Steinhilber W, Dittié A, Lütcke H, Kern H F, Tooze J
Department of Anatomy and Cell Biology, University of Marburg, Federal Republic of Germany.
Eur J Cell Biol. 1990 Apr;51(2):242-51.
We separated by two-dimensional (2D) gel electrophoresis the content of isolated rat zymogen granules and from the gel excised a protein of apparent MW 77,500 and an isoelectric point of about 4.7. A rabbit antiserum against this previously uncharacterized rat zymogen granule protein recognized two cDNA clones in a rat pancreas expression library. The cDNA inserts of these two clones had sequences showing perfect homology to the published cDNA sequence of rat pancreatic lysophospholipase. The antiserum recognized only a single protein, lysophospholipase, on one and two-dimensional immunoblots of rat pancreas homogenates and isolated zymogen granules. The antiserum did not react with any protein in homogenates of rat liver, spleen, adrenal, parotid, and prostate tissue. The zymogen granule protein of the guinea pig, previously identified as Lipase 1, was recognized specifically by the antiserum against rat lysophospholipase. This guinea pig protein can now be regarded as lysophospholipase. The same protein was demonstrated in the transformed rat acinar cell line AR4-2J, where both the rate of total enzyme synthesized and the amount of mRNA increased following treatment with dexamethasone. Immunogold labeling established that pancreatic lysophospholipase is restricted exclusively to exocrine cells where it occurs only in compartments of the exocytotic pathway. It could also be detected in pancreatic juice in the ducts of the tissue. Finally, we have shown that lysophospholipase is not related to the zymogen granule membrane protein GP2. This work establishes that lysophospholipase is a normal member of the set of soluble enzymes and proenzymes that are stored in zymogen granules and secreted into pancreatic juice.
我们通过二维（2D）凝胶电泳分离了分离出的大鼠酶原颗粒的成分，并从凝胶中切下了一种表观分子量为77,500且等电点约为4.7的蛋白质。针对这种先前未鉴定的大鼠酶原颗粒蛋白制备的兔抗血清在大鼠胰腺表达文库中识别出两个cDNA克隆。这两个克隆的cDNA插入片段的序列与已发表的大鼠胰腺溶血磷脂酶的cDNA序列显示出完全同源性。该抗血清在大鼠胰腺匀浆和分离的酶原颗粒的一维和二维免疫印迹上仅识别单一蛋白质溶血磷脂酶。该抗血清与大鼠肝脏、脾脏、肾上腺、腮腺和前列腺组织匀浆中的任何蛋白质均无反应。豚鼠的酶原颗粒蛋白，先前被鉴定为脂肪酶1，被抗大鼠溶血磷脂酶的抗血清特异性识别。这种豚鼠蛋白现在可被视为溶血磷脂酶。在转化的大鼠腺泡细胞系AR4-2J中也证实了相同的蛋白，在用地塞米松处理后，该细胞系中总酶合成速率和mRNA量均增加。免疫金标记证实胰腺溶血磷脂酶仅局限于外分泌细胞，且仅存在于胞吐途径的区室中。在该组织导管的胰液中也可检测到它。最后，我们表明溶血磷脂酶与酶原颗粒膜蛋白GP2无关。这项工作证实溶血磷脂酶是储存在酶原颗粒中并分泌到胰液中的可溶性酶和酶原中的正常成员。
