Phospholipase A(2) with platelet aggregation inhibitor activity from Austrelaps superbus venom: protein purification and cDNA cloning.
Four phospholipase A(2) (PLA(2)) enzymes (Superbins a, b, c, and d) with varying platelet aggregation inhibitor activities have been purified from Austrelaps superbus by a combination of gel filtration, ion-exchange, and reversed-phase high-pressure liquid chromatography. Purity and homogeneity of the superbins have been confirmed by high-performance capillary zone electrophoresis and mass spectrometry. The electron spray ionization mass spectrometry data showed that their molecular masses range from 13,140 to 13,236 Da. Each of the proteins has been found to be basic and exhibit varying degrees of PLA(2) activity. They also displayed different platelet aggregation inhibitory activities. Superbin a was found to possess the most potent inhibitory activity with an IC(50) of 9.0 nM, whereas Superbin d was found to be least effective with an IC(50) of 3.0 microM. Superbins b and c were moderately effective with IC(50) values of 0.05 and 0.5 microM, respectively. The amino-terminal sequencing confirmed the identity of these superbins. cDNA cloning resulted in the identification of 17 more PLA(2) isoforms in A. superbus venom. It has also provided complete information on the precursor PLA(2). The precursor PLA(2) contained a 27-amino-acid signal peptide and 117- to 125-amino-acid PLA(2) (molecular mass ranging from 13,000 to 14,000 Da). Two of these PLA(2) enzymes resembled more closely (87%) Superbin a in structure. Two unique PLA(2) enzymes containing an extra pancreatic loop also have been identified among the isoforms.
Singh
S B
SB
Department of Biochemistry, National University of Singapore, 10 Medical Drive, Singapore, 119260.
Armugam
A
A
Kini
R M
RM
Jeyaseelan
K
K
D017836:Agkistrodon / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001315:Australia; D001483:Base Sequence; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology; D003001:Cloning, Molecular; D016384:Consensus Sequence / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004546:Elapid Venoms / Q000201:enzymology* / Q000235:genetics; D054499:Group IA Phospholipases A2; D006801:Humans; D020128:Inhibitory Concentration 50; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D030162:Reptilian Proteins
D018076:DNA, Complementary; D004546:Elapid Venoms; D007527:Isoenzymes; D010975:Platelet Aggregation Inhibitors; D011498:Protein Precursors; D021382:Protein Sorting Signals; D030162:Reptilian Proteins; D010741:Phospholipases A; D054499:Group IA Phospholipases A2; C406281:superbin