Purification, characterization, and cDNA cloning of acidic platelet aggregation inhibiting phospholipases A(2) from the snake venom of Vipera lebetina (Levantine viper).
Two novel acidic phospholipase A(2)s (PLA(2)) were isolated by size exclusion chromatography and reversed-phase chromatography from the crude Vipera lebetina venom. The molecular masses of VLPLA(2)-1 (13,704 Da) and VLPLA(2)-2 (13,683 Da) and their internal tryptic peptides were determined by MALDI-TOF mass-spectrometry. When tested in human platelet-rich plasma, both enzymes showed a potent inhibitory effect on aggregation induced by ADP and collagen. Chemical modification with p-bromophenacylbromide abolished the enzymatic activity of PLA(2); its anti-platelet activity was fully inhibited in case of collagen as inducer and partially inhibited in case of ADP as inducer. The complete cDNAs encoding PLA(2) were cloned from a single venom gland cDNA library. Complete amino acid sequences of the VLPLA(2) were deduced from the cDNA sequences. The full-length cDNA sequences of the VLPLA(2) possess 615bp and encode an open reading frame of 138 amino acids that include signal peptide (16 amino acids) and mature enzyme (122 amino acids). The VLPLA(2)s have significant sequence similarity to many other phospholipase A(2)s from snake venoms. The phylogenetic analysis on the basis of the amino acid sequence homology demonstrates that VLPLA(2)s grouped with other Asp49 PLA(2)s and they appear to share a close evolutionary relationship with the European vipers.
Vija
Heiki
H
National Institute of Chemical Physics and Biophysics, Laboratory of Bioorganic Chemistry, Akadeemia tee 23, Tallinn 12618, Estonia.
Samel
Mari
M
Siigur
Ene
E
Aaspõllu
Anu
A
Trummal
Katrin
K
Tõnismägi
Külli
K
Subbi
Juhan
J
Siigur
Jüri
J
D000098:Acetophenones / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003094:Collagen / Q000494:pharmacology; D018076:DNA, Complementary / Q000737:chemistry; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D054467:Phospholipases A2 / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D010802:Phylogeny; D010974:Platelet Aggregation / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014757:Viper Venoms / Q000737:chemistry / Q000201:enzymology*; D017819:Viperidae / Q000235:genetics / Q000378:metabolism*
D000098:Acetophenones; D018076:DNA, Complementary; D014757:Viper Venoms; D003094:Collagen; D054467:Phospholipases A2; C006821:4-bromophenacyl bromide