Biochemistry Department, Science College, King Saud University, P.O. Box 22452, Riyadh 11495, Saudi Arabia; Laboratory of Plant Biotechnology Applied to Crop Improvement, Faculty of Science of Sfax, University of Sfax, Sfax 3038, Tunisia.
Int J Biol Macromol. 2013 Nov;62:537-42. doi: 10.1016/j.ijbiomac.2013.10.003. Epub 2013 Oct 10.
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 (sPLA2-IIA) and IB (sPLA2-IB) phospholipases previously purified from pancreas and intestine, respectively. Group V phospholipase (sPLA2-V) was purified to homogeneity by heat treatment, ammonium sulphate precipitation and RP-HPLC. The N-terminal sequence of the purified sPLA2-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 °C was 52 U/mg. Like sPLA2-IB and sPLA2-IIA, the sPLA2-V is found to be stable between pH 3 and 11 after 30 min of incubation. The purified sPLA2-V retained 65% of its activity after 10 min of incubation at 70 °C and it absolutely requires Ca(2+) for enzymatic activity. In addition it displayed high tolerance to organic solvents. Kinetic parameters Kmapp, kcat and the deduced catalytic efficiency (kcat/Kmapp) of the purified group-V, -IB and -IIA PLA2s 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.
在本研究中，我们从软骨鱼黄貂鱼的心脏中纯化了第五组磷脂酶，并将其生化性质与先前分别从胰腺和肠道中纯化的第二 A 组（sPLA2-IIA）和第一 B 组（sPLA2-IB）磷脂酶进行了比较。第五组磷脂酶（sPLA2-V）通过热处理、硫酸铵沉淀和反相高效液相色谱法（RP-HPLC）纯化至均一性。纯化的 sPLA2-V 的 N 末端序列与哺乳动物的高度同源。该酶被发现为单体，分子量估计为 14 kDa。在 pH 8 和 37°C 下测量的纯化酶的比活性为 52 U/mg。与 sPLA2-IB 和 sPLA2-IIA 一样，sPLA2-V 在孵育 30 分钟后在 pH 3 到 11 之间稳定。在 70°C 孵育 10 分钟后，纯化的 sPLA2-V 保留了 65%的活性，并且绝对需要 Ca(2+)才能发挥酶活性。此外，它对有机溶剂具有高耐受性。使用磷脂酰乙醇胺（PE）、磷脂酰胆碱（PC）或磷脂酰丝氨酸（PS）作为底物，测定了纯化的第五组、第一 B 组和第二 A 组 PLA2s 的 Kmapp、kcat 和推导的催化效率（kcat/Kmapp）的动力学参数。三种酶更有效地水解两性离子 PE 和 PC 底物，而不是阴离子 PS 底物。
