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The intraspecific geographical venom variations of Calloselasma rhodostoma from Malaysia (CR-M), Indonesia (CR-I), Thailand (CR-T) and Vietnam (CR-V) were investigated through 1D SDS-PAGE and nano-ESI-LCMS/MS. The venom antigenicity, procoagulant activities and neutralization using Thai C. rhodostoma Monovalent Antivenom (CRMAV) were also investigated. SDS-PAGE patterns of the venoms were relatively similar with minor variations. Proteomic analysis revealed that snake venom metalloproteinases (SVMPs, particularly P–I class), serine proteases (SVSPs) and snaclecs dominated the venom protein composition (68.96–81.80%), followed by L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2) (7.37–11.08% and 5.18–13.81%, respectively), corroborating C. rhodostoma envenoming effects (hemorrhage, consumptive coagulopathy, thrombocytopenia and local tissue necrosis). Other proteins of lower abundances (2.82–9.13%) identified include cysteine-rich secretory proteins (CRISP), phospholipase B, phosphodiesterase, nerve growth factor, 5′-nucleotidase, aminopeptidase and hyaluronidase. All four venoms exhibited strong procoagulant effects which were neutralized by CRMAV to different extents. CRMAV immunoreactivity was high toward venoms of CR-M, CR-I and CR-T but relatively low for CR-V venom. Among the venom samples from different locales, CR-V venom proteome has the smallest SVMP composition while SVSP, PLA2 and phosphodiesterase were more abundant in the venom. These variations in C. rhodostoma venom protein composition could partly explain the differences seen in immunoreactivity. (198 words).

Phospholipase-B-like (SVPLB-like) enzymes are present in relatively small amounts in a number of venoms, however, their biological function and mechanisms of action are un-clear. A three-dimensional model of the SVPLB-like enzyme from Crotalus adamanteus was generated by homology modeling based on the crystal structures of bovine Ntn-hydrolyases and the modeled protein possesses conserved domains characteristic of Ntn-hydrolases. Molecular dynamics simulations indicate that activation by autocatalytic cleavage results in the removal of 25 amino acids which increases accessibility to the active site. SVPLB-like enzymes possess a highly reactive cysteine and are hence amidases that to belong to the N-terminal nucleophile (Ntn) hydrolase family. The Ntn-hydrolases (N-terminal nucleophile) form a superfamily of diverse enzymes that are activated autocatalytically; wherein the N-terminal catalytic nucleophile is implicated in the cleavage of the amide bond.

Two species of giant hornet phospholipase B (PLB), alpha and beta, were purified from the venom of Vespa mandarinia. The purification procedure was simplified by two steps of column chromatographies, Sephadex G-100 and SP-Sepharose. The molecular sizes of PLB alpha and beta were 29.5 and 26.0 kDa, respectively. The isoelectric point of alpha and beta enzymes were pH 10.6 and 10.7, respectively. The temperature optimum for egg yolk lecithin was a broad peak at 40-60 degrees C for both enzymes. Amino acid compositions of both enzymes were high contents of aspartic acid, glycine, leucine, lysine and other aliphatic amino acids. Cystine was similar amounts to other species of phospholipases (PLs). The K(m) values of alpha and beta enzymes were 8.29 and 7.53 mg/ml for egg yolk lecithin, respectively. In the catalytic specificity for L-alpha-phosphatidylcholine-beta-oleoil-gamma-palmitoil, the K(m) values of alpha enzyme for gamma-palmitoil and beta-oleoil residues were 0.528 and 1.392 mM, respectively. While the K(m) values of beta enzyme for gamma-palmitoil and beta-oleoil residues were 7.91 and 2. 68 mM, respectively. Both alpha and beta enzymes were inhibited strongly by cepharanthine. The lecithin hydrolysis of alpha enzyme was competitively inhibited, but beta enzyme was uncompetitive. Cepharanthine also inhibited noncompetitively PLA(2)s of bovine pancreas, bee venom and Naja mossambica mossambica.

Phospholipases A and B activities of the oriental hornet (Vespa orientalis) venom and venom apparatus. Toxicon15, 141–156, 1977.—Oriental hornet venom is a rich source of both phospholipase A (PhA) and phospholipase B (PhB) activities. This was shown by incubating venom with egg yolk or with pure lecithin and lysolecithin. Activity was measured by titrating liberated fatty acids and by phosphorus analyses of separated phospholipids from thin-layer chromatographic plates. Both lecithin and lysolecithin were rapidly hydrolyzed by venom at pH 4 and 8. With egg yolk as substrate, the optimum Ph activity was observed at pH 5 although considerable activity was observed from pH 3·5 to 9·5. In contrast purified substrates showed greater activity at an alkaline pH, whether assay was in the presence of collidine-acetate or Tris buffer or in the presence or absence of ether. Ether dramatically changed the optimal pH for Ph activity, with egg yolk as substrate, from acidic to alkaline. It is not known whether these PhA and PhB activities are dual activities of a single enzyme or activities of two separate enzymes. The venom has neither PhC nor lipase activities. PhA and PhB activities were observed not only in pure venom (V), but also in venom sacs (VS) where the venom is stored, in the acid (venom) glands (H+) where the venom is produced, and in the alkaline (Dufours) gland (OH−) whose function is unknown. The release of free fatty acids from egg yolk at pH 5 was in the ratios of 1 (V): 0·13 (VS): 0·04 (H+): 0·30 (OH−). Measurements of Ph activities in combinations of the above preparations showed that strong activators or inhibitors of the enzyme are not present. The mid-gut, fat body and hemolymph of the hornet showed Ph activity only equal to about one-half of one per cent of that of the OH− gland. Antisera produced against V, VS, H+ and OH− were cross-reactive and inhibited to varying degrees the Ph activities of each of the above preparations. The highest titre antisera were produced in rabbits injected with V or VS, in contrast to the low titres produced with H+ and OH− glands. Low Ph activity is detectable in the venom sacs 2 days prior to emergence, with 20-fold higher activities being observed at 5 days of age. The oriental hornet may be extremely useful as a rich source of not only PhA but also PhB activities. The effects of this latter enzyme on biological systems have not been thoroughly evaluated. The drastic disruption of phospholipid structure and hydrophobic binding forces between phospholipid and protein, by the combined PhA plus PhB activities, may be responsible for some of the pharmacological actions of hornet venom.

One single protein species with phospholipase activity has been isolated from Bothrops alternatus venom by a procedure involving gel-filtration on Sephadex G-50 (Step 1), chromatography on SP-Sephadex C-50 (Step 2) and gel-filtration on Sephadex G-75 (Step 3). The purified sample behaved as a homogeneous, monodisperse protein with a molecular weight of 15,000 and isoelectric point of 5.04. The yield in enzyme activity was 48% of the starting material and the apparent purification was 51-fold. When assayed on 1,2-diheptanoyl- or 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine, fatty acids and lysolecithins were the only reaction products, in accordance with the predicted stoichiometry. Studies on positional specificity suggested that the enzyme is a phospholipase A2. The enzyme requires Ca2+ ions for activity and exhibited stereochemical specificity, since the enantiomeric 2, 3-diheptanoyl-sn-glycero-1-phosphorylcholine was not hydrolyzed. Under the experimental conditions employed, reaction products representative of either phospholipase B or C activities could not be detected. After Step 1, the phospholipase activity recovered was higher than the total activity in the crude venom sample, which is explained by the separation of an inhibitor during enzyme purification. The inhibitor was responsible for the initial lag period that characterized the kinetics of the enzyme reaction with crude venom acting on aggregated substrates (lipoprotein, vesicles or micelles), while the rate of hydrolysis of monomeric lecithins was not affected.

Two phospholipases B (Vx I and Vx II) were purified from the venom of the Japanese yellow hornet, Vespa xanthoptera, by sequential chromatography on Sephadex G-100, SP-Sephadex and Mono S columns. They are very similar to each other in molecular and enzymatic properties, though the specific activity of Vx I was one-fifth that of Vx II. They hydrolyze the acyl ester bonds at the 1-position of phosphatidylcholine and lysophosphatidylcholine, therefore, their enzymatic specificities were of the A1 and L1 types.

Phospholipase A and B activity of several Egyptian snake venoms and scorpion toxin was determined by employing the rates of hydrolysis of lecithin and lysolecithin indicating phospholipase A activity and phospholipase B activity, respectively. Phospholipase A showed maximal activity at pH range 7·0–9·0. Marked activation of the enzyme occurred upon the addition of ether, sodium deoxycholate, Ca 2+ and Mg 2+ , while inhibition occurred upon the addition of EDTA, or a combination of sodium deoxycholate and calcium. The enzyme phospholipase B showed maximal activity at pH range 8·0–10·0 and was inhibited by EDTA and ether.

Each of six cobra (Naja naja) venom fractions possesses phospholipase A2 activity—hydrolysing 1,2 diacyl-sn-glycerophosphorylcholine, e.g. lecithin, in the 2-position, as well as phospholipase B activity, hydrolyzing 1-monoacyl-sn-glycerophosphorylcholine, e.g. lysolecithin. For all fractions the pH optimum was 9 for lecithin hydrolysis and above 10 for lysolecithin hydrolysis. The enzymatic activity of all fractions on lecithin was several orders of magnitude higher than that on lysolecithin. The Km values for lysolecithin hydrolysis by the various Naja naja phospholipase isoenzymes at pH 10 and 37° were about 1 mM.

TIB3 cENERic term phospholipase designates a class of enzymes which specifically hydrolyze phospholipids. Four types of phospholipases are known-phospholipase A, B, C, D ; they are individually characterized by a specific site ofaction on the phospholipid molecule. Phospholipase A (phosphatide aryl hydrolyze, EC 3.1 .1 .4) catalyzes the hydrolysis of one ester bond in phosphatide molecules with formation of lysophosphatides and release of free fatty acids. The other phospholipases attack the phosphatide molecules at different site. Phospholipase B (EC 3.1 .1 .E splits off the fatty acid of lysophosphatide ; phospholipase C (EC 3.1 .4 .3) effects hydrolytic rupture of the phosphorylated nitrogen base with formation of a, s diglyceride, and phospholipase D (EC 3.1 .4.4) splits the nitrogen basephophoric acid ester bond with production of phosphatidic acid . Snake and bee venoms are the most potent known sources of phospholipase. As far as known they contain phospholipase A only. Phospholipase A has been identified in all snake venoms tested, whether beloning to theViperidae, Elapidae or Crotalidae families [1,2,3,4]. Phospholipase A has also been detected in bacteria [5] and in body tissues and fluids . Ox and human pancreas are especially rich in phospholipase A [6,7] and the presence of the enzyme in duodenal content, pancreatic juice, bile, serum [8,9] and in brain [10] has been demonstrated .

Rabbit erythrocyte phospholipase B (lysolecithin acyl-hydrolase E.C. 3.1.1.5) hydrolyses the lysophospholipids produced in Ringhals cobra venom—treated rabbit erthrocytes and osmotic ghosts by the action of the venom phospholipase A (phosphatide acyl-hydrolase E.C. 3.1.1.4). Raising the incubation temperature of the osmotic hemolysate-venom mixture to 60° leads to inactivation of the erythrocyte phospholipase B with preservation of the lysocompounds formed due to the action of the heat-stable venom phospholipase A. Subjecting the rabbit erythrocytes to various hemolytic agents, such as hypotonic medium, saponin, digitonin or cobra venom-direct lytic factor (DLF), causes liberation of phospholipase B into the particle-free supernatants, as indicated by hydrolysis of added lysolecithin.