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 (p15-1) 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. 1.5μM) 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.