Purification and characterization of L-amino acid oxidase from Agkistrodon blomhoffii ussurensis snake venom of Changbai Mountains

The L-a. a, oxidase of Agkistrodon blomhof fii ussurensis of Changbai Mountains in northeast of China has been separated by using ion-exchange and gel filtration techniques, This enzyme is composed of two subunits, the molecular weight of one subunit is about 36 000, the another is about 57 000, determined by sodium dodecyl sulfate-polyacryamide gel electrophoresis and matrix assisted laser desorption ion/time of flight mass spectrometry, The activity of L-a, a. oxidase determined using L-Leu as substrate. The optimal pH of the enzyme is 4. 5 similar to 5. 5 and 8 similar to 9. The UV-Visible absorption spectrum of L-a, a. oxidase shows the characteristics of flavor-proteins.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of phospholipase A(2) and fibrinolytic enzyme, two enzymes obtained from Chinese Agkistrodon blomhoffii Ussurensis venom

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to analyze two enzymes, phospholipase AZ and fibrinolytic enzyme isolated from Chinese Agkistrodon blomhoffii Ussurensis venom. Using sinapinic acid as the matrix, positive ion mass spectra of the enzymes were obtained, In addition to the dominant protein [M+H](+) ions, multimeric and multiply charged ions were also observed in the mass spectra, The higher the concentration of the enzymes, the more multiply charged polymer and multimeric ions were detected, Our results indicate that MALDI-TOFMS can provide a rapid and accurate method for molecular weight determination of snake venom enzymes, Mass accuracies of 0.1 and 0.3 % were achieved by analysis of highly dialyzed phospholipase A2 and fibrinolytic enzyme, and these results are much better than those obtained using sodium dodecyl sulfate-palyacrylamide gel electrophoresis. MALDI-TOFMS thus provides a reliable method to determine the purity and molecular weight of these enzymes, which are of potential use as therapeutants, Copyright (C) 1999 John Wiley & Sons, Ltd.

Isolation and characterization of venom from nematocysts of jellyfish Rhopilema esculentum Kishinouye

The present work is the first report of the biochemical characterization of the venom from nematocysts of the jellyfish Rhopilema esculentum Kishinouye. The nematocysts were isolated by autolysis and centrifugation and separated by flow cytometry. Four types of nematocysts were identified: mastigophores, euryteles, and atrichous and holotrichous isorhiza. SDS-PAGE and amino acid analyses demonstrated that most of the proteins in the nematocyst extract were between 10 kDa and 40 kDa, and that glutamic acid was the main amino acid. A hemolytic activity assay showed that the activity of the nematocyst venom (RNV) was strongest in Tris-HCl buffer (50 mmol/L, pH 7.8, 5% glycerol, 0.5 mmol/L EDTA, 0.1 mol/L NaCl). The hemolytic activity was related to protein concentration and the HU50 against chicken erythrocytes was 0.91 A mu g/mL.

Comparative analysis of methods for concentrating venom from jellyfish Rhopilema esculentum Kishinouye

In this study, several methods were compared for the efficiency to concentrate venom from the tentacles of jellyfish Rhopilema esculentum Kishinouye. The results show that the methods using either freezing-dry or gel absorption to remove water to concentrate venom are not applicable due to the low concentration of the compounds dissolved. Although the recovery efficiency and the total venom obtained using the dialysis dehydration method are high, some proteins can be lost during the concentrating process. Comparing to the lyophilization method, ultrafiltration is a simple way to concentrate the compounds at high percentage but the hemolytic activities of the proteins obtained by ultrafiltration appear to be lower. Our results suggest that overall lyophilization is the best and recommended method to concentrate venom from the tentacles of jellyfish. It shows not only the high recovery efficiency for the venoms but high hemolytic activities as well.

Factors affecting the protease activity of venom from jellyfish Rhopilema esculentum Kishinouye

In this paper, the effects of some chemical and physical factors such as temperature, pH values, glycerol, and divalent metal cations on the protease activity of venom from jellyfish, Rhopilema esculentum Kishinouye, were assayed. Protease activity was dependent on temperature and pH values. Zn2+, Mg2+, and Mn2+ in sodium phosphate buffer (0.02 M, pH 8.0) could increase protease activity. Mn2+ had the best effects among the three metal cations and the effect was about 20 times of that of Zn2+ or Mg2+ and its maximal protease activity was 2.3 x 10(5) U/mL. EDTA could increase protease activity. PMSF had hardly affected protease activity. O-Phenanthroline and glycerol played an important part in inhibiting protease activity and their maximal inhibiting rates were 87.5% and 82.1%, respectively. (c) 2005 Elsevier Ltd. All rights reserved.

Insecticidal activity of proteinous venom from tentacle of jellyfish Rhopilema esculentum Kishinouye

Insecticidal activity of proteinous venom from tentacle of jellyfish Rhopilema esculentum Kishinouye was determined against three pest species, Stephanitis pyri Fabriciusa, Aphis medicaginis Koch, and Myzus persicae Sulzer. R. esculentum full proteinous venom had different insecticidal activity against S. pyri Fabriciusa, A. niedicaginis Koch, and M. persicae Sulzer. The 48 It LC50 values were 123.1, 581.6, and 716.3 mu g/mL, respectively. Of the three pests, R. esculentuin full proteinous venom had the most potent toxicity against S. pyri Fabriciusa, and the corrected mortality recorded at 48 It was 97.86%. So, S. pyri Fabriciusa could be a potential target pest of R. esculentum full proteinous venom. (c) 2005 Elsevier Ltd. All rights reserved.

Factors influencing hemolytic activity of venom from the jellyfish Rhopilema esculentum Kishinouye

In this study, hemolytic activity of venom from the jellyfish Rhopilema esculentum Kishinouye and some factors affecting it were assayed. The HU50 of R. esculentum full venom (RFV) against chicken erythrocytes was 3.40 mu g/ml and a Hill coefficient value was 1.73 suggesting at least two molecules participated in hemolytic activity. The hemolytic activity of RFV was affected by some chemical and physical factors such as divalent cations, EDTA, (NH4)(2)SO4, pH and temperature. In the presence of Mg2+, Cu2+, Zn2+, Fe2+, Ca2+ ( >= 2 mM), Mn2+ (>= 1 mM), EDTA (>= 2 mM) and (NH4)(2)SO4, the hemolytic activity of RFV was reduced. RFV had strong hemolytic activity at the pH 6-10 and the hemolytic ratios were 0.95-1.19. Hemolytic activity was temperature-sensitive and when RFV was pre-incubated at temperatures over 40 degrees C, it was sharply reduced. (c) 2007 Elsevier Ltd. All rights reserved.

Partial characterization of the hemolytic activity of the nematocyst venom from the jellyfish Cyanea nozakii Kishinouye

Using a recently developed technique to extract jellyfish venom from nematocysts, the present study investigated the hemolytic activity of Cyanea nozakii Kishinouye nematocyst venom on chicken erythrocytes. Venom extract caused a significant concentration-dependent hemolytic effect. The extract could retain its activity at -80 degrees C but was unstable when kept at 4 degrees C and -20 degrees C for 2 days. The hemolytic activity was inhibited by heating within the range of 37-100 degrees C. The extract was active over a pH range of 5.0-8.63 and the pH optima for the extract was 7.8. Incubation of the venom with sphingomyelin specially inhibited hemolytic activity by up to 70%. Cu2+ and Mn2+ greatly reduced the hemolytic activity while Mg2+, Sr2+ and Ba2+ produced a relatively low inhibiting effect on the hemolytic activity. Treatment with Ca2+ induced a concentration-dependent increase in the hemolytic activity. In the presence of 5 mM EDTA, all the hemolytic activity was lost, however, the venom containing 1.5 mM EDTA was stable in the long-term storage. PLA(2) activity was also found in the nematocyst venom of C. nozakii. These characteristics provide us a fundamental knowledge in the C. nozakii nematocyst venom which would benefit future research. (C) 2010 Published by Elsevier Ltd.

Isolation and cloning of exendin precursor cDNAs from single samples of venom from the Mexican beaded lizard (Heloderma horridum) and the Gila Monster (Heloderma suspectum)

Reptile venoms are complex cocktails of bioactive molecules, including peptides. While the drug discovery potential of most species remains unrealized, many are endangered and afforded protection under international treaties. In this study, we describe how potential clinically important bioactive peptides and their corresponding mRNAs can be structurally characterized from single, small samples of reptile venom. The potential type-2 diabetes therapeutics, exendin-3 and exendin-4, from the Mexican beaded lizard (Heloderma horridum) and the Gila monster (Heloderma suspectum), respectively, have been characterized at both protein and nucleic acid levels to illustrate the efficacy of the technique and its contribution to biodiversity conservation.

Biotransformation of maximakinin, a bradykinin-related nonadecapeptide from toad venom, by mammalian kallikrein and salivary proteases

Maximakinin is an N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the venom of a Chinese toad (Bombina maxima) that displays highly selective activity at mammalian arterial smooth muscle receptors. In this study, we report that incubation of maximakinin with either kallikrein or human saliva generates catabolites with enhanced bioactivity that retain the tissue selective effects of the parent molecule. In addition, we have observed that kallikrein rapidly cleaves the C-terminal arginyl residue of both maximakinin and bradykinin – a cleavage hitherto considered to be performed by a carboxypeptidase that facilitates selective bradykinin receptor targeting. Maximakinin has thus evolved as a `smart' defensive weapon in the toad with inherent resistance to the signal-terminating protease hardware in the potential predator. Thus, natural selection of amphibian skin peptides for antipredator defence, through interspecies delivery by an exogenous secretory mode, produces subtle structural stabilization modifications that can potentially provide new insights for the design of orally active and selectively targeted peptide therapeutics.

Molecular cloning of a novel putative potassium channel-blocking neurotoxin from the venom of the North African scorpion, Androctonus amoreuxi

Scorpion venoms are a particularly rich source of neurotoxic proteins/peptides that interact in a highly specific fashion with discrete subtypes of ion channels in excitable and non-excitable cells. Here we have employed a recently developed technique to effect molecular cloning and structural characterization of a novel putative potassium channel-blocking toxin from the same sample of venom from the North African scorpion, Androctonus amoreuxi. The deduced precursor open-reading frame is composed of 59 amino acid residues that consists of a signal peptide of approximately 22 amino acid residues followed by a mature toxin of 37 amino acid residues. The mature toxin contains two functionally important residues (Lys27 and Tyr36), constituting a functional dyad motif that may be critical for potassium channel-blocking activity that can be affirmed from structural homologs as occurring in the venoms from other species of Androctonus scorpions. Parallel proteomic/transcriptomic studies can thus be performed on the same scorpion venom sample without sacrifice of the donor animal.

The smooth muscle pharmacology of maximakinin, a receptor-selective, bradykinin-related nonadecapeptide from the venom of the Chinese toad, Bombina maxima.

Structural homologues of vertebrate regulatory peptides found in defensive skin secretions of anuran amphibians often display enhanced bioactivity and receptor binding when compared with endogenous mammalian peptide ligands. Maximakinin, a novel N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the skin venom of a Chinese toad (Bombina maxima), displays such activity enhancement when compared with bradykinin but is additionally highly selective for mammalian arterial smooth muscle bradykinin receptors displaying a 50-fold increase in molar potency in this smooth muscle type. In contrast, a 100-fold decrease in molar potency was observed at bradykinin receptors in intestinal and uterine smooth muscle preparations. Maximakinin has thus evolved as a “smart” defensive weapon in the toad with receptor/tissue selective targeting. Natural selection of amphibian skin venom peptides for antipredator defence, through inter-species delivery by an exogenous secretory mode, produces subtle structural stabilisation modifications that can potentially provide new insights for the design of selectively targeted peptide therapeutics.

Isolation of scorpion (Androctonus amoreuxi) alpha-neurotoxins and parallel cloning of their respective cDNAs from a single sample of venom

The venoms of buthid scorpions are known to contain basic, single-chain protein toxins (alpha toxins) consisting of 60–70 amino acid residues that are tightly folded by four disulfide bridges. Here we describe isolation and sequencing of three novel putative alpha toxins (AamH1-3) from the venom of the North African scorpion, Androctonus amoreuxi, and subsequent cloning of their precursor cDNAs from the same sample of venom. This experimental approach can expedite functional genomic analyses of the protein toxins from this group of venomous animals and does not require specimen sacrifice for cloning of protein toxin precursor cDNAs.