Two related thrombin-like enzymes present in Bothrops atrox venom

This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical Km on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80 percent homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii

Two related thrombin-like enzymes present in Bothrops atrox venom.

This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical K(m) on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80% homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii.

Edema induction by the disintegrin-like/cysteine-rich domains from a Bothrops atrox hemorrhagin.

Viperine and crotaline snake venoms contain one or more hemorrhagic metalloproteases called hemorrhagins. The most potent hemorrhagins belong to the P-III class and have, in addition to the protease domain, disintegrin-like and cysteine-rich domains. Although proteolytic degradation of vascular endothelium basement membrane has been established to be the main factor responsible for hemorrhage, several studies reveal other factors that actually do facilitate this process. Recent evidence has shown that the nonprotease domains of the P-III class hemorrhagins are able to inhibit the platelet aggregation by blocking essential procoagulant integrins on platelets. In this study we report the identification of a hemorrhagin from Bothrops atrox venom. This enzyme, a P-III class metalloprotease, undergoes an apparent spontaneous degradation, releasing a proteic fragment containing the disintegrin-like/cysteine-rich domains. This fragment shows the capability to induce an edematogenic process, suggesting the existence of a still unknown nonenzymatic mechanism of vascular permeability increase.

Urate protects a blood-sucking insect against hemin-induced oxidative stress.

Urate at high concentrations (up to 5 mM) is found in the hemolymph of the blood-sucking bug, Rhodnius prolixus. Increased urate levels are observed in the days following a blood meal. Injecting hemin into the hemocoel increases both urate titer and TBARS formation in the hemolymph. The urate response to hemin injection seems to arise from increased synthesis by the fat body as urate secretion by this organ is stimulated in vitro by incubation with hemin, and markedly counteracted by allopurinol. Allopurinol injection also results in increased TBARS formation in the hemolymph. High O2 atmospheric conditions also increases hemolymph urate levels, confirming that urate release represents an antioxidant response. Urate concentrations at the range reported here might account for almost all free radical scavenging activity of the hemolymph, as deduced from TRAP assay experiments, indicating that this is the major low molecular weight protection of this insect against oxidative insult. Since large amounts of hemin are produced in the midgut following blood digestion, increased urate hemolymph levels are suggested to be an important protective biochemical adaptation to allow blood feeding.

Isolation of a calcium-binding phosphoprotein from the oocytes and hemolymph of the blood-sucking insect Rhodnius prolixus.

A novel calcium-binding phosphoprotein was isolated from the oocytes of the blood-sucking bug Rhodnius prolixus. This protein exhibits an apparent molecular mass of 18 kDa on gel filtration, but migrates as an 8-kDa band on N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine/SDS-polyacrylamide gels. It has a high content of serine (24% of the total number of residues), and phosphoserine is the sole amino acid phosphorylated in vivo. A similar protein was partially purified from the hemolymph. It resembles the oocyte form of the protein in its NH2-terminal sequence and its ability to be taken up by growing ovaries. 45Ca binding to the oocyte phosphoprotein was determined after SDS-polyacrylamide gel electrophoresis followed by blotting on nitrocellulose membranes. Titration of Ca2+-binding sites shows a high capacity (approximately 50 mol/mol of protein), but a low affinity (K0.5 congruent with 10(-3) M). Based on these characteristics, we have named this protein Rhodnius calcium-binding phosphoprotein. It resembles phosvitin, a phosphoprotein present in the oocytes of nonmammalian vertebrates.

Molecular and biochemical characterization of the Dio-9-resistant pma1-1 mutation of the H(+)-ATPase from Saccharomyces cerevisiae.

The plasma-membrane H(+)-ATPase gene PMA1 was sequenced in four Dio-9-resistant strains of Saccharomyces cerevisiae, isolated independently. The same amino acid substitution Ala608----Thr was found in the four mutated strains. The mutant ATPase activity was decreased while the Km value for MgATP was increased. The ATPase efficiency (V/Km) of the mutant was reduced by a factor of 25 under acid conditions (pH 5.5), and by a factor of 10 at physiological pH (pH 6.6). The mutation also strongly reduces the inhibition by vanadate of ATPase activity, suggesting that the altered amino acid is involved in phosphate binding and/or in the E1-E2 transition.

Modification of ATP regulatory function in sarcoplasmic reticulum Ca2(+)-ATPase by hydrophobic molecules.

The effects of the three hydrophobic molecules triphenylphosphine, trifluoperazine and 3-nitrophenol on Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles was investigated. When ATP was the substrate, triphenylphosphine (3 microM) increased the amount of Ca2+ accumulated by the vesicles. At high concentrations triphenylphosphine inhibited Ca2+ uptake. This effect varied depending on the ATP concentration and the type of nucleotide used. With ITP there was only inhibition and no activation of Ca2+ uptake by triphenylphosphine. On the other hand, trifluoperazine inhibited Ca2+ accumulation regardless of whether ATP or ITP was used as substrate. When 5 mM oxalate was included in the medium in order to avoid binding of Ca2+ to the low-affinity Ca2(+)-binding sites of the enzyme, both stimulation by triphenylphosphine and inhibition by trifluoperazine were reduced. In leaky vesicles at low Ca2+ concentrations, triphenylphosphine and 3-nitrophenol were competitive inhibitors of ATPase activity at the regulatory site of the enzyme (0.1-1 mM ATP). A striking difference was observed when both the high- and low-affinity Ca2(+)-binding sites were saturated. In this condition, triphenylphosphine and 3-nitrophenol promoted a 3-4-fold increase in the apparent affinity for ATP at its regulatory site.

Activation of Ca2+ uptake and inhibition of reversal of the sarcoplasmic reticulum Ca2+ pump by aromatic compounds.

The effects of aromatic compounds in sarcoplasmic reticulum Ca2+-ATPase were investigated. The solubility of the drugs in various organic solvents and water was measured. The ratio between the solubility in organic solvents and that in water (distribution coefficient) was used as an index of their hydrophobicity. The order found was triphenylphosphine greater than diphenylamine greater than 3-nitrophenol greater than 4-nitrophenol greater than 1,3-dihydroxybenzene. The effects observed on the Ca2+-ATPase were correlated with hydrophobicity of the drugs, activation and inhibition being obtained at a lower concentration the greater the distribution coefficient of the drug into organic solvent. In leaky vesicles, the effects of each compound on the ATPase activity varied depending on the Ca2+ concentration in the medium: it inhibited in the presence of 5 microM Ca2+ and activated when the Ca2+ concentration was raised to 2 mM. In intact vesicles, 3- and 4-nitrophenol, diphenylamine, and triphenylphosphine enhanced both the rate of ATP hydrolysis and the amount of Ca2+ accumulated by the vesicles. These four drugs inhibited Ca2+ uptake when ITP was used as substrate. 1,3-Dihydroxybenzene enhanced the amount of Ca2+ accumulated by the vesicles regardless of whether ATP or ITP was the substrate. All five compounds inhibited the phosphorylation of the enzyme by Pi, the efflux of Ca2+, and the synthesis of ATP measured during the reversal of the Ca2+ pump. The results indicate that the hydrophobic character of various organic compounds determines their access to sensitive domains of the membrane-bound calcium pump. Additional specific effects are then produced, depending on the structure of each compound.

Contribution of water to free energy of hydrolysis of pyrophosphate.

The energy of hydrolysis of phosphate compounds varies depending on whether they are in solution or bound to the catalytic site of enzymes. With the purpose of simulating the conditions at the catalytic site, the observed equilibrium constant for pyrophosphate hydrolysis (Kobsd) was measured in aqueous mixtures of dimethyl sulfoxide, ethylene glycol, or polymers of ethylene glycol. The reaction was catalyzed by yeast inorganic pyrophosphatase at 30 degrees C. All the cosolvents used promoted a decrease of Kobsd. Polymers of ethylene glycol were more effective than dimethyl sulfoxide or ethylene glycol in decreasing Kobsd. The higher the molecular weight of the polymer, the lower the value of Kobsd. A decrease in Kobsd from 346 M (delta G degree obsd = -3.5 kcal mol-1) to 0.1 M (delta G degree obsd = 1.3 kcal mol-1) was observed after the addition of 50% (w/v) poly(ethylene glycol) 8000 to a solution containing 0.9 mM MgCl2 and 1 mM Pi at pH 8.0. The association constants of Pi and pyrophosphate for H+ and Mg2+ were measured in presence of different ethylene glycol concentrations in order to calculate the Keq for hydrolysis of different ionic species of pyrophosphate. A decrease in all the Keq was observed. The results are interpreted according to the concept that the energy of hydrolysis of phosphate compounds depends on the different solvation energies of reactants and products.