Higgs H N, Han M H, Johnson G E, Glomset J A
Howard Hughes Medical Institute, Department of Medicine, University of Washington, Seattle, Washington 98195-7370, USA.
J Biol Chem. 1998 Mar 6;273(10):5468-77. doi: 10.1074/jbc.273.10.5468.
We report the molecular cloning and expression of a phosphatidic acid-preferring phospholipase A1 from bovine testis. The open reading frame encoded an 875-amino acid protein with a calculated molecular mass of 97,576 daltons and a pI of 5.61. The sequence included a region similar to a lipase consensus sequence containing the putative active site serine and also included a potential, coiled-coil-forming region. Expression of the open reading frame in COS1 cells resulted in a 20-44-fold increase in phosphatidic acid phospholipase A1 activity over that of control cells. Mutation of the putative active site serine (amino acid 540) demonstrated that it was essential for this increase in enzyme activity. Northern blot analysis revealed at least five different messages with the highest overall message levels in mature testis, but detectable message in all tissues examined. Two possible alternately spliced regions in the open reading frame also were identified. Finally, a search of the data base identified six related proteins: a potential counterpart of the phospholipase A1 in Caenorhabditis elegans, two putative lipases in yeast, and three proteins separately encoded by the Drosophila retinal degeneration B gene and its mouse and human homologues.
我们报道了从牛睾丸中克隆并表达一种偏好磷脂酸的磷脂酶A1。该开放阅读框编码一个含有875个氨基酸的蛋白质，计算分子量为97,576道尔顿，等电点为5.61。该序列包含一个与脂肪酶共有序列相似的区域，其中含有推定的活性位点丝氨酸，还包含一个潜在的卷曲螺旋形成区域。在COS1细胞中表达该开放阅读框导致磷脂酸磷脂酶A1活性比对照细胞增加20 - 44倍。推定的活性位点丝氨酸（第540位氨基酸）的突变表明，它对于酶活性的这种增加至关重要。Northern印迹分析显示至少有五种不同的转录本，在成熟睾丸中的总体转录本水平最高，但在所检测的所有组织中均可检测到转录本。还在开放阅读框中鉴定出两个可能的可变剪接区域。最后，数据库搜索鉴定出六种相关蛋白质：秀丽隐杆线虫中磷脂酶A1的一个潜在对应物、酵母中的两个推定脂肪酶，以及分别由果蝇视网膜变性B基因及其小鼠和人类同源物编码的三种蛋白质。
