Separation of phospholipase A2 in Habu snake venom by glycyrrhizin (GL)-affinity column chromatography and identification of a GL-sensitive enzyme.
By means of glycyrrhizin (GL)-affinity and Mono S column chromatographies (HPLC), at least four GL-binding proteins (p25, p17, p15-1 and p15-2) in the two Superdex fractions (P-II and P-III fractions) from Habu snake venom were selectively purified. By determination of their N-terminal partial amino acid sequences, a metalloprotease (p25) and three GL-binding phospholipases A2 (gbPLA2s) [PA2Y (p17), PA21 (p15-1) and PA2B (p15-2)] were identified. PA2B (lysine-49 PLA2) was found to be the most sensitive to GL because (i) it strongly bound to a GL-affinity column; and (ii) its enzyme activity was selectively inhibited by low dose (ID50 = approx. 1.5 microM) of GL, but not by GA. Furthermore, these three gbPLA2s were phosphorylated by casein kinase II (CK-II) in vitro and GL inhibited the CK-II-mediated stimulation of their enzyme activities in vitro.
Ohtsuki
K
K
Laboratory of Genetical Biochemistry, Kitasato University School of Allied Health Sciences, Sagamihara, Japan.
Abe
Y
Y
Shimoyama
Y
Y
Furuya
T
T
Munakata
H
H
Takasaki
C
C
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D047390:Casein Kinase II; D002846:Chromatography, Affinity; D003435:Crotalid Venoms / Q000737:chemistry* / Q000201:enzymology; D019695:Glycyrrhizic Acid / Q000378:metabolism*; D054505:Group V Phospholipases A2; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017841:Trimeresurus / Q000378:metabolism*; D014757:Viper Venoms
D002352:Carrier Proteins; D003435:Crotalid Venoms; D014757:Viper Venoms; C490099:glycyrrhizin-binding protein, Trimeresurus flavoviridis; C086010:glycyrrhizin-binding proteins; D019695:Glycyrrhizic Acid; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D010741:Phospholipases A; D054505:Group V Phospholipases A2; D054467:Phospholipases A2; C101424:phospholipase A2-V, Trimeresurus