Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom.
An acidic (pI approximately 4.5) phospholipase A(2) (BthA-I-PLA(2)) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an approximately 13.7kDa single chain Asp49 PLA(2) with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 1SLWQFGKMINYVM-GESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC(51). Crystals of this acidic protein diffracted beyond 2.0A resolution. These crystals are monoclinic and have unit cell dimensions of a=33.9, b=63.8, c=49.1A, and beta=104.0 degrees. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3-4 times more active than BthTX-II, a basic D49 myotoxic PLA(2) from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA(2) caused a hypotensive response in the rat and inhibited platelet aggregation. Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA(2), while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA(2) homologues and that either of these two activities may exist alone. BthA-I-PLA(2), in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies.
Andrião-Escarso
Sílvia H
SH
Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo, 14049-900 Ribeirão Preto-SP, Brazil.
Soares
Andreimar M
AM
Fontes
Marcos R M
MR
Fuly
André L
AL
Corrêa
Fernando M A
FM
Rosa
José C
JC
Greene
Lewis J
LJ
Giglio
José R
JR
D000595:Amino Acid Sequence; D000818:Animals; D000959:Antihypertensive Agents / Q000737:chemistry / Q000378:metabolism; D017837:Bothrops; D003435:Crotalid Venoms / Q000737:chemistry* / Q000201:enzymology; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D000959:Antihypertensive Agents; D003435:Crotalid Venoms; D010975:Platelet Aggregation Inhibitors; D010741:Phospholipases A