Iijima N, Uchiyama S, Fujikawa Y, Esaka M
Faculty of Applied Biological Science, Hiroshima University, Higashihiroshima, Japan.
Lipids. 2000 Dec;35(12):1359-70. doi: 10.1007/s11745-000-0653-4.
Phospholipase A2 (PLA2) activity was investigated in various tissues of male and female red sea bream. In both male and female fishes, the specific activity of PLA2 in the gills was 70 times higher than that in other tissues, such as the adipose tissue, intestine, and hepatopancreas. Therefore, we tried to purify PLA2 from the gill filaments of red sea bream to near homogeneity by sequential chromatography on Q-Sepharose Fast Flow, Butyl-Cellulofine, and DEAE-Sepharose Fast Flow columns, and by reversed-phase high-performance liquid chromatography. Two minor and one major PLA2, tentatively named G-1, G-2 and G-3 PLA2, were purified, and all showed a single band with an apparent molecular mass of approximately 15 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The exact molecular mass values of G-1, G-2, and G-3 PLA2 were 14,040, 14,040 and 14,005 Da, respectively. G-1, G-2, and G-3 PLA2 had a Cys 11 and were all identical in N-terminal amino acid sequences from Ala-1 to Glu-56. A full-length cDNA encoding G-3 PLA2 was cloned by reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends methods, and G-3 PLA2 was found to be classified to group IB PLA2 from the deduced amino acid sequence. G-1, G-2, and G-3 PLA2 had a pH optimum in an alkaline region at around pH 9-10 and required Ca2+ essentially for enzyme activity, using a mixed-micellar phosphatidylcholine substrate with sodium cholate. These results demonstrate that three group I PLA2, G-1, G-2, and G-3 PLA2, are expressed in the gill filaments of red sea bream.
研究了雌雄真鲷不同组织中的磷脂酶A2（PLA2）活性。在雄鱼和雌鱼中，鳃中PLA2的比活性均比其他组织（如脂肪组织、肠道和肝胰腺）高70倍。因此，我们尝试通过在Q-Sepharose Fast Flow、Butyl-Cellulofine和DEAE-Sepharose Fast Flow柱上进行连续色谱分离以及反相高效液相色谱法，从真鲷鳃丝中纯化PLA2至接近均一状态。纯化得到了两种次要的和一种主要的PLA2，暂时命名为G-1、G-2和G-3 PLA2，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，它们均呈现出一条表观分子量约为15 kDa的单一蛋白条带。G-1、G-2和G-3 PLA2的确切分子量分别为14,040、14,040和14,005 Da。G-1、G-2和G-3 PLA2均含有Cys 11，并且从Ala-1到Glu-56的N端氨基酸序列完全相同。通过逆转录酶-聚合酶链反应和cDNA末端快速扩增方法克隆了编码G-3 PLA2的全长cDNA，从推导的氨基酸序列发现G-3 PLA2属于IB组PLA2。使用含有胆酸钠的混合胶束磷脂酰胆碱底物时，G-1、G-2和G-3 PLA2在pH 9 - 10左右的碱性区域具有最佳pH值，并且酶活性基本需要Ca2+。这些结果表明，真鲷鳃丝中表达了三种I组PLA2，即G-1、G-2和G-3 PLA2。
