Separation of phospholipase A2 in Habu snake venom by glycyrrhizin (GL)-affinity column chromatography and identification of a GL-sensitive enzyme

Abstract

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-l) 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 μM) of GL, but not by GA. Furthermore, these three gbPLA2s were phosphorylated by casein kinase 11 (CK-II) in vitro and GL inhibited the CK-II-mediated stimulation of their enzyme activities in vitro.