Amino acid sequence and circular dichroism of Indian cobra (Naja naja naja) venom acidic phospholipase A2

The full amino acid sequence of the acidic phospholipase A2 from Indian cobra (Naja naja naja) venom was determined and its tertiary structure examined by circular dichroism (CD). The sequence was aligned with other sequences of secreted phospholipase A2 from snakes of the genus Naja, using the progressive alignment method of Feng and Doolittle (J. Mol. Evol. (1987) 25, 351-360). The primary sequence of Naja naja naja phospholipases A2 shows up to 85% identity with the other acidic Naja phospholipase A2. CD studies indicate a 40-50% alpha-helical content in a tertiary structure which resists denaturation at high temperature, with or without chaotropic salts.     

Characterization and immunological comparison of isoenzymes of phospholipases A2 from snake venoms of different genera and families.

Phospholipases A2 (PLA2) were isolated and purified from the Taiwan cobra (Naja naja atra) and several snake species of the same or different genera by semipreparative cation-exchange and reversed-phase HPLC. They were shown to possess different enzymatic activity toward the synthetic substrate L-alpha-lecithin by the fatty-acid titration method. The immunological cross-reactivity of these structurally similar isotoxins was investigated using immunodiffusion, precipitin reaction and enzyme-linked immunosorbent assay (ELISA). PLA2 from the venoms of the same species such as the Taiwan cobra (Naja naja atra) and the Indian cobra (Naja naja naja) showed a high degree of antigenic resemblance whereas no immunoreactivity was observed among those PLA2 from different genera. Quantitative immunoreactivity assays by ELISA revealed the partial cross-reactivity between the antibody against PLA2 of Taiwan cobra and those isoenzymes from snakes of remotely-related species. The immunological relatedness between PLA2 of the representative snake species of different genera and families is shown to be correlated with the extent of sequence homology among these enzymes.     

Amino-acid sequences of four cytotoxins (cytotoxins I, II, III and IV) purified from the venom of the Thailand cobra, Naja naja siamensis

Four cytotoxins, designated as cytotoxins I, II, III and IV, were isolated from the venom of the Thailand cobra (Naja naja siamensis) by gel filtration on Sephadex G-75 followed by CM-cellulose chromatography. The amino-acid sequences were determined by a combination of conventional methods. Cytotoxins I, II, III and IV were each composed of 60 amino-acid residues and their molecular weights were calculated to be 6693, 6646, 6709 and 6739, respectively. The amino-acid sequences were compared with those of cytotoxins from other cobra venoms already sequenced.     

Sequence characterization of venom toxins from Thailand cobra

Several toxins with distinct pharmacological properties were isolated from the venom of Thailand cobra (Naja naja siamensis) by cation-exchange chromatography. Two neurotoxins and one basic toxin with cardiotoxic activity were further purified and sequenced. The neurotoxins characterized were closely similar to the previously reported long- and short-chain neutrotoxins. The complete sequences of one minor neurotoxin and one cardiotoxin analogue were determined with the automatic protein sequencer in non-stop single runs of Edman degradation coupled with C-terminal sequence determination with carboxypeptidase digestion. The minor neurotoxin consists of 62 amino-acid residues with 8 cysteine residues and is found to be almost identical to cobrotoxin, a major toxic component of Formosa cobra (Naja naja atra). The sequence comparison of the 60-residue cardiotoxin with other reported cytotoxins of snake venoms indicates that 8 cysteine residues at the positions 3, 14, 21, 38, 42, 53, 54, and 59 are invariant among all sequences, with only two conservative changes at other positions along the sequence. The upshot of this report exemplified the facile sequence analysis of venom toxins by the application of pulsed-liquid phase protein sequencer and also revealed new analogues of a minor neurotoxin and one major cardiotoxin reported previously on the same species of Thailand cobra.     

中华眼镜蛇神经毒素cDNA的克隆及表达

通过ＲＴ－ＰＣＲ的方法,从广西产中华眼镜蛇总ＲＮＡ中克隆到３个新的神经毒素ｃＤＮＡ序列,命名为ＮＬ１、ＮＬ２和ＮＬ３。它们编码的蛋白质序列与台湾眼镜蛇神经毒素ｃｏｂｒｏｔｏｘｉｎ的同源性分别为７７％、７２％和９８％,均具有维持ｃｏｂｒｏｔｏｘｉｎ结构与功能必需的残基Ｔｙｒ＾２５、Ｌｙｓ＾２７、Ｔｒｐ＾２９、Ａｒｇ＾３３和Ｌｙｓ＾４７。ＮＬ３克隆于ｐＥＴ２８ｂ＋表达载体内,转化至ＢＬ２１（ＤＥ３）中     

Amino acid sequence of nerve growth factor purified from the venom of the Formosan cobra Naja naja atra

Nerve growth factor (NGF) was isolated from the venom of the Formosan cobra (Naja naja atra). The amino acid sequence was determined by a combination of conventional methods. The total number of amino acid residues was 116, giving a molecular mass of 13,057 Da. The sequence was identical with that deduced from the nucleotide sequence of an NGF cDNA from the venom gland of Naja naja siamensis, reported by Selby et al. [J. Neurosci. Res., 18, 293-298 (1987)].     

Purification, characterization, and partial amino acid sequence of nerve growth factor from cobra venom.

The nerve growth factor (NGF) from Naja naja (cobra) venom has been purified and its structure compared to the NGF from mouse submaxillary gland. A two-step purification procedure has been devised, consisting of a gel filtration step in 1 M acetic acid followed by chromatography of the active pool on carboxymethylcellulose at pH 5. The molecular weight of the native protein was found to be 28000, and this value was reduced by approximately one-half under denaturing conditions. These values are comparable to those obtained for mouse 2.5S or betaNGF. Tryptic peptide maps of S-[14C]carboxymethyl NGF gave the number of labeled peptides expected for a structure composed of two identical or very similar subunits. Thus, the quaternary structures of mouse and cobra NGF are the same. Cyanogen bromide (CNBr) treatment of Naja naja NGF produced three fragments, of which two were purified to homogeneity. These fragments and the whole protein were analyzed in the automated protein Sequencer. The amino-terminal CNBr fragment of the protein was also subjected to digestion by thermolysin and the resultant peptides were purified and characterized. These data plus those from the characterization of the tryptic peptides provided the basis of the construction of a tentative primary structure of Naja naja NGF which is approximately 60% identical with mouse NGF.     

Isolation and characterization of a lethal phospholipase A2 (NN-IVb1-PLA2) from the Indian cobra (Naja naja naja) venom.

Indian cobra (Naja naja naja) venom is reported to contain multiple forms of phospholipase A2. Only a couple of them have been isolated and characterized. A lethal phospholipase A2 (NN-IVb1-PLA2) from Naja naja naja venom has been purified in three steps involving CM-Sephadex C-25, Sephadex G-50 and rechromatography on CM-Sephadex C-25 columns. It is a basic protein with pl value between 7-7.5 and has molecular weight between 11,000-11,500. The LD50 of NN-IVb1-PLA2 is 1.2 mg/K g body weight. It induces neurotoxic symptoms in the experimental mice and is devoid of myotoxic, anticoagulant, edema inducing and direct hemolytic activities.     

Primary structure and functional properties of cobra (Naja naja naja) venom Kunitz-type trypsin inhibitor.

A trypsin inhibitor from the venom of the cobra Naja naja naja has been isolated by a single step of reverse-phase high-performance liquid chromatography. The protein strongly inhibits trypsin (Ki = 3.5 pM). The primary structure was determined by peptide analysis of the [14C]carboxymethylated inhibitor. The 57-residue polypeptide chain belongs to the family of Kunitz-type inhibitors, and exhibits 42% residue identity with bovine pancreatic trypsin inhibitor. The structure shows only 70% identity with the corresponding peptide from the Capa cobra (Naja nevia), establishing that the inhibitor molecule exhibits extensive variations. Functionally, a basic residue at position P3' correlates with strong inhibition.     

Characterization of a cytotoxin-like basic protein from the cobra (Naja naja naja) venom

A cytotoxin-like basic protein has been isolated from the venom of the nominate race of cobra (Naja naja naja from Pakistan) by a single step of high-performance liquid chromatography. The primary structure was determined and consists of 62 amino acid residues in a single polypeptide chain. It is highly similar to that of the cytotoxin-like basic proteins isolated from other Naja species, but differs in two of the SS-loop structures from that of cytotoxins.     

广东眼镜蛇蛇毒与眼镜王蛇蛇毒冻干粉的鉴别及质量控制的研究 V．广东眼镜蛇蛇毒与眼镜王蛇蛇毒的氨基酸分析

本文采用日立８３５－５０型氨基酸自动分析仪测定了广东眼镜蛇蛇毒与眼镜王蛇蛇毒的氨基酸成分,结果表明两种蛇毒的氨基酸组成基本相同,但多种氨基酸的含量存在明显差异,为蛇毒鉴别和质控提供实验依据。     

Isolation and characterization of an acidic lethal phospholipase Az from Malayan cobra (Naja naja sputatrix) venom

An acidic, lethal phospholipase Az was purified to electrophoretic homogeneity from the venom of the Malayan cobra (Naja naja sputatrix). The enzyme has an isoelectric point of 5.58, a molecular weight of 12000, and a medium lethal dose (LD50) of 0.86 micrograms/g in mice by intravenous injection. The enzyme also exhibited weak anticoagulant and edema-forming activities. The amino acid composition of the enzyme is similar to those of other cobra venom phospholipases Az.     

A simple method to obtain a covalent immobilized phospholipase A(2).

In the present work, we obtained an immobilized phospholipase A(2) system through covalent coupling by using an acrylic polymer Eupergit C as support. The immobilized enzyme from cobra venom (Naja naja naja) showed good retention activity and excellent stability. Both properties are of great importance for biomedical applications such as hypercholesterolemia treatments.     

Activation, aggregation, and product inhibition of cobra venom phospholipase A2 and comparison with other phospholipases

The kinetics of phospholipid hydrolysis by cobra venom phospholipase A2 were examined and compared to those of phospholipase A2 from porcine pancreas, Crotalus adamanteus (rattlesnake) venom, and bee venom. Only the enzyme from Naja naja naja (cobra) venom was found to be activated significantly by phosphorylcholine-containing compounds when hydrolyzing phosphatidylethanolamine. The cobra venom enzyme was also the only one in which these activators induced protein aggregation. The parallel specificity for activators and aggregators suggests that these two phenomena are linked. Product effects were also shown to vary between these four phospholipases. These effects manifest themselves in nonlinear time courses, in changes in steady state velocity, and in the differential effects of serum albumin on reaction rates. Different effects were even seen for the same enzyme when acting on different substrates. A model is presented to account for these observations; its main features are enzyme activation by an activator molecule, whose specificity depends on the enzyme, and an activator-induced aggregation of the enzyme.     

蛇神经毒素的表达和鉴定

抽提中华眼镜蛇毒腺总RNA,通过反转录PCR扩增Cobrotoxin cDNA,克隆并测序。该cDNA编码83个氨基酸,包括21个氨基酸的信号肽和62个氨基酸的成熟蛋白。该成熟蛋白的氨基酸序列和通过蛋白测序从台湾眼镜蛇鉴定的Cobrotoxin完全一致。PCR扩增编码Cobrotoxin的DNA,并亚克隆到表达载体pMAL-P₂。此外,通过合成寡核苷酸片段,拼接成完整的CM11基因,并将其克隆至pMAL-P₂。经IPTG诱导,两种神经毒素基因在大肠杆菌中都得到高效的可溶性融合表达。表达产物通过SDS-PAGE和蛋白印迹杂交加以鉴定。表达的融合蛋白经过Sepharose 6Bamylose亲和色谱和DEAESepharose FF离子交换色谱得到有效纯化。经Xa因子酶切后得到的两种重组神经毒素都具有小白鼠体内毒性。     

Kinetics of the hydrolysis of micellar substrates catalyzed by snake venom phospholipases A2.

Effects of Ca2+ on the kinetic parameters for the hydrolysis of mixed micelles of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (diC16PC) with Triton X-100, catalyzed by a cobra (Naja naja atra) (Group I) and a Habu (Trimeresurus flavoviridis) (Group II) PLA2s, were studied and compared with the results reported for other Group I and II enzymes. The substrate bindings to Group I enzymes were independent of the Ca2+ binding, whereas the substrate bindings to Group II enzymes were facilitated more than 10 times by the Ca2+ binding to the enzymes. The result for Group II enzymes, but not Group I enzymes, seemed compatible with the hypothesis for interpreting the catalytic mechanism that an intermediate complex should be stabilized by the coordination of the bound Ca2+ with the phosphoryl group and the carbonyl oxygen atom of the ester bond at the sn-2 position of the bound substrate molecule [Verheij et al. (1980) Biochemistry 19, 743-750 and (1981) Rev. Physiol. Biochem. Pharmacol. 91, 91-203]. The pH dependence of the kinetic parameters for the hydrolysis of the mixed micellar diC16PC, catalyzed by the cobra (N. naja atra) (Group I) and Habu (T. flavoviridis) (Group II) PLA2s, was also studied. The pK values of the catalytic group, His 48, and Tyr 52 for N. naja atra PLA2, shifted from 7.25 to 7.70 and from 10.30 to 10.85, respectively, and the corresponding values for T. flavoviridis PLA2 shifted from 5.80 to 6.95 and from 10.10 to 10.76, respectively, on binding of the micellar substrates to the enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparative study of cobra (Naja) venom enzymes

1. The L-amino acid oxidase, hyaluronidase, alkaline phosphomonoesterase, protease, phosphodiesterase, acetylcholinesterase, phospholipase A and 5'-nucleotidase activities of 47 samples of venoms from all the six species of cobra (Naja), including five subspecies of Naja naja, were examined. 2. The results demonstrated interspecific differences in the venom contents of phospholipase A, acetylcholinesterase, hyaluronidase and phosphodiesterase. These differences in venom enzyme contents can be used for the differentiation of species of the genus Naja. 3. Thus, our results revealed a correlation between the enzyme composition of venom and the taxonomic status of the snake at the species level for the genus Naja.     

The anticoagulant activity of Malayan cobra (Naja naja sputatrix) venom and venom phospholipase A2 enzymes

Malayan cobra (Naja naja sputatrix) venom was found to exhibit an in vitro anticoagulant activity that was much stronger than most common cobra (genus Naja) venoms. The most potent anticoagulants of the venom are two lethal phospholipase A2 enzymes with pI's of 6.15 and 6.20, respectively. The anticoagulant activity of the venom is due to the synergistic effect of the venom phospholipase A2 enzymes and polypeptide anticoagulants. Bromophenacylation of the two phospholipase A2 enzymes reduced their enzymatic activity with a concomitant drop in both the lethal and anticoagulant activities.     

Major complement inhibiting factors from the venom of the Central Asian cobra Naja naja oxiana

The properties of two anticomplementic factors isolated by CM-Sepharose chromatography from the basic non-adsorbed on DEAE-Sepharose fraction of the Central Asian cobra Naja naja oxiana venom, were studied. Of these three factors (CFB-I, CFB-II and CFB-III) the latter had been characterized earlier. CFB-I was shown to be a protein with an N-terminal Asp and a molecular mass of about 39 kDa (data from gel chromatography); its content in the venom is 3.6 mg/g of dry venom. The protein inhibits mainly the classical pathway of the complement activation, being bound to component C4 (Ki = 9 nM). CFB-I seems to be analogous to the CI inhibitor from the venom of the Naja haje cobra. An analysis of the N-terminal sequence of CFB-II showed it to be identical to the earlier characterized cytotoxin I. CFB-I inhibits the formation of C3 convertase with Ki = 2.2-2.8 microM by way of binding to C4b and thus interfering with the component C2 sorption.