Studies on phospholipase A in Trimeresurus flaoviridis venom. III. Purification and some properties of phospholipase A inhibitor in Habu serum.

Phospholipase A [EC 3.1.1.4] inhibitor was purified from Habu (Trimeresurus flavivurudls) serum by gel filtration on Sephadex G-200, chromatography on DE-23 cellulose and affinity chromatography on a Sepharose 4B-phospholipase A column. By these procedures, a 31-fold increase in specific activity was attained with a yield of 15%. The purified material was homogeneous as judged by cellulose acetate and polyacrylamide gel electrophoresis. It had an apparent molecular weight of 100,000 as measured by gel filtration on Sephadex G-200. The purified inhibitor was stable for 20 min at 80 degrees and was unstable below pH 6. It migrated before albumin in cellulose acetate electrophoresis and did not form any precipitin line with the crude venom or with purified phospholipase A in immunodiffusin tests. An 8-fold excess of the purified inhibitor by weight was required to inhibit completely both the egg yolk clearing action and the hemolytic action of phospholipase A.     

Crystallization and preliminary X-ray study of blood coagulation factor IX/factor X-binding protein with anticoagulant activity from Habu snake venom.

Crystals of a blood anticoagulant from the venom of the Habu snake, Trimeresurus flavoviridis, have been obtained using ammonium sulfate by the vapor diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2 with cell dimensions a = 172 A, b = 86 A, c = 65 A, and diffract to at least 4.0 A resolution.     

Assay for phospholipase A activity of snake venom with asolectin as substrate

A reliable, rapid, and relatively inexpensive assay for phospholipase A activity in Naja naj venom is described. Asolectin, a mixture of soybean phospholipids, is used as the substrate instead of the more expensive, natural substrate l-α-lecithin. Phospholipase A activity of snake venom, monitored by hydroxamate formation, obeys Michaelis-Menten kinetics and is proportional to time and to venom concentration. The pH profile of activity shows a broad plateau from pH 7.0 to 9.5 and a shoulder between pH 5 and 6.     

A novel blood coagulation factor IX/factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake): isolation and characterization

Using affinity chromatography on a column of factor X-Cellulofine, we have isolated a novel blood coagulation factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake). This anticoagulant protein was also purified by chromatography on Sephadex G-75 and S-Sepharose Fast Flow. The yield of the purified protein was approximately 16 mg from 400 mg of crude venom. The purified protein gave a single band on both analytical alkaline disc-gel electrophoresis and SDS-PAGE. This protein had a relative molecular weight (Mr) after SDS-PAGE of 27,000 before reduction of disulfide bonds and 14,000 after reduction of disulfide bonds. The protein prolonged the clotting time induced by kaolin or factor Xa. In the presence of Ca2+, it formed a complex with factor X, the molar ratio being 1 to 1. Similar complex formation was observed with factor Xa and factor IX/factor IXa, but not with other vitamin K-dependent coagulation factors, i.e., prothrombin, factor VII, protein C, protein S, and protein Z. The interaction of this anticoagulant protein with factor IX/factor X was dependent on gamma-carboxyglutamic acid (Gla) domains, since Gla-domainless derivatives of factor X and factor IXa beta' did not interact with this anticoagulant protein.     

Mild pressure induces resistance of erythrocytes to hemolysis by snake venom phospholipase A2.

It is generally assumed that mild pressure of a few atmospheres, such as that applied to blood cells during routine centrifugation, does not affect cell function. The results of the present study refute this notion. To explore the effect of mild pressure on cell function we examined its effect on the susceptibility of red blood cells (RBC) to hemolysis by snake venom phospholipase A2 (PLA2). Rat RBC were subjected to pressure of up to five atmospheres, returned to ambient pressure and interacted with PLA2 to induce hemolysis. The hemolysis was markedly decreased with increasing the pressure applied before induction of hemolysis. Application of such a pressure induces the shedding of a chemical factor, as yet uncharacterized, which facilitates the action of PLA2 on RBC.     

Viscous macromolecules inhibit erythrocyte hemolysis induced by snake venom phospholipase A2

The effect of medium viscosity on lysis of red blood cells (RBC) induced by snake venom phospholipase A2 (PLA2) was examined. The medium viscosity was modified by the addition of various macromolecules which differ in their chemical nature and in their capacity to increase fluid viscosity. PLA2 and Ca++ were applied to cells suspended in viscous medium to induce hemolysis. It was found that the hemolysis is inhibited in direct proportion to increasing viscosity of the extracellular fluid. This phenomenon was observed with aggregated as well as disaggregated RBC. To examine whether the viscosity interferes with the accessibility of the enzyme to the cell, the medium viscosity was modified after binding of the enzyme to the cells; PLA2 was added to a RBC suspension in the presence of Ba++ which binds the enzyme to the cell membrane but does not activate it. The cell-enzyme complex was separated by gel filtration and suspended in viscous medium in the presence of Ca++ which activates the reaction. Also in this case RBC lysis was inhibited as the medium viscosity was increased. It is proposed that the action of PLA2 on RBC membrane is regulated by the viscosity of the cell surface aqueous environment.     

A permanent magnetic field alters the phospholipase activity in the snake venom from Vipera lebetina

It was found that at the exposure of Vipera lebetina snakes (during 10 days for 30 min daily) to SMF (0.15 T1) the specific activity of venom phospholipase A1, A2 and phosphodiesterase C increased by 20.6 +/- 2.8; 31.7 +/- 3.2 and 32.7 +/- 1.3% correspondingly. The above mentioned changes of venom enzyme activity were accompanied with the decrease of its total protein amount by 31.6 +/- 2.2%. It could be supposed that the described changes are able to cause significant changes in the total metabolic activity of cells and the organism as a whole.     

The action of snake venom, phospholipase A and trypsin on purified myelin in vitro.

1. Purified myelin was incubated with snake venom or phospholipase A in the presence of or absence of trypsin at 37 degrees C, pH7.4, for different times. 2. Analysis of the myelin pellet obtained after centrifugation of the myelin sample incubated with snake venom or phospholipase A alone showed conversion of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine into their corresponding lyso compounds. No significant loss of myelin protein was observed in these samples. 3. A marked digestion of basic proteins and proteolipid protein was observed from the myelin pellet when trypsin was present in the incubation mixture. 4. The digestion of basic protein and particularly of proteolipid from myelin suggest that phospholipases may make protein more exposed to proteolytic enzyme for its digestion. 5. The relevance of the co-operative effect of phospholipases and proteinases as a model system of the mechanism of myelin breakdown in degenerative brain diseases is discussed.     

Quercetin as an inhibitor of snake venom secretory phospholipase A2

As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors.     

Toxicity domain in presynaptically toxic phospholipase A2 of snake venom

About 42 complete amino-acid sequences of phospholipases A2 (phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) are known, including those of 13 presynaptically toxic enzymes, but the structural features responsible for the neurotoxicity and distinguishing the toxins from the non-neurotoxic enzymes are far from being clear. In this study, we examined the charged-residue distributions and hydrophobic characteristics based on the sequence data and the predicted tertiary structure and proposed a possible toxicity domain. We found that the presynaptically toxic enzymes have three or four more basic amino-acid residues than the non-neurotoxic enzymes at positions 59, 60, 65, 70-73 and 97 or 98. These residues appear to cluster near the surface region at the N-terminal side. The cationic nature of this basic cluster in the toxin is enhanced by the alpha-amino group of the N-terminus and the dipole moment of helices 96-110 and 1-10. Moreover, these toxic-site residues are usually associated with hydrophobic regions at 1-7, 64-81 and 97-109.     

Isolation, characterization and biological activity of acidic phospholipase A2 isoforms from Bothrops jararacussu snake venom

Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIB diffracted beyond 1.8 A resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 A, b = 54.2 A and c = 90.7 A. The crystal structure has been refined to a crystallographic residual of 16.1% (R(free) = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities.     

Structure and function comparison of Micropechis ikaheka snake venom phospholipase A2 isoenzymes

Comparison of the crystal structures of three Micropechis ikaheka phospholipase A2 isoenzymes (MiPLA2, MiPLA3 and MiPLA4, which exhibit different levels of pharmacological effects) shows that their C-terminus (residues 110-124) is the most variable. M-Type receptor binding affinity of the isoenzymes has also been investigated and MiPLA4 binds to the rabbit M-type receptor with high affinity. Examination of surface charges of the isoenzymes reveals a trend of increase in positive charges with potency. The isoenzymes are shown to oligomerize in a concentration-dependent manner in a semi-denaturing gel. The C-termini of the medium (MiPLA4) and highly potent (MiPLA2) isoenzyme molecules cluster together, forming a highly exposed area. A BLAST search using the sequence of the most potent MiPLA2 results in high similarity to Staphylococcus aureus clotting factor A and cadherin 11. This might explain the myotoxicity, anticoagulant and hemoglobinuria effects of MiPLA2s.