have strong hemorrhagic activity. Partial characterization is reported for this major hemorrhagic toxin in C. v. viridis venom.     

Interaction of proflavine with 2-hydroxy-5-nitrobenzylated papain

Interaction of proflavine with papain, modified by hydroxynitrobenzylation of Trp-177 (HNB-papain), is characterized by a dissociation constant about twice that of proflavine's binding to native papain. Kinetic analyses revealed that proflavine's activation of papain-catalyzed hydrolysis of benzoyl-L-arginine ethyl ester persists with the HNB-enzyme; however, hydroxynitrobenzylation of papain precludes proflavine's inhibition of benzoyl-L-arginine p-nitroanilide (BzArgNan) hydrolysis. Yet, proflavine noncompetitively inhibits hydrolysis of BzPheValArgNan by both native and HNB-papain to about the same extent. Thus, proflavine appears to reduce nonproductive binding of smaller substrates, but may also interfere with conformational repositioning necessary for anilide hydrolysis.     

Characterization of Ac3-proteinase from the venom of Agkistrodon acutus (hundred-pace snake)

Ac3-Proteinase from the venom of Agkistrodon acutus was isolated in a homogeneous form by a previously published method. Ac3-Proteinase possessed lethal, hemorrhagic, caseinolytic, azocaseinolytic, dimethylcaseinolytic and hide powder azure hydrolytic activities. These activities were inhibited when Ac3-Proteinase was incubated with the metal chelators ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), tetraethylenepentamine (TEP), 1,10-phenanthroline, phosphoramidon or beta-mercaptoethanol. The toxin also hydrolyzed the oxidized A and B chains of both insulin and fibrinogen. The cleavage sites in the oxidized B chain of insulin were identified as His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25). The A alpha chain of fibrinogen was digested first followed by hydrolysis of the B beta chain. Toxicological and biochemical properties of Ac3-Proteinase were investigated further and are reported in this paper.     

Hemorrhagic toxin from the venom of Agkistrodon bilineatus (common cantil)

1. Hemorrhagic toxin was isolated from Agkistrodon bilineatus (Common cantil) venom using a three-step purification procedure to obtain 32.8 mg of purified hemorrhagic toxin from 700 mg of crude venom. 2. The purified toxin was homogeneous by disc polyacrylamide gel electrophoresis at pH 8.3, and by isoelectric focusing. 3. Hemorrhagic toxin possessed lethal, hemorrhagic and proteolytic activities. These activities of this toxin were inhibited by ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis-(beta-aminoethylether)N,N'-tetraacetic acid (EGTA), but not by cysteine or soybean trypsin inhibitor (SBTI). 4. Its molecular weight was approximately 48 kDa and the isoelectric point was 4.2. 5. Purified preparation hydrolyzed the Asn(3)--Gln(4), His(10)--Leu(11), Ala(14)--Leu(15), Tyr(16)--Leu(17), Arg(22)--Gly(23) and Phe(24)--Phe(25) bonds of oxidized insulin B. chain. 6. The A alpha chain of fibrinogen was first split and B beta chain was cleaved later by this toxin. 7. Hemorrhagic toxin contains 1 mol of zinc and 2 mol of calcium per mol of protein.     

Characterization of Ac1-Proteinase from the venom of Agkistrodon acutus (Hundred-pace snake) from Taiwan

1. Ac1-Proteinase from the venom of Agkistrodon acutus was isolated in a homogeneous form by a previously published method. 2. Ac1-Proteinase possessed lethal, hemorrhagic, caseinolytic, azocaseinolytic, azoalbumin hydrolytic and hide powder azure hydrolytic activities. 3. The toxin also hydrolyzed the oxidized B chain of insulin and fibrinogen. The cleavage sites in the oxidized B chain of insulin were identified as Ala(14)-Leu(15) and Tyr(16)-Leu(17). The A alpha chain of fibrinogen was digested. 4. Biological properties of Ac1-Proteinase were investigated further and are reported in this paper.     

Trypsin-like protease from soybean seeds. Purification and some properties

An enzyme was purified from soybean seeds mainly by repeated ion-exchange chromatography using benzoyl-L-arginine p-nitroanilide (BAPA) as a substrate. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The molecular weight was estimated as 59,000 by gel filtration. The enzyme was most active toward BAPA between pH 8 and 10. The enzyme was inactive toward protein substrates but hydrolyzed synthetic substrates and oligopeptides exclusively at the carboxyl side of L-arginine and L-lysine. Kinetic studies using synthetic substrates showed that, on the basis of Vmax/Km, the enzyme preferentially hydrolyzed amide substrates over ester substrates. Benzoyl-L-arginine 4-methylcoumaryl-7-amide (Bz-Arg-MCA) was the best substrate. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), tosyl-L-lysine chloromethyl ketone (Tos-Lys-CH2Cl), leupeptin, and antipain. p-Chloromercuribenzoate (PCMB) was only partially inhibitory. Various protein inhibitors of trypsin such as soybean trypsin inhibitor were ineffective. From the primary specificity and susceptibility to chemicals, the enzyme can be said to be a trypsin-like serine protease. Although the physiological role of the enzyme is unclear, it seems likely that it is involved in limited hydrolysis of certain physiological peptides during processing.     

Use of an IgG fragment prepared with particulate plasmin to study the C1 binding and activation.

Facb, fragment antigen and complement binding (this last property is shown when the fragment is involved in an immune complex); Fc, C-terminal half of the heavy-chain dimer; pFc′, major C-terminal fragment released from IgG by pepsin or plasmin digestion; DFP, diisopropylphosphofluoridate; ATEE, N-acetyl-L-tyrosine ethyl ester; BAEE, N-benzoyl-L-arginine ethyl ester; TAME, p-toluene sulfonyl-L-arginine methyl ester; SDS, sodium dodecyl sulfate; SBTI, soybean trypsin inhibitor.The nomenclature of the complement components (C1, C1q, C1r, C1s) follows the W.H.O. recommendations. Enzymatic activities are expressed in nanokatals (nkat.) as recommended in the Enzyme Nomenclature (1973).     

Heat-stable extracellular proteolytic enzyme produced by Thermus caldophilus strain GK24, an extremely thermophilic bacterium

Proteolytic activity was detected in the culture supernatant of a newly isolated, extremely thermophilic bacterium belonging to the genus Thermus, and tentatively named T. caldophilus sp. n. strain GK24. The enzyme activity continued to increase for at least three days after cells reached the stationary phase of growth. Purification of the proteolytic enzyme was tried with ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. The most purified enzyme fraction thus obtained appeared to be homogeneous in a chromatographic analysis, but still had seven bands of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Treatment of the protease with denaturing reagents or organic solvents did not alter the chromatographic profile and the purified enzyme sample showed a large sedimentation coefficient of about 11S. The optimal pH of the hydrolytic activity of the enzyme was observed at around 7.8 for casein and 7.2 for N-carbobenzoxy-L-leucyl-L-tyrosinamide (Z-Leu-Tyr-NH2). The enzyme was stable in the pH range of 5 to 11 for 1 day at 4 degrees C or for 1 h at 70 degrees C. The enzyme sample showed a maximal activity at 90 degrees C and had an extreme stability toward treatment by heat and denaturing reagents. The enzyme sample was inactivated almost completely by diisopropyl fluorophosphate (DFP), but not by ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA). From these results, the enzyme seems to be a serine protease, and not to be a metallo-enzyme such as thermolysin. The enzyme also was hydrolytic active toward an ester compound, N-benzoyl-L-tyrosine ethyl ester (BTEE), but not toward N-benzoyl-L-arginine ethyl ester (BAEE).     

Isolation of human thrombin using affinity chromatography on gramicidin C-silochrome C 80

Thrombin purification is conducted by biospecific chromatography on gramicidin C-silochrome C 80. Preparations possessing the fibrinogen-coagulating activity of 2500-3200 NIH units per 1 mg of protein and containing 98% of active sites are obtained. Data obtained from electrophoresis in PAAG with the presence of DS-Na show the alpha-thrombin content to be 96%; the admixture of beta-thrombin possessing no coagulating activity does not exceed 4%. The kinetic constants are presented for thrombin hydrolysis of tosyl-L-arginine methyl ester (TAME), benzoyl-L-arginine ethyl ester (BAEE) and chromogenic substrate S-2238. The addition of isopropanol increases sharply the stability of thrombin when storing it in the aqueous-salt solutions.     

Identification of serine esterases in tissue homogenates

Serine esterases react with [3H]diisopropylphosphofluoridate ([3H]DFP) to produce radioactive adducts that can be resolved by denaturing slab gel electrophoresis. To identify an esterase or its catalytic subunit, a potential substrate was included in the reaction mixture with the expectation that it would suppress the enzyme's reaction with [3H]DFP. The nature of the enzyme could be inferred from the character of the substrates that suppress labeling. The validity of this analytical method was tested with two serine proteases, trypsin and alpha-chymotrypsin, and two serine esterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and several of their natural or model substrates or inhibitors. Application of the method to complex biological systems was tested with chicken embryo brain microsomes. Trypsin labeling with [3H]DFP was suppressed by alpha-N-benzoyl-l-arginine ethyl ester (BAEE) and poly-l-lysine but not by benzoyl-l-tyrosine ethyl ester (BTEE). [3H]DFP labeling of chymotrypsin was suppressed by both BAEE and BTEE. Labeling of AChE and BuChE was suppressed by their natural and some related substrates and inhibitors. [3H]DFP reacted with brain microsomes to produce nine distinct radioactive bands. When the relevant substrates and inhibitors of AChE were included in the reaction mixtures, labeling of only the 95-kDa band was suppressed, implicating it as AChE. Labeling of the 85- and 79-kDa bands was inhibited by butyrylcholine, suggesting that these proteins have BuChE activity.     

The requirement for esterase activation in the anti-immunoglobulin-triggered movement of B lymphocytes.

In this paper we bring evidence suggesting that there is activation of an esterase upon reaction of anti-immunoglobulin antibodies (anti-Ig) with murine B lymphocytes. B lymphocytes upon exposure to anti-Ig cap the ligand-receptor complexes and immediately afterward become briefly motile. It is this latter step which is inhibitable by exposure to di-isopropyl phosphofluoridate (DFP). Various experimental manipulations indicated that treatment with anti-Ig activates the cell for motility which, however, is not manifested until the temperature is raised to 37 degrees C. The cell incubated with anti-Ig at cold temperatures becomes susceptible to the effect of DFP, suggesting that the antibody-treated cells are activated up to but not beyond the DFP inhibitable step. Exposure of cells to DFP and removal of it before their treatment with anti-Ig does not affect the anti-Ig-induced response. Four lines of evidence indicate that the reduction of lymphocyte movement of DFP is due to the inhibition of an esterase activated by the combination of antibody and cell: 1) The inhibition by DFP is irreversible; once DFP has reacted it can be washed away and the antibody-treated cell is still inhibited. 2) The inhibition increases with time of contact of lymphocytes and DFP and with the concentration of DFP. 3) A very poorly phosphorylating phosphonate, phenyl ethyl pentylphosphonate is completely inactive under conditions where an excellent phosphorylating phosphonate, p nitrophenyl ethyl pentylphosphonate maximally inactivated the cells' movement. 4) The amino acid esters, tosyl L arginine methyl ester and benzoyl arginine methyl ester specifically prevent the inactivation by DFP. The last finding suggests that tosyl L arginine methyl ester and benzoyl L arginine methyl ester might be substrates for the putative antibody-induced lymphocyte esterase. Lymphocytes incubated with antibody in the cold for more than 30 min lose their ability to move when the temperature is raised, suggesting that there is a time-dependent deactivation of the cell.     

Thermodynamic bases for fatty acid ethyl ester synthase catalyzed esterification of free fatty acid with ethanol and accumulation of fatty acid ethyl esters

Myocardial homogenates rapidly synthesize fatty acyl ethyl esters from nonesterified fatty acid and ethanol in the absence of coenzyme A or ATP, and the enzyme catalyzing this reaction, fatty acid ethyl ester synthase, has been purified 5400-fold to homogeneity [Mogelson, S., & Lange, L. G. (1984) Biochemistry (preceding paper in this issue)]. To define the factors permitting this de novo synthesis of ester bonds and the consequent accumulation of fatty acyl ethyl esters in myocardium, we determined thermodynamic parameters relevant to the kinetics and equilibria of this reaction and specifically characterized (1) the rates of synthesis of ethyl oleate, in both the presence and absence of purified enzyme catalyst, and (2) the physical properties of the product, ethyl oleate, in an aqueous milieu. Compared to the reaction of ethanol and oleate in the absence of catalyst, fatty acid ethyl ester synthase enhanced the rate of ethyl oleate synthesis by reducing the free energy of activation (delta G) from 32.5 to 19.9 kcal/mol, effected in large part by a positive entropy shift, delta Senz - delta S uncat = 23.9 cal/(mol.deg). Rate constants in the presence and absence of enzyme at 37 degrees C were 6 X 10(-2) s-1 and 7.8 X 10(-11) M-1 s-1, respectively, indicating a catalytic power of at least 10(8)M for this enzyme. Kinetic data indicated an enzymatic Vmax of 1.25 nmol/(mg.s) (37 degrees C). The equilibrium constant was calculated for the reaction oleate + ethanol in equilibrium ethyl oleate and was 0.095 M-1 at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on urinary kallikreins. I. Purification and characterization of human urinary kallikreins.

Human urinary kallikrein [EC 3.4.21.8] (HUK) was purified about 200-fold with an overall yield of 40 percent from crude powder by DEAE-cellulose chromatography, acetone fractionation, Sephadex G-100 gel filtration and DEAE-Sephadex A-50 chromatography. Its activity was 200 kallikrein units (KU) per A280. HUK from active fractions obtained by DEAE-Sephadex A-50 chromatography was separated into three active components showing isoelectric points of 3.9 (HUK-1), 4.0 (HUK-2), and 4.2 (HUK-3) by isoelectric focusing: each HUK component was homogeneous on disc electrophoresis. The approximate molecular weights of HUK-1, -2 and -3 were estimated to be 2.7 X 10(4), 2.7 X 10(4), and 2.9 X 10(4), respectively, by gel filtration on a Sephadex G-100 column. The optimum pH's of HUK-1, -2, and -3 in esterolytic action were found to be 8.0, 8.3, and 7.5, respectively, and they were fairly heat stable in comparison with other glandular kallikreins. The three components of HUK were weakly inhibited by Trasylol, but were not affected by soybean and ovomucoid trypsin inhibitors. They were strongly resistant to treatment with urea and weakly resistant to treatment with guanidine. The activation energies of HUK-1, -2, and -3 were found to by 1.17 X 10(4), 5.1 X 10(3), and 1.45 X 10(4) cal per mole, respectively. The Km values were estimated toward N-alpha-tosyl-L-arginine methyl ester (TAME), N-alpha-benozyl-L-arginine ethyl ester (BAEE), and N-alpha-benozyl-L-arginine methyl ester (BAME).     

Comparative study of Agkistrodon acutus venoms from China and Taiwan

Three hemorrhagic toxins (Ac1-, Ac2- and Ac3-proteinases) were isolated from the lyophilized venom of Agkistrodon acutus from China using gel filtration on a Sephadex G-75 column, followed by chromatography on diethylaminoethyl (DEAE)-Sephadex A-50, DEAE-Sephacel and DEAE-cellulose. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis. Three hemorrhagic toxins possessed both lethal and proteolytic activities. These activities were inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol (beta-aminoethyl)N,N,N',N'-tetraacetic acid (EGTA), o-phenanthroline or cysteine, but not by soybean trypsin inhibitor (SBTI), p-chloromercuribenzoate (PCMB) or diisopropyl fluorophosphate (DFP). Molecular weight of hemorrhagic toxins (Ac1-, Ac2- and Ac3-proteinases) were determined to be 24,500, 25,000 and 57,000, respectively. It was found that hemorrhagic toxins have considerable similarity to hemorrhagic toxins isolated by Nikai et al. (1977) and Sugihara et al. (1978, 1979), but Ac2- and Ac3-proteinases from China and Taiwan are electrophoretically unrelated.     

Purification and characterization of alpha-toxin of Clostridium oedematiens type A

The alpha-toxin of Clostridium oedematiens type A was purified from culture filtrate by two steps of column chromatography and repeated gel filtration. The purified alpha-toxin proved homogeneous in polyacrylamide gel electrophoresis and agar gel double diffusion. The molecular weight of the alpha-toxin was estimated at 280,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and at 260,000 by gel filtration on a Sephadex G-200 column. The isoelectric point determined by isoelectric focusing polyacrylamide gel electrophoresis was 6.1. No dissociation of the purified alpha-toxin into subunits was demonstrated in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 50% lethal and edematizing doses per mg protein of the purified alpha-toxin were 5.9 X 10(4) and 5.9 X 10(5), respectively. The L +/50 doses per mg protein of the toxin was 4.6 X 10(3). The purified alpha-toxin, when injected intradermally into the rabbit skin, induced increased vascular permeability. The toxin contained little or no hemolytic or lecithinase activity. These results attest that the lethal, edematizing and vascular permeability-enhancing activities elicited by C. oedematiens type A culture reside on the same protein molecule.