This proposal comprises two subproposals that use similar methods. 1. Mammalian dipeptidyl peptidase IV (DPP IV) is a highly glycosylated serine protease, that releases N-terminal dipeptides from oligopeptides. It is both an integral membrane protein (CD26) and a plasma protein, the physiological role of which is unknown (Hosono et at., 1999). Among its diverse physiological roles, DPP IV removes X-Pro, His-Ala or tyr-Ala dipeptides from the N-termini of hormones such as neuropeptide Y and substance P. It was discovered in snake venoms by Jorge da Silva and Aird (2000). DPP IVs role in venom may be to prevent a hypertensive response on the part of the envenomated prey by inactivating vasoconstrictive peptidyl hormones (Aird, 2002). It may also contribute to the persistent hypotension seen in human envenomation and to inflammation. The present study should shed light on the role of human soluble DPP IV. 2. Snake venom enzymes that cleave phosphate esters were first discovered when venom phosphodiesterase (PDE) was reported by Gulland and Jackson (1938). Despite this long history, PDE's role in envenomation remained enigmatic until Aird (2002) proposed that its function might be to release endogenous purines, which would immobilize the prey via hypotension and suppression of neurotransmitter release. Snake venom PDE has attracted great interest because of its utility in nucleic acid studies (>2,400 Medline citations). Despite its near reagent status, its structure is unknown. Most forms of PDE are membrane-bound or cytosolic, whereas venom PDE is extremely soluble, so as to function in the extracellular fluid of prey organisms. It has broad substrate specificity [Razzell and Khorana, 1959;Laskowski, 1980], making it well suited to the rapid liberation of adenosine nucleotides from various oligonucleotide precursors, a central theme in envenomation (Aird, 2002). It also has pyrophosphatase activity [Laskowski, 1980], releasing nucleotides and pyrophosphate from nucleoside triphosphates. Nucleotides are rapidly degraded to nucleosides by 5'-nuc!eotidase, present in both venom and prey tissues. Venom PDE was the first enzyme reported to have an active site threonine forming a covalent (phosphorylated) intermediate [Burgers et al., 1979;Gulp and Butler, 1986]. These functional characteristics suggest that it is likely to be structurally unique.
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