Biological and biochemical characterization of two new PLA2 isoforms Cdc-9 and Cdc-10 from Crotalus durissus cumanensis snake venom.
This work reports the purification, biological characterization and amino acid sequence of two new basic PLA(2) isoforms, Cdc-9 and Cdc-10, purified from the Crotalus durissus cumanensis venom by one step analytical chromatography reverse phase HPLC. The molecular masses of the PLA(2) were 14,175+/-2.7 Da for Cdc-9 and 14,228+/-3.5 Da for Cdc-10 both deduced by primary structure and confirmed by MALDI-TOF. The isoforms presented an amino acid sequence of 122 amino acid residues, being Cdc-9: SLVQFNKMIK FETRKSGLPF YAAYGCYCGW GGQRPKDATD RCCFVHDCCY GKVAKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLS TYKNEYMFYP DSRCREPPEY TC with pI value of 8.25 and Cdc-10: SLLQFNKMIK FETRKSGVPF YAAYGCYCGW GGRRPKDPTD RCCFVHDCCY GKLTKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLN TYKNEYMFYP DSRCRGPPEY TC with a pI value of 8.46, showing highly conserved Ca(2+)-binding and catalytic sites. The PLA(2) activity decreased when the isoforms Cdc-9 and Cdc-10 were incubated with 4-bromophenacyl bromide (p-BPB), anhydrous acetic acid and p-nitrobenzene sulfonyl fluoride (NBSF) when compared with the activity of both native isoforms. In mice, the PLA(2) isoforms Cdc-9 and Cdc-10 induced myonecrosis and edema. Myotoxic and edema activities were reduced after treatment of the isoforms with p-BPB; acetylation of the lysine residues and the treatment of PLA(2) with NBSF have also induced edema reduction. However, p-BPB strongly diminishes the local and systemic myotoxic effects.
Romero-Vargas
Frey Francisco
FF
Department of Biochemistry, Institute Biology CP 6109, State University of Campinas, Campinas, SP, Brazil.
Ponce-Soto
Luis Alberto
LA
Martins-de-Souza
Daniel
D
Marangoni
Sergio
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D017124:Conserved Sequence; D003435:Crotalid Venoms / Q000737:chemistry / Q000201:enzymology* / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D017839:Crotalus; D004305:Dose-Response Relationship, Drug; D004487:Edema / Q000139:chemically induced; D004795:Enzyme Stability; D007527:Isoenzymes; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000187:drug effects / Q000473:pathology; D009336:Necrosis; D054467:Phospholipases A2 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D011489:Protein Denaturation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003435:Crotalid Venoms; D007527:Isoenzymes; C547196:CDC-10 protein, Crotalus durissus; C547195:CDC-9 protein, Crotalus durissus; D054467:Phospholipases A2; D002118:Calcium