Liu Xiao-Long, Pan Hua, Yang Guan-Zhen, Wu Xiang-Fu, Zhou Yuan-Cong
Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 1999;31(1):41-45.
Synthetic oligonucleotides were used to amplify phospholipase A(2) (PLA(2)) gene by RT-PCR from total RNA of snake Agkistrodon acutus venom gland. The PCR products were subcloned and positive clones were screened with acidic PLA(2) gene from Agkistrodon halys Pallas. Finally, four cDNAs of PLA(2) isoenzymes were isolated. Their complete sequences were determined by bidirectional sequencing and their amino acid sequences were deduced. They were designated as A.aAPLA(2)I A.aAPLA(2)II A.aBPLA(2) and A.aLys(49)-PLA(2) according to their isoelectric points calculated by computer and special structure characteristics respectively. The amino acid sequence of 1 10 residues of A.aAPLA(2)I deduced from the cDNA is identical to that of acidic PLA(2) which had been isolated from Agkistrodon acutus. A.aLys(49)-PLA(2) is unique because of the usual Asp(49) is replaced by Lys(49), which may lower its enzymatic activity. Their similarity scores were calculated and compared by computer. The successful cloning of these isoenzymes genes may provide more information for the study on structure-function relationship of PLA(2) family.
用合成的寡核苷酸从尖吻蝮蛇毒腺总RNA中通过RT-PCR扩增磷脂酶A(2)(PLA(2))基因。将PCR产物亚克隆，并用白眉蝮蛇的酸性PLA(2)基因筛选阳性克隆。最终，分离出4种PLA(2)同工酶的cDNA。通过双向测序确定其完整序列，并推导其氨基酸序列。根据计算机计算的等电点和特殊结构特征，它们分别被命名为A.aAPLA(2)I、A.aAPLA(2)II、A.aBPLA(2)和A.aLys(49)-PLA(2)。从cDNA推导的A.aAPLA(2)I的1至10个残基的氨基酸序列与从尖吻蝮蛇中分离出的酸性PLA(2)的氨基酸序列相同。A.aLys(49)-PLA(2)是独特的，因为通常的Asp(49)被Lys(49)取代，这可能会降低其酶活性。通过计算机计算并比较它们的相似性得分。这些同工酶基因的成功克隆可能为PLA(2)家族结构-功能关系的研究提供更多信息。
