Effects of nerve growth factors from mouse salivary glands and snake venom on the sympathetic ganglia of neonatal and developing mice.
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1. Quantitative histological analysis has been made of the effects of nerve growth factor (NGF) from mouse submaxillary gland and from the venom of Vipera russelli on superior cervical ganglia of neonatal mice. 2. The hypertrophic and hyperplastic effects reported by other workers have been confirmed. 3. The hypertrophic effect arises from an increase in the rate at which the sympathetic neurones attain their mature size. The size at maturity is never exceeded. 4. The hyperplastic effect arises from an increase in the rate of production of neurones from less differentiated cells. In the developmental period, the number of neurones can exceed that found at maturity. If injection of NGF is discontinued the excess neurones disappear. 5. If injection of NGF is continued to maturity, the excess number of neurones is maintained. (+info)
Purification and characterization of a new RGD/KGD-containing dimeric disintegrin, piscivostatin, from the venom of Agkistrodon piscivorus piscivorus: the unique effect of piscivostatin on platelet aggregation.
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Piscivostatin, a novel dimeric disintegrin containing Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD) sequences, was isolated from the venom of Agkistrodon piscivorus piscivorus. The molecule consisted of two chains designated as the alpha and beta chains, comprising 65 and 68 amino acid residues, respectively. Piscivostatin had two binding motifs recognized by platelet glycoprotein IIb/IIIa (GPIIb/IIIa), and the biological activity of dimeric disintegrin piscivostatin toward platelet aggregation differed from those of other monomeric disintegrins such as trimestatin and echistatin. We measured platelet aggregation by the laser light scattering method during the process of ADP-induced platelet aggregation. Both dimeric and monomeric disintegrins inhibited the formation of small (9 to 25 microm in diameter), medium-sized and large aggregates (25 to 70 microm in diameter) in a dose-dependent manner. The platelet aggregates disaggregated after reaching a maximal number on either treatment with ADP alone or monomeric disintegrin/ADP. However, the small aggregates did not disaggregate on treatment with piscivostatin/ADP even when applied over time. When washed platelets were incubated with an anti-GPIIb/IIIa monoclonal antibody, PT25-2, which induces conformational changes of GPIIb/IIIa to a form accessible to fibrinogen and other adhesion proteins without platelet activation, piscivostatin induced a platelet shape change alone with no aggregate formation. The present study indicated that piscivostatin has two unique contradictory activities; acting as a double inhibitor of platelet aggregation and platelet aggregate dissociation. (+info)
Solution structure of a Kunitz-type chymotrypsin inhibitor isolated from the elapid snake Bungarus fasciatus.
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Bungarus fasciatus fraction IX (BF9), a chymotrypsin inhibitor, consists of 65 amino acid residues with three disulfide bridges. It was isolated from the snake venom of B. fasciatus by ion-exchange chromatography and belongs to the bovine pancreatic trypsin inhibitor (BPTI)-like superfamily. It showed a dissociation constant of 5.8 x 10(-8) m with alpha-chymotrypsin as measured by a BIAcore binding assay system. The isothermal titration calorimetry revealed a 1:1 binding stoichiometry between this inhibitor and chymotrypsin and apparently no binding with trypsin. We further used CD and NMR to determine the solution structure of this venom-derived chymotrypsin inhibitor. The three-dimensional NMR solution structures of BF9 were determined on the basis of 582 restraints by simulated annealing and energy minimization calculations. The final set of 10 NMR structures was well defined, with average root mean square deviations of 0.47 A for the backbone atoms in the secondary structure regions and 0.86 A for residues The side chains of Phe(23), Tyr(24), Tyr(25), Phe(35), and Phe(47) exhibited many long-range nuclear Overhauser effects and were the principal components of the hydrophobic core in BF9. To gain insight into the structure-function relationships among proteins in the BPTI-like superfamily, we compared the three-dimensional structure of BF9 with three BPTI-like proteins that possess distinct biological functions. These proteins possessed similar secondary structure elements, but the loop regions and beta-turn were different from one another. Based on residues at the functional site of each protein, we suggest that the flexibility, rigidity, and variations of the amino acid residues in both the loop and beta-turn regions are related to their biological functions. (+info)
Amino acid sequence of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake).
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The complete amino acid sequence of notexin, a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake), has been elucidated. The protein consists of a single chain of 119 amino acids cross-linked by seven disulfide bridges and has a formula weight of 13,578. The main fragmentation of the peptide chain was accomplished with a staphylococcal protease specific for glutamoyl bonds. A cyanogen bromide fragment and tryptic peptides were used to align the five major staphylococcal protease peptides. The sequence was determined by Edman degradation using the direct phenylthiohydantoin method and with carboxypeptidase A. Notexin is shown to be homologous to both porcine pancreatic phospholipase A and a phospholipase A from the venom of Naja melanoleuca. (+info)
Activation of human prothrombin by a procoagulant fraction from the venom of Echis carinatus. Identification of a high molecular weight intermediate with thrombin activity.
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In the presence of a procoagulant fraction (Echis carinatus procoagulant) isolated from the venom of the saw-scaled viper Echis carinatus sochureki, purified human prothrombin (P1) is completely converted to thrombin. The first step is the removal of an NH2-terminal peptide (F1) representing approximately one-third of the prothrombin molecule. The remaining peptide (P2) is then cleaved by the action of E.c. procoagulant to yield a two-chain, disulfide-bridged protein (P'2) which has the same molecular weight as P2. P'2 has enzymic (thrombin) activity, as evidence by incorporation of radiolabeled diisopropylphosphate into its heavy chain (TB), hydrolysis of p-toluenesulfonylarginine methyl ester, and clotting of fibrinogen. Relative to thrombin, its esterolytic activity greatly exceeds its clot-promoting activity. Examination of the polypeptide chains obtained by reducing P'2 has shown that its larger chain (TB) is indistinguishable from the heavy chain of thrombin. Its other chain (F2TA) consists of the light chain (TA) of thrombin bound by peptide linkage to the protion of the prothrombin molecule which had been adjacent to F1. Removal of this portion (F2) is catalyzed by thrombin (and, evidently, by P'2), but not by the E.c. procoagulant. When F2 is removed from P'2, the remaining two-chian protein is indistinguishable from thrombin by any of the criteria applied--molecular weight, subunit chain composition, or enzymic activity. Polyacrylamide gel electrophoresis was carried out in sodium dodecyl sulfate before and after disulfide reduction of samples generated in the presence and in the absence of diisopropylphosphorofluoridate, which inhibits thrombin but not the E.c. procoagulant. Such experiments showed that thrombin (and probably P'2), as well as E.c. procoagulant, catalyzes the release of F1. Furthermore, thrombin brings about the cleavage of F1 to yield a two-chain, disulfidebridged protein (F'1). These observations, particularly those made in the course of characterizine P'2, have led to the conclusion that cleavage of the peptide bond connecting the TA and TB portions of the prothrombin molecule (or its derivatives) produces a serine active center and, hence, a molecule possessing thrombin activity. This cleavage is catalyzed by the E.c. procoagulant but not by thrombon itself. (+info)
Interaction of lanthanide ions with bovine factor X and their use in the affinity chromatography of the venom coagulant protein of Vipera russelli.
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The substitution of trivalent lanthanide ions for Ca(II) in the Ca(II)-DEPENDENT ACTIVATION OF BOVINE Factor X by the coagulant protein of Russell's viper venom was studied at pH 6.8. Factor X contains two high affinity metal binding sites which bind Gd(III), Sm(III), and Yb(III) with a Kd of about 4 X 10-7 M and four to six lower affinity metal binding sites which bind Gd(III), Sm(III) with a Kd of about 1.5 X 10-5M. In comparison, 1 mol of Factor X binds 2 mol of Ca(II) with a Kd of 3 X 10-4M and weakly binds many additional Ca(II) ions. No binding of Gd(III) to the venom protein was observed. Dy(III), Yb(III), Tb(III), Gd(III), Eu(III), La(III), AND Nd(III) cannot substitute for Ca(II) in the Ca(II)-dependent activation of Factor X by the venom protein at pH 6.8. Kinetic data consistent with the models of competitive inhibition of Ca(II) by Nd(III) yielded a Ki of 1 to 4 X 10-6M. The substitution of lanthanide ions for Ca(II) to promote protein complex formation of Factor X-metal-venom protein without the activation of Factor X facilitated the purification of the coagulant protein from crude venom by affinity chromatography. Using a column containing Factor X covalently bound to agarose which was equilibrated in 10 mM Nd(III), Tb(III), Gd(III), or La(III), the coagulant protein was purified 10-fold in 40% yield from crude venom and migrated as a single band on gel electrophoresis in sodium dodecyl sulfate. These data suggest that lanthanide ions complete with Ca(II) for the metal binding sites of Factor X and facilitate the formation of a nonproductive ternary complex of venom protein-Factor X-metal. Tb(III) fluorescence, with emission maxima at 490 and 545 nm, is enhanced 10,000-fold in the presence of Factor X. The study of the participation of an energy donor intrinsic to Factor X in energy transfer to Tb(III) may be useful in the characterization of the metal binding sites of Factor X. (+info)
Chemotactic activity derived from interaction of factors D and B of the properdin pathway with cobra venom factor or C3B.
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Interaction of D (the activated form of D) and B, factors of the properdin pathway, with C3b (the major cleavage fragment of C3) generates a convertase, C3B, which cleaves C3 and initiates the terminal complement sequence C5-C9. A functionally analogous more stable C3 convertase, CoVFB, ir formed by substituting cobra venom factor (CoVF) for C3b. Mixtures of highly purified CoVF, B, and D were chemotactic for human neutrophil polymorphonuclear leukocytes as assessed in Boyden chambers either by microscopic enumeration of migrating cells or by counting of 51Cr-labeled cells. Control mixtures containing CoVF, B, and D, reacted in the absence of Mg++, were hemolytically inactive and devoid of chemotactic activity. Over a range of doses, the chemotactic activity of mixtures yielding CoVFB correlated with their hemolytic activity. Pretreatment of neutrophils with mixtures containing CoVFB rendered them unresponsive to subsequent chemotactic stimulation by kallikrein of C5a, indicating cross-deactivation to other chemotactic factors. Similar neutrophil deactivation occurred after exposure to a mixture of C3b, B, and D in which C3B was formed; with short incubation times and high cell concentration C3B also exhibited some chemotactic activity. The chemotactic activity of C3B and CoVFB is an example of a biologic function arising from interactions among factors of the properdin pathway per se, as distinguished from the capacity of this pathway to activate C3 and the terminal complement sequence. (+info)
Studies on the inhibition of C56-initiated lysis (reactive lysis). V. The roleof C567-INH in the regulation of complement-dependent haemolysis initiated by cobravenom factor.
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Activation of the alternative pathway of complement by a factor from cobra venom (CVF) can lead to lysis of unsensitized erythrocytes (E) of some species. In these studies we observed that alterations in CVF-induced lysis could be produced by manipulation of C567-INH, a naturally occurring inhibitory activity which acts on fluid phase C567 complexes. Venom lysis of sheep and guinea-pig E was markedly inhibited by serum fractions having C567-INH activity. Microgram quantities of poly-L-lysine (PLL), molecular weight 180,000, a polycation which is a functional antagonist to C567-INH in serum, potentiated CVF lysis of sheep and guinea-pig E, and permitted the lysis of human E, which are otherwise not suscepticle to CVF lysis. The potentiation of venom lysis by PLL seemed not to be due to alterations in the target cell membrane; furthermore, it in turn was reversed by substances with C567-INH activity. This suggests that the generation of fluid phase C567 complexes contributes to the CVF-induced lysis of erythrocytes of these species, and that the haemolytic potential of fluid phase C567 generated during alternative pathway activation by this means is regulated by C567-INH. (+info)