Enzymatic properties of stingray Dasyatis pastinaca group V, IIA and IB phospholipases A2: A comparative study

In the present study, we have purified the group V phospholipase from the heart of cartilaginous fish stingray Dasyatis pastinaca and compared its biochemical properties with group IIA (sPLA(2)-IIA) and IB (sPLA(2)-IB) phospholipases previously purified from pancreas and intestine, respectively. Group V phospholipase (sPLA(2)-V) was purified to homogeneity by heat treatment, ammonium sulphate precipitation and RP-HPLC. The N-terminal sequence of the purified sPLA(2)-V exhibits a high degree of homology with those of mammal. The enzyme was found to be monomeric with a molecular mass estimation of 14 kDa. The specific activity of the purified enzyme, measured at pH 8 and 37 degrees C was 52 U/mg. Like sPLA(2)-IB and sPLA(2)-IIA, the sPLA(2)-V is found to be stable between pH 3 and 11 after 30 mm of incubation. The purified sPLA(2)-V retained 65% of its activity after 10 mm of incubation at 70 degrees C and it absolutely requires Ca2+ for enzymatic activity. In addition it displayed high tolerance to organic solvents. Kinetic parameters K-mapp, k(cat) and the deduced catalytic efficiency (k(cat)/K-mapp) of the purified group-V, -IB and -IIA PLA(2)s were determined using phosphatidylethanolamine (PE), phosphatidylcholine (PC) or phosphatidylserine (PS) as substrate. The three enzymes hydrolyze the zwiterionic PE and PC substrates more efficiently than anionic PS substrate. (C) 2013 Elsevier B.V. All rights reserved.