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1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, hyaluronidase, alkaline phosphomonoesterase, 5'-nucleotidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 26 samples of venoms of 13 taxa of Vipera were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. The results indicate the presence of certain common characteristics among the venoms, particularly if V. russelli is excluded from the comparison. The results also support the recently proposed reassignment of V. russelli to a separate genus. 3. The data show that information on venom biological properties can be used for differentiation of venoms of many species of Vipera. Particularly useful for this purpose are the protease, phosphodiesterase, phospholipase A and the procoagulant activities and the Sephadex G-75 gel filtration patterns of the venoms.

We studied the interaction of the antipsoriatic compound anthralin (1.8-dihydroxy-9-anthrone), and its metabolites anthraquinone (1.8-dihydroxy-9.10-anthraquinone) and anthralin dimer (1.8.1'.8'.-tetrahydroxy-10.10'-bis-9[10]-dianthrone) with the inner mitochondrial membrane. Mitochondrial membrane functions such as ubiquinone redox equilibria, redox status of iron sulfur clusters, cyanide-sensitive and cyanide-insensitive oxygen consumption, adenosine triphosphate (ATP) synthesis, ATP hydrolysis, and adenine nucleotide content of mitochondria were analyzed. Anthralin is an inhibitor of mitochondrial oxygen uptake in the presence of ADP and substrate (cyanide-sensitive respiration), inhibits ATP synthesis without affecting ATP hydrolysis, and depletes mitochondria of ATP. Anthralin dimer is a much weaker inhibitor of mitochondrial functions and anthraquinone is almost inactive. Anthralin, but not anthraquinone and anthralin dimer, reverses uncoupler stimulated oxygen consumption, stimulates cyanide-insensitive respiration, reduces mitochondrial ubiquinone-9 and -10 to the corresponding ubiquinols and reduces mitochondrial iron sulfur clusters. Anthralin may induce formation of reactive oxygen species by enhancing autoxidation of mitochondrial components and/or by catalyzed oxidation of anthralin. Taken together, anthralin acts as an electron donor to inner mitochondrial membrane associated redox components, inhibits the electron transport chain, and has an oligomycin-like effect. Anthralin dimer and anthraquinone do not function as electron donors and act by a different reaction mechanism. Respiratory measurements in human keratinocytes revealed similar results as obtained with isolated mitochondria. We suggest that modulation of membrane redox status may be a common concept of anthralin action in target cells such as keratinocytes and neutrophils.

The influence of pH and temperature on the kinetic properties of esterase E-II from Bitis gabonica venom has been studied. The pH profiles for the hydrolysis of N alpha-benzoyl-L-arginine ethyl ester showed that a group with pK approximately 7 must be ionized for activity. Measurement of N alpha-benzyl-L-arginine-p-nitroanilide hydrolysis reveals that the pK of the active site is significantly lowered (i.e. to approximately 6.3) in the enzyme-substrate complex. The temperature effect on the pK suggests the presence of a carboxylate in the active site of the enzyme. This suggestion is corroborated by the influence of organic solvent perturbations of the pK indicating a catalytic group of the neutral acid type. Values for the thermodynamic parameters associated with activation are reported. The transition state for ester degradation is at lower delta G and delta H levels than that for amide degradation. delta H and delta S for ester hydrolysis were noted to compensate each other with a compensation temperature Tc = 339K. Compensation can probably be related to weak bonding effects.

Binding studies with the structurally similar vasoconstrictor peptides 125I-endothelin and 125I-sarafotoxin b, the former of mammalian origin and the latter derived from snake venom, reveal their mutually exclusive binding to rat atrium and various regions of the rat brain. In these tissues endothelin, like sarafotoxin, induces phosphoinositide hydrolysis which is in part Ca2+-independent. It is suggested that endothelins and sarafotoxins share common binding sites and mechanisms of action.

The intracellular transport of venom proteins has been studied in active and resting venom glands of the snake Vipera palaestinae by electron microscope radioautography after an intra-arterial injection of [3H]leucine. In the active gland, most of the label is initially (10 min) found over the RER. By 30 min, the relative grain density of the Golgi complex reaches its maximum, with concomitant increase in the labeling of the condensing vacuoles. Later on, a steep increase in radioactivity of the secretory granules is observed. At 3 h, these granules, which comprise about 2% of the cell volume, contain 22% of the total grains. At the following hour, their labeling declines and at the same time the radioactivity of the secreted venom is increased. It is concluded that, in the active cell, venom proteins are transported via the Golgi apparatus into membrane-bounded granules which are the immediate source of the secreted venom. An alternative pathway, which involves the RER cisternae as a storage compartment, seems unlikely, since incorporated label does not accumulate in this compartment after prolonged postpulse intervals. The route of intracellular transport of proteins in the resting glands is similar to that of the active ones, but the rate of synthesis and transport is much slower. The present results and earlier data, thus, show that the increase in the rate of secretion after initiation of a new venom regeneration cycle is the result of accelerated rates of both synthesis and transport.

Several phospholipases A could be isolated from the venom of the European viper, Vipera ammodytes, having different specific activities toward egg lecithin and different lethalities. The most lethal of these enzymes is fraction "k2" having an intravenous LD50 for white mice of 0.021 mg per kg and a specific activity of 280 microM/min mg at 40 degrees C. The enzyme could be labeled with 131I without loosing its enzymatic activity and lethality. The passage of this enzyme from blood into cerebrospinal fluid (CSF) was followed in anesthetized cats. Approximately 1% of the blood level of the enzyme was found in CSF indicating the ability of this protein to penetrate the blood-brain barrier. Although the lethality of fraction "k2" becomes as low as 0.085 microgram/kg when applied intraventricularly, it is not very likely that the central effects of this fraction are of major importance in envenomation since the distribution pattern of the labeled enzyme shows that most of the protein remains in liver, lungs and kidneys, presumably non-selectively bound to membranes and only 0.2% of the injected fraction can reach the brain. Relatively high amount of enzyme was also found in the diaphragm. The penetration of the blood-brain barrier of the radiolabeled phospholipase is within the limits for the proteins of this size (M.w. 14500).

Puff adder venom contains a protease capable of cleaving the gamma-chain of cross-linked D-dimer, derived from the plasmin digestion of fibrin, into apparently symmetrical monomers. The cross-linked gamma-chains are separated in the process without apparent loss of mass and without loss of the substituent at the glutamine cross-link site, if fluorescent D-dimer (the lysine analogue dansylcadaverine used as substituent) is used as substrate [Purves, L. R., Purves, M., Lindsey, G. G., & Linton, N. J. (1986) S. Afr. J. Sci. 82, 30]. The gamma-chain from puff adder venom digested D-monomer was isolated and cleaved by cyanogen bromide, and the carboxy-terminal peptide was isolated and sequenced. The carboxy-terminal peptide composition indicated a lower content of histidine, leucine, and glycine than expected. Manual microsequencing by gas-phase Edman degradation demonstrated that two amino-terminal ends were present. By use of the known sequence of the human fibrinogen gamma-chain, the sequencing data could be resolved into a dipeptide cross-linked between lysine-406 and either glutamine-398 or -399 (residues 6 and 13 or 14 from the carboxy-terminal end of the gamma-chain) with the loss of residues 401-404 that occur between the cross-link sites of both antiparallel cross-linked gamma-chains. D-dimer is therefore separated into monomers by cleavage of the gamma-chain between the cross-link sites. Two symmetrical fragments are produced consisting of a cross-linked dipeptide with the loss of four amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) family were characterized in newborn rat heart myocytes using human and rat endothelins (ET-1 and ET-3, respectively), SRTX-b and SRTX-c. Binding studies in intact cells and homogenates revealed significantly higher affinities of ET-1 and SRTX-b than of ET-3 and SRTX-c towards these receptors. This binding profile of ET/SRTX peptides points to their interaction with the receptor subtype designated E-S alpha. All four peptides induced time- and dose-dependent phosphoinositide hydrolysis with the following rank order of potency: ET-1 greater than SRTX-b greater than SRTX-c greater than ET-3. Thus, ET-3 which possesses an intermediate affinity toward the receptor was the least effective with regard to this response. These results confirm and extend our earlier report that the ET/SRTX peptides interact with a newly characterized receptor(s) associated with phosphoinositide metabolism and Ca2+ mobilization. The initiation of inositol phosphate formation is largely independent of extracellular Ca2+, verapamil and nifedipine, indicating that the ET/SRTX peptides are not agonists for the voltage-dependent Ca2+-channels.

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) families were detected in the rat uterus. These receptors specifically bind 125I-SRTX-b (Bmax = 220 fmol/mg protein), as well as ET-1, ET-3 and SRTX-c (IC50's 10, 5, 300 and 780 nM, respectively). Activation of the uterine ET/SRTX receptors induced dose-dependent phosphoinositide (PI) hydrolysis and three typical contractile responses: 1) increase in the muscle tonic tension; 2) increase in frequency of the spontaneous rhythmic contractions; 3) decrease of relaxation in each spontaneous rhythmic cycle. All three effects appeared at doses as low as 0.5-1 nM. Dose responses yield ED50 values of 5.5, 30 and 680 nM for ET-1, SRTX-b and ET-3, respectively. SRTX-c was the least effective peptide in achieving decrease in relaxation. In view of these results, and since the uterine responses to the peptides were almost immediate and reversible, we suggest that the functional ET/SRTX receptor of the rat uterus that is coupled to PI hydrolysis may be of physiological significance.

The total yield of venom (desiccated) in 25 adult V. russelli (mean length 111 +/- 1.8 cm (S.E.) ranges from 21-268 mg (127 +/- 13 mg, mean +/- S.E.) and that of 13 juvenile snakes (mean length 79 +/- 2.8 cm) is 8-79 mg (45 +/- 7 mg). The venom yield per milking correlated well with the length of the snake. The average amount of venom injected in the first bite of 31 adults (mean length 107 +/- 1.4 cm) is 63 +/- 7 mg (range 6-147 mg) and by 17 juvenile snakes (mean length 83 +/- 1.1 cm) is 41 +/- 8 mg (range 3-138 mg). Adults inject 45% of the glands' content in the first bite. More than 75 mg of venom (desiccated) was injected by 11 out of 31 adults and 2 out of 17 juvenile snakes. This indicates that in a substantial proportion of cases of envenomation, 40 ml of Burma Pharmaceutical Industry monovalent antivenom, which is commonly used, may not be adequate.

The indirect immunofluorescent antibody technique was applied to the study of Echis carinatus pyramidum venom antigens in venom gland tissue using semi-thin frozen sections. A total of four rabbit antisera, two monoclonal antibodies active against E. carinatus venom, two monoclonal antibodies active against the rodent malaria parasite, Plasmodium chabaudi, and two monoclonal antibodies active against the human malaria parasite, Plasmodium falciparum, were investigated. The results of this study suggest that each secretory cell within the main part of the gland produces all the venom constituents. The resultant venom is therefore considered to be produced as a single package by each individual secretory cell. The different constituents of the venom studied are not produced at the same time or at the same rate throughout the secretory cycle, some being produced at the beginning and others at a later stage.

Preparations of phosphodiesterase from Vipera lebetina venom immobilized on agarose were obtained. The kinetic properties for the hydrolyses of various substrates of soluble and immobilized phosphodiesterase, e. g. effects of pH, temperature, substrate concentrations, etc., were compared. The values of Km, V and activation energy for the substrate hydrolyses were determined.

We have recently shown that the rat atrium and brain contain specific high affinity receptors for the novel snake vasoconstrictor peptide sarafotoxin-b (SRTXb), and demonstrated toxin-induced phosphoinositide hydrolysis. Here we report on the characteristics of 125I-SRTXb receptors and their regional distribution in rat brain. 125I-SRTX receptors in the rat brain bind the toxin rapidly and with high affinity. The binding was not inhibited by ligands of known neurotransmitter receptor and ion channels. 125I-SRTX receptors have a distinctive regional distribution. The highest densities were observed in the cerebellum, thalamus and hypothalamus (850, 550 and 450 fmol/mg protein, respectively) and the lowest densities in the caudate and cerebral cortex (82 and 62 fmol/mg protein, respectively). Taken together our results suggest that mammalian brains contain a hitherto undetected neuroreceptor that may operate in neurotransmission with a "SRTX-like" brain peptide, similar to the SRTX homologous vasoconstrictor peptide of the mammalian endothelium endothelin.

Sarafotoxins, a group of 21-residue cardiotoxic peptides from snake venom that induce coronary vasoconstriction, show high-affinity binding to rat atrial and brain membranes and activate the hydrolysis of phosphoinositides. Neither their binding nor their activity is affected by blockers or activators of known receptors and ion channels, suggesting that sarafotoxins act either directly on the phosphoinositide phosphodiesterase system or on a novel receptor. Their amino acid sequence shows a high degree of homology with that of endothelin, a recently described 21-residue vasoconstrictor peptide found in porcine aortic endothelium. This is remarkable, since endothelin is a natural compound of the mammalian vascular system while sarafotoxins are highly toxic components of snake venom.

The interaction of snake venom phospholipases A2 with phospholipids has been studied by intrinsic fluorescence. This has been performed in order to understand why some enzymes possess anticoagulant properties while others have no action on blood clotting. We show that phospholipases A2 can be distinguished according to their binding properties to phospholipid vesicles. Strong inhibitors of coagulation interact with phospholipids with a significant change of their fluorescence while poor inhibitors have little or no effect. Strong inhibitors have a great affinity toward phosphatidylserine and do not require Ca2+ for interaction. Similar results are obtained with phosphatidylcholine-phosphatidylserine 1:1 mixtures. The diether analogue of phosphatidylcholine shows that formation of the complex is promoted by Ca2+ and can occur whenever the lipids are in crystal or fluid phase. Inactivation of anticoagulant phospholipase A2 decreased the affinity of enzyme for the phospholipids. The change in the intrinsic fluorescence of the phospholipases A2 on binding indicates a modification of the environment of the tryptophan residues. This is discussed in terms of the so-called interface recognition site as seen in the case of pancreatic phospholipase A2. It is concluded that the phospholipases may inhibit coagulation by competing with clotting proteins for the lipid surface. Although not considered in this study, the consequence of the hydrolysis of lipids remains to be estimated.

Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated 125I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa-tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of 125I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. Kallikrein did not cleave the zymogen. Nonactivation cleavage was noted by thrombin, but only in the absence of calcium. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In contrast, incubation of factor IX with elastase (Takaki A et al, J Clin Invest 71:1706, 1983) or chymotrypsin did not lead to generation of an antithrombin III-binding site, despite their digestion of 125I-factor IX into heavy and light chain-sized fragments. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.

The kinetics of I125-labelled venom and antivenom of Russell's viper (Vipera russelli) were determined following i.v. administration in mice. The kinetic profiles follow a two-compartment open model with mean distribution half-lives of 15.93 and 15.36 min, and mean elimination half-lives of 8.88 and 5.37 hr for venom and antivenom, respectively. Antivenom follows closely that of venom in its mode of distribution while its elimination is almost twice as fast as that of venom. The steady-state distribution ratio between blood and tissue indicates a greater affinity of the venom to the tissues as compared to antivenom. The primary route of elimination of venom and antivenom or their degraded products was via the urine. Small amounts of trichloroacetic acid-precipitable radioactivity and radioimmunoassayable venom and antivenom recovered in the urine suggest that very little intact venom or antivenom was excreted in the urine. Kinetic studies using unlabelled venom produced values comparable to those obtained with the labelled venom. The results emphasize the importance of maintaining optimal serum antivenom levels, by timely and repeated administration instead of a single bolus dose, in the management of snake envenoming.

A gene (Ecs) encoding a platelet aggregation inhibitor, echistatin (Ecs), has been chemically synthesized. Met at position 28 of the native protein was replaced by Leu in the recombinant Ecs. To express this synthetic gene in Escherichia coli, an expression vector, pJC264, was constructed by inserting portions of the E. coli cheB and cheY gene complex into the plasmid pUC13. High-level expression of the synthetic [Leu-28]Ecs was achieved by its fusion with the E. coli cheY gene in the expression vector. Recombinant [Leu-28]Ecs was liberated from the fusion protein by CNBr cleavage at the Met inserted between the CheY protein and [Leu-28]Ecs. The recombinant [Leu-28]Ecs was purified to homogeneity by reverse-phase high-performance liquid chromatography. The refolded [Leu-28]Ecs was identical to native Ecs in inhibiting platelet aggregation, suggesting that Met at position 28 is not essential for the biological activity of this platelet aggregation inhibitor.

Puff adder venom, which has been pretreated with phenylmethylsulphonyl fluoride and extensively dialysed, is capable of destroying selectively proteinase inhibitory activity in human plasma by an action of an EDTA-sensitive venom proteinase. We found that incubation of 1/5 vol. of such venom with human plasma at 25 degrees C leads to a concomitant increase in renin to 4.4 times control by 5 h. The activation of inactive renin was abolished by 10 mM EDTA and the rate of activation was reduced by 50% in the presence of 5 mM phenylmethylsulphonyl fluoride and by 90% when 0.32 mg/ml soybean trypsin inhibitor and 5 mM N-ethylmaleimide were added as well. The venom proteinase thus appears to activate inactive renin via an activation of endogenous plasma proteinases. This may be accomplished either by activation of proteinase precursors or action on proteinase inhibitor-proteinase complexes. By destroying proteinase inhibitors at the same time as it activates endogenous proteinases, Bitis arietans metalloproteinase would appear to be particularly useful for studies of the activation of inactive renin in human plasma, since endogenous proteinases are then free to activate inactive renin without subsequent inhibition by endogenous proteinase inhibitors.

Incubation of human plasma inter-alpha-trypsin inhibitor with crotalid, viperid, colubrid or elapid venoms resulted in random cleavage of the intact inhibitor (200,000 mol. wt) and formation of inhibitor of 130,000, 77,000, 58,000, and 38,000 mol. wt, along with several minor products. The overall patterns of digestion varied among the venoms studied. However, a 77,000 mol. wt inhibitor cleavage product was formed by all venoms tested, and this fragment was resistant to proteolysis even after a 24 hr incubation with the venoms. Venom pre-treated with phenylmethylsulfonyl fluoride digested inter-alpha-trypsin inhibitor; however, pre-treatment with EDTA completely stopped the reaction, indicating that venom metalloproteinases were responsible for the inhibitor digestion. The inhibitor cleavage products retained the ability to inhibit trypsin, but had no inhibitory activity against venom proteinases.

A kinin-releasing enzyme was isolated from Bitis arietans (puff adder) venom by Sephadex G-100 and DEAE-cellulose column chromatographies. The kinin-releasing enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion. Its molecular mass is approximately 45 kDa with an isoelectric point of 6.5. Kinin-releasing enzyme possesses proteolytic activity which hydrolyzes the Leu6-Cys7, His10-Leu11 and Ala14-Leu15 bonds of the B chain of oxidized insulin and the A alpha and B beta chain of fibrinogen. Kinin-releasing and benzoyl-L-arginine ethyl ester hydrolytic activities of this enzyme were inhibited by diisopropyl fluorophosphate, suggesting that the serine hydroxyl group is involved in enzymatic activities.