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A Gloydius blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences from known ecto-5'-nucleotidases (ecto-5'-NTs). Molecular cloning of ecto-5'-NT from G. blomhoffi brevicaudus venom predicted that it was a glycosyl phosphatidylinositol-anchored membrane protein containing 588 amino acid residues with 7 potential N-linked glycosylation sites. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian ecto-5'-NT sequences. This is the first report of the primary structure of ecto-5'-NT from a reptile. Gel-filtration chromatography of fresh venom from Gloydius blomhoffi blomhoffi, a subspecies of G. blomhoffi, revealed that at least a part of ecto-5'-NT is bound to exosome-like vesicles.

Exosomes are small membrane vesicles (30-100 nm) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We provide here the first evidence for the presence of exosome-like vesicles in snake venom. We isolated vesicles from fresh venom from Gloydius blomhoffii blomhoffii by gel-filtration. We found that the vesicles showed a typical exosome-like size and morphology as analyzed by electron microscopy. We observed that the vesicles contained dipeptidyl peptidase IV, aminopeptidase A, ecto-5'-nucleotidase and actin. Vesicle preparations truncated bioactive peptides such as angiotensin II, substance P, cholecystokinin-octapeptide, glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1. The role of these vesicles is still unknown, but they may affect blood pressure and glucose homeostasis following envenomation.

The aminopeptidase activities of snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis, Bothrops jararaca and Crotalus atrox were investigated. Aminopeptidase A (APA), aminopeptidase B and aminopeptidase N activities were present in all snake venoms. The strongest APA activity was found in venom from G. blomhoffi brevicaudus. The susceptibility to metallopeptidase inhibitors and the pH optimum of the partially purified enzyme from G. blomhoffi brevicaudus venom were similar to those of known APAs from mammals. A G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences in known APAs. Molecular cloning of APA from G. blomhoffi brevicaudus venom predicted that it was a type II integral membrane protein containing 958 amino acid residues with 17 potential N-linked glycosylation sites. It possessed a His-Glu-Xaa-Xaa-His-(Xaa)(18)-Glu zinc binding motif that allowed the classification of this protein as a member of the M1 family of zinc-metallopeptidases, or gluzincins. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian APA sequences. This is the first study to report the primary structure of APA from a reptile.

The major lethal toxins present in the venoms of the red-headed krait, Bungarus flaviceps, and the Malayan krait, Bungarus candidus, have both been purified. Each consists of two polypeptide chains, A and B, joined by a disulfide bond. In the present study, primary structures of these toxins were determined by Edman degradation and by nucleotide sequencing of the cDNA clones. Amino acid sequencing of the N-terminus and enzymatically digested peptides revealed that the A and B chains were highly homologous to those of beta-bungarotoxins (beta-Bgts) from Bungarus multicinctus, respectively. We isolated cDNA clones encoding the A and B chains from both B. flaviceps and B. candidus venom gland cDNA libraries using probes designed based on the cDNA sequence of beta-Bgt from B. multicinctus. Two isoforms of the A chain and one isoform of the B chain were obtained from B. flaviceps, and one isoform of the A chain and two isoforms of the B chain were obtained from B. candidus. Both of the two A chains from B. flaviceps are made up of 119 amino acids and comprise 15 cysteine residues, while the A chains of beta-Bgt from other Bungarus species including B. candidus comprise 13 cysteine residues. The B chains from both species are composed of 59 amino acid residues and comprise seven cysteines. In conclusion, the lethal toxin from B. flaviceps is considered to be a novel isoform of beta-Bgt, which has a different pattern of cysteine residues from known beta-Bgts.

The sequence of a long-chain neurotoxin (71 amino acid residues, 10 half-cystines) from the venom of the African banded water cobra (Boulengerina annulata) was determined by Edman degradation. It exhibits high sequence similarity with long-chain neurotoxins from the venoms of four species of African cobras (genus Naja), which are collectively more similar to the Boulengerina toxin than to those of Asian Naja species. These results are discussed in the light of phylogenetic hypotheses on the relationships of African cobras.

A novel peptide has been isolated from the venom of Agkistrodon halys blomhoffii using a bioassay that monitors the stimulant effect on rat stomach fundus. The 11-amino acid peptide, named blomhotin, was purified to homogeneity by gel-filtration column chromatography and reverse-phase HPLC. The amino acid sequence of blomhotin was determined to be pGlu-Gly-Arg-Pro-Pro-Gly-Pro-Pro-Ile-Pro-Arg, which is similar to that of bradykinin-potentiating peptides which themselves cause no contraction of smooth muscle. The contraction induced by blomhotin showed homologous desensitization, implicating the involvement of a blomhotin-specific site in the response.