Affinity chromatography of phospholipase A2 from Naja naja naja (Indian cobra) venom

A rapid and improved purification procedure is described for phospholipase A2 from the Indian cobra, Naja naja naja. The procedure is based on affinity chromatography of the venom through Affi-Gel Blue to obtain a 9-fold purification in one step. However, as there are multiple forms of the enzyme in the venom and other proteins do bind to Affi-Gel Blue, further purification is achieved by DE-11 cellulose. Finally, a minor contaminant is removed by Sulfopropyl Sephadex C-25 chromatography. The resulting product was found to be pure by analytical isoelectric focusing and double diffusion precipitin tests. An isoelectric focusing column run with venom collected from a single snake gave a similar profile to that obtained with venom pooled from several snakes.     

Purification and characterization of a myotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom

A major phospholipase A2 (NN-XIII-PLA2) which constitutes 20% of the whole Naja naja naja venom was purified to homogeneity on CM-Sephadex C-25 column chromatography. NN-XIII-PLA2 is a basic protein with a mol. wt of 11,200 by SDS-PAGE. This enzyme has low enzymatic activity but is more toxic to mice than the whole venom. The LD50 value (i.p.) of NN-XIII-PLA2 is 2.4 mg/kg body weight (whole venoms LD50 is 2.8 mg/kg body weight). It induces neurotoxic-like signs in experimental animals. It induces myotoxicity when injected i.m. into the thigh muscle of mice and edema when injected into the foot pads of mice. This enzyme has a fluorescence maxima between 310-316 nm which is typical of tyrosine residues.     

Purification and characterization of a neurotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom.

Snake venoms contain multimolecular forms of phospholipase A2 which are diverse with respect to their pharmacological properties. A neurotoxic PLA2 from Naja naja naja venom has been purified in two steps. (1) The whole venom was fractionated on CM-Sephadex C-25 column; 4.6% of the total PLA2 activity recovered was found in the NN-V fraction. (2) The NN-Vb-PLA2 fraction was purified to homogeneity by gel filtration of fraction NN-V on Sephadex G-50. It is a basic protein with a mol. wt between 10,500 and 11,000, and is more toxic than other basic PLA2s purified from Naja naja naja venom. The LD50 of NN-Vb-PLA2 is 0.27 mg/kg body wt. It induced neurotoxic symptoms in experimental mice and is devoid of myotoxic, anticoagulant and edema-inducing activities.     

Effect of cobra venom (Naja haje) on insulin release by rat pancreas in vitro

Additions of cobra venom at 10, 20 and 30 μg per ml to incubation media of rat pancreas caused no significant effect on insulin release. Additions of the venom at a concentration of 60 μg per ml caused a significant inhibition of glucose induced insulin secretion at low (60 mg per 100 ml) and high (300 mg per 100 ml) glucose concentrations. The possibilities of local release of adrenergic transmitter, impaired glucose metabolism by the beta cells and diminished intracellular levels of cyclic AMP in the beta cells, by venom are discussed.     

A neurotoxic phospholipase A2 variant: isolation and characterization from eastern regional Indian cobra (Naja naja) venom.

CM-Sephadex C-25 column chromatography profile of Indian cobra (Naja naja) venom from eastern region showed a distinct and a dominant phospholipase peak, peak-10, while it was not seen in either southern or western venom samples. Peak-10 was subjected to CM-Sephadex C-25 and Sephadex G-50 column chromatography to isolate NN-X-PLA(2). NN-X-PLA(2) is a single chain protein with the relative molecular weight of 10kDa by SDS-PAGE. It was toxic to mice with an LD(50) value 0.098 mg/kg body weight (i.p.) and the mice exhibited acute neurotoxic symptoms. Upon indirect stimulation, it inhibited the twitching of frog's gastrocnemius muscle in a dose dependent manner. NN-X-PLA(2) was weakly anticoagulant and devoid of cytotoxicity, myotoxicity, hemorrhage, edema inducing, and directlytic activities and effects on platelet aggregation process. Upon chemical modification independently with p-bromophenacyl bromide and acetic anhydride, NN-X-PLA(2) lost both enzymatic and toxic properties.     

Neuromuscular effects of nigexine, a basic phospholipase A2 from Naja nigricollis venom

Nigexine is a basic phospholipase A2 from the venom of the spitting cobra Naja nigricollis. In addition to its anticoagulant and cytolytic properties, nigexine also affects neuromuscular transmission in vitro. On chick biventer cervicis preparations, 1.5 microM nigexine caused a slowly developing block of responses to nerve stimulation, and a progressive loss of postjunctional sensitivity. Nigexine was at least 10 times less potent than notexin. On frog cutaneous pectoris preparations, nigexine caused a transient facilitation of evoked acetylcholine release, followed by a block. Spontaneous release was not abolished, and nigexine induced the release of abnormally large packets of transmitter. Nigexine also caused contracture of muscle fibres, accompanied by depolarization and degeneration. Nigexine appears to be able to cause prejunctional blockade and direct muscle damage to isolated skeletal muscle preparations.     

Chemical modification of lysine and histidine residues in phospholipase A2 from the venom of Naja naja atra (Taiwan cobra)

The major phospholipase A₂ was isolated from Naja naja atra venom by successive chromatography on SP-Sephadex C-25, DEAE-Sephacel, CM-Sephadex C-25 and SP-Sephadex C-25 columns. The homogeneity was verified by disc electrophoresis and the isoelectric point determined to be 5.2. The specific activity was 3400 U/mg protein, and the ld₅₀ 8 mg/kg mouse. The purified phospholipase A₂ was subjected to lysine modification with cyanate at pH 8.0. After suitable periods (3 and 16 hr), the carbamylated derivatives were separated on a column of DEAE-Sephacel and eight fractions were obtained (DE-1 to DE-8). The results of amino acid analysis showed that one to five Lys-residues were modified. Associated with modification of increasing numbers of Lys-residues were progressive decreases in pI values and marked decreases (3 to > 30-fold) in ld₅₀ values. However, the decrease in enzymatic activity was slight and antigenic specificity was unaffected. The results show a clear dissociation between enzymatic activity and lethal toxicity. The enzyme was also subjected to chemical modification with p-bromophenacyl bromide. Alkylation of the only His-47 at the active site of the phospholipase A₂ destroys both catalytic activity and lethal toxicity, whereas the antigenicity remained unchanged. Although all the native, Lys-modified and His-modified phospholipases A₂ were perturbed by the presence of Ca²⁺ and the difference spectra of Lys-modified DE-6 was similar to that of native phospholipase A₂, the difference spectra of His-modified enzyme differed greatly from that of the native enzyme. The emission intensity of 8-anilinonaphthalenesulfonate-enzyme complex was altered by increasing concentrations of Ca²⁺, and different results were observed at different pH values of the buffer solution, indicating that Ca²⁺ causes pH-dependent conformational changes. The Scatchard plots showed only one kind of specific interaction between 8-anilinonaphthalenesulfonate and native or Lys-modified enzyme (DE-6), and the dissociation constant of Lys-modified DE-6 was similar to that of the native enzyme. On the other hand, the His-modified enzyme lost the ability to bind 8-anilinonaphthalenesulfonate.     

Phospholipid hydrolysis in serum lipoproteins by a basic phospholipase A2 from Naja nigricollis snake venom and an acidic phospholipase A2 from Naja naja atra snake venom

Apparent Km and Vmax values for PC and PE hydrolysis were determined following exposure of HDL, LDL, and VLDL to a basic phospholipase A2 from N. nigricollis snake venom and an acidic phospholipase A2 from N. nigricollis snake venom and an acidic phospholipase A2 from N. n. atra snake venom. Both enzymes hydrolyzed the lipoprotein phospholipids approximately as fast as they hydrolyzed pure phospholipids in mixed micelles, however, the N. nigricollis enzyme, which has a much stronger anticoagulant effect than the N. n. atra enzyme, had lower apparent Vmax values. These values were highest for phospholipids in VLDL and lowest for HDL, however, the differences between the lipoproteins were relatively small with the N. nigricollis enzyme while the differences were much larger with the N. n. atra enzyme. Fractions of the two enzymes in which varying numbers of lysines were carbamylated showed much larger differences in relative rates of phospholipid hydrolysis in HDL, LDL and VLDL. Triton X-100 eliminates these differences in rates of hydrolysis. These results are discussed in terms of the differences in the organized structure of the lipoprotein classes and in the penetration ability of the phospholipases.     

Effects of cobra (Naja haje) venom on blood glucose, blood phosphate and plasma insulin-like activity in dogs

Studies were made on the effects of i.v. injection of cobra venom on blood glucose and phosphate levels and plasma insulin-like activity (ILA) during i.v. glucose tolerance test in dogs. Dogs treated with a lethal dose (0·1 mg/kg) of the venom showed a type of animal diabetes characterized by hyperglycaemia, diminished utilization of glucose increased blood phosphate levels and diminished insulin activity. Animals treated with a sublethal dose (0·04 mg/kg) of venom had a significantly delayed clearance of glucose. It is suggested that these effects are due to a primary action of the venom on insulin secretion and hepatic disposal of glucose and secondarily due to stimulation of the adrenal medulla and release of epinephrine.     

Purification and characterization of an anticoagulant phospholipase A(2) from Indian monocled cobra (Naja kaouthia) venom.

An anticoagulant, non-toxic phospholipase A(2) was isolated from the venom of Indian monocled cobra (Naja kaouthia) by a combination of ion-exchange chromatography on CM-Sephadex C-50 and gel filtration on Sephadex G-50. This purified protein named NK-PLA(2)-I, had a subunit molecular mass of 13.6 kDa and migrated as a dimer under non-reduced condition in SDS-PAGE. NK-PLA(2)-I was a highly thermostable protein requiring basic pH optima for its catalytic activity and showed preferential hydrolysis of phosphotidylcholine. This protein exhibited higher anticoagulant, indirect hemolysis, liver and heart tissue damaging activity but exerted less toxicity, direct hemolysis, edema and lung tissue damaging activity as compared to whole venom. Treatment of NK-PLA(2)-I with rho-BPB, TPCK, PMSF, antivenom and heating had almost equal effect on PLA(2), and other pharmacological properties except in vitro tissue damaging activity. Current investigation provides a fairly good indication that NK-PLA(2)-I induces various pharmacological effects by mechanisms, which are either dependent or independent of its catalytic activity.     

眼镜蛇毒分泌型磷脂酶A_2的研究进展

眼镜蛇毒分泌型磷脂酶A2(sPLA2)作为眼镜蛇毒中的一个重要成分,有着广泛的生物学活性,近年来的研究热点正日益从眼镜蛇粗毒转向毒素中的某个单体,如sPLA2的研究。该文简要综述了眼镜蛇毒sPLA2的结构特征、分离纯化以及分子生物学特性,着重论述了sPLA2多样的药理毒理作用,并对其作用机制和发展前景进行了探讨。     

Isolation and characterization of a toxic phospholipase A2 in the spitting cobra (Naja mossambica mossambica) venom.

N. Martin-Moutot and H. Rochat. Isolation and characterization of a toxic phospholipase A₂ in the spitting cobra (Naja mossambica mossambica) venom. Toxicon17, 127–136, 1979.—A phospholipase A₂ from the spitting cobra (Naja mossambica mossambica) was purified by chromatographic procedures involving gel filtration on Sephadex G-50 and ion exchange on Amberlite IRC-50. The purified enzyme was homogeneous according to physico-chemical criteria as well as chemical purity tests. This protein was characterized by amino acid composition, determination of N- and C- terminal amino acid sequences, isoelectric pH, molecular weight. It exhibits phospholipase activity and toxicity to mice. Positional specificity towards phosphatidylcholine showed that it belongs to the A₂ type. Optimal activity was observed at an alkaline pH and calcium was required for phospholipase activity.     

Effects of Naja haje (Egyptian cobra), Naja naja (hooded cobra), Naja nigricollis (spitting cobra) and Naja mossambica mossambica (Mozambique spitting cobra) venoms on the isolated guinea-pig tracheal muscle

Venoms from N. haje, N. naja, N. nigricollis and N. mossambica were tested on the isolated guinea-pig trachea. The four venoms (1-30 micrograms/ml) contracted the tracheal smooth muscle after a delay of 40-60 sec. A second challenge with the venoms caused either no or a much reduced contraction or a relaxant effect. The contraction could be prevented by pretreatment with antihistaminics, but not by atropine, methysergide or indomethacin, indicating that it is due to histamine release by the venoms. This release requires extracellular Ca2+, as it could be prevented by pretreatment with verapamil. Under conditions which prevented histamine release or its effect, each of the four venoms resulted in a reproducible relaxant effect which was not blocked by propranolol. It is concluded that the venoms have one or more component(s) causing histamine release which masks the relaxation caused by another component(s) of the venoms.     

Convulsant activity of Naja Naja venom and its phospholipase A component

Naja naja (hooded cobra) venom and its purified components were injected intraventricularly into rats. Purified cobrotoxin (50 μg) and cardiotoxin (125 μg) caused piloerection, lacrimation and death but no convulsive activity. Crude cobra venom (5 μg) and a partially purified phospholipase A (2·5 μg) isolated from Naja naja venom induced convulsions. Analysis of brain phospholipids after injection of 330 μg phospholipase A demonstrated an increase in lysophosphatides. Hydrolysis of phospholipids was greater at the onset of convulsions than $\text{1}\text{2}$hr before onset. Lysolecithin (100 μg) and stearic acid (500 μg), hydrolysis products of phospholipase A action, did not induce convulsions after intraventricular injection. Phospholipase C in doses as low as 1 μg caused convulsions when injected intraventricularly. Examination of brain phospholipids at the onset of convulsant activity after injection of 250 μg of the enzyme showed a reduction in total lipid phosphorus and a corresponding increase in total aqueous phosphorus and diglyceride as a result of the enzyme's action. The possible involvement of phospholipase A in convulsant activity is discussed.     

Effects of phospholipase A2 from cobra and bee venom on the presynaptic membrane

L. G. Magazanik, I. M. Gotgilf, T. I. Slavnova, A. I. Miroshnikov and U. R. Apsalon. Effects of phospholipase A₂ from cobra and bee venom on the presynaptic membrane. Toxicon17, 477–488, 1979.—Phospholipases A₂ from bee venom and cobra venom have been isolated and studied. A parallelism was found between enzymatic activity and the ability to block spontaneous miniature end-plate potentials (m.e.p.p.'s) or end-plate potentials (e.p.p.'s) induced by nerve stimulation in the frog sartorius muscle. Different experimental procedures affected both enzymatic activity and blocking ability in qualitatively the same way. Thus, modification of the histidine residue in cobra venom phospholipase by bromophenacyl bromide or the removal of Ca-ions from the medium abolished both activities. Replacement of Ca²⁺ by Sr²⁺ inhibited both the enzymatic and presynaptic effects of cobra venom phospholipase, but did not inhibit the presynaptic action of bee venom phospholipase and decreased its enzymatic activity only 6-fold. Irreversible binding of cobra and bee venom phospholipase to the presynaptic membrane was found in Ca-free solution but Ca-ions were essential for the presynaptic blocking effect induced by these phospholipases. A reduction in the effect of high K⁺ on m.e.p.p. frequency was observed after cobra venom phospholipase treatment. The similar effects of hypertonic sucrose solution and the mitochondrial poison TTFB (4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole) were changed only slightly by bee and cobra venom phospholipase pretreatment. It is concluded that the mechanism of presynaptic blockade induced by bee venom and cobra venom phospholipase consists mainly of damage to sites of release at the presynaptic membrane. There are also some signs of disturbances of depolarization-secretion coupling and of the process of formation of new quanta. The possible functional role of enzymatic activity in the presynaptic effect is discussed.     

Purification and characterization of a cytotoxic neurotoxin-like protein from Naja haje haje venom that induces mitochondrial apoptosis pathway

This study reported the purification and characterization of a cytotoxic, neurotoxin-like protein derived from the venom of the Egyptian cobra Naja haje haje, Elapidae family, and explored their mechanistic role in the cell death. The protein purification was performed through ion-exchange chromatography and gel-filtration chromatography and was characterized by SDS?CPAGE, amino acid sequencing, and mass spectrum analysis. The antitumor activity of Naja haje venom (NHV) and its fractions (NHVI, NHV-Ia, NHV-Ib, NHV-Ic, NHV-II, NHV-III, and NHV-IV) were tested against different human cancer cell lines. The molecular cascade of cell death was explored through evaluation of apoptosis/necrosis ratio, DNA fragmentation, histone deacetylase (HDAC) activity, mitochondrial transmembrane potential (????_m), cytochrome c release, total caspases, caspase-3, caspase-9, and cell cycle analysis by flow cytometry. Most of the separated fractions possessed variable cytotoxic effect against different cancer cells. The most potent antitumor fraction was NHV-Ic due to its ability to induce DNA damaging and fragmentation that was associated with a significant induction of apoptosis via mitochondrial pathway and disturbed cell cycle phases as well as an inhibition of HDAC activity. NHV-Ic induced the mitochondrial pathway initially by the impairment of ????_m besides the DNA damage and in response to that the mitochondria-released cytochrome c that may in turn activated total caspases, caspase-3 and caspase-9 in lymphoblastic leukemia 1301 cells. The partial amino acid sequencing of NHV-Ic revealed 100, 95.65, and 91.3% homology with the Long neurotoxin 1 from Naja haje anchietae (Angolan cobra), Naja haje haje (Egyptian cobra), and Boulengerina annulata annulata (banded water cobra), respectively.