Purification and characterization of a cysteine-rich secretory protein from Philodryas patagoniensis snake venom.

Cysteine-rich secretory proteins (CRiSPs) are widespread in reptile venoms, but most have functions that remain unknown. In the present study we describe the purification and characterization of a CRiSP (patagonin) from the venom of the rear-fanged snake Philodryas patagoniensis, and demonstrate its biological activity. Patagonin is a single-chain protein, exhibiting a molecular mass of 24,858.6 Da, whose NH(2)-terminal and MS/MS-derived sequences are nearly identical to other snake venom CRiSPs. The purified protein hydrolyzed neither azocasein nor fibrinogen, and it could induce no edema, hemorrhage or inhibition of platelet adhesion and aggregation. In addition, patagonin did not inhibit contractions of rat aortic smooth muscle induced by high K(+). However, it caused muscular damage to murine gastrocnemius muscle, an action that has not been previously described for any snake venom CRiSPs. Thus, patagonin will be important for studies of the structure-function and evolutionary relationships of this family of proteins that are widely distributed among snake venoms.

Purification and characterization of a cysteine-rich secretory protein from Philodryaspatagoniensis snake venom

Cysteine-rich secretory proteins (CRiSPs) are widespread in reptile venoms, but most have functions thatremain unknown. In the present study we describe the purification and characterization of a CRiSP(patagonin) from the venom of the rear-fanged snake Philodryas patagoniensis, and demonstrate itsbiological activity. Patagonin is a single-chain protein, exhibiting a molecular mass of 24,858.6 Da, whoseNH2-terminal and MS/MS-derived sequences are nearly identical to other snake venom CRiSPs. The purifiedprotein hydrolyzed neither azocasein nor fibrinogen, and it could induce no edema, hemorrhage or inhibitionof platelet adhesion and aggregation. In addition, patagonin did not inhibit contractions of rat aortic smoothmuscle induced by high K+. However, it caused muscular damage to murine gastrocnemius muscle, an actionthat has not been previously described for any snake venom CRiSPs. Thus, patagonin will be important forstudies of the structure-function and evolutionary relationships of this family of proteins that are widelydistributed among snake venoms.

Pharmacologic and molecular characterization of the vascular ETA receptor in the venomous snake Bothrops jararaca.

Endothelins (ETs) and sarafotoxins (SRTXs) are active isopeptides that have very similar structures and functions. All isoforms interact with two specific G-protein-coupled receptors, ET(A) and ET(B). To characterize functional vascular ET receptors in the poisonous snake, Bothrops jararaca, cumulative concentration-response curves to ETs and SRTXs were performed in isolated aortic rings, in the absence and presence of selective ET receptor antagonists. Vascular expression of ET receptor messenger RNA (mRNA) was evaluated by reverse transcriptase (RT) polymerase chain reaction (PCR) analysis, and a fragment of the ET(A) receptor was cloned and sequenced. In vivo, ET-1 induced a dose-dependent biphasic response on anesthetized B. jararaca snakes. In vitro, ET-1, SRTX-b, ET-3, SRTX-c, and IRL-1620 induced concentration-dependent vasoconstriction, with a potency order suggesting the presence of typical ET(A) receptors. BQ-123, a selective ET(A) antagonist, inhibited contractions induced by ET-1 and SRTX-b with expected negative log of the dissociation constant, K(B), (pK(B)) values for mixed ET(A)/ET(B) receptor populations. The nonselective ET(A)/ET(B) receptors antagonist, PD-142893, produced similar inhibition. The ET(B) antagonist, IRL-1038, potentiated contractile responses to SRTX-c. ET-1 and SRTX-c responses were also potentiated when aortic rings were pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME) plus indomethacin. Processing of the B. jararaca aortic first-strand complementary DNA, by RT-PCR with primers designed from the Gallus gallus ET(A) receptor sequence, enabled isolation, purification, cloning, and sequencing of a single band. The partial sequence of the B. jararaca ET(A) receptor showed a very high sequence similarity with ET(A) receptor sequences from chicken, rat, human, and Xenopus. In conclusion, vascular responses to SRTXs/ETs in the B. jararaca aorta are mediated predominantly, but not exclusively, by typical ET(A) receptors.