Shen F S, Roberts S F, Lindberg I
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112.
J Biol Chem. 1989 Sep 15;264(26):15600-5.
A putative processing enzyme for proenkephalin, with activity directed toward basic residues, was purified over 2000-fold from washed bovine adrenal medullary chromaffin granule membranes. The molecular mass of this membrane-bound adrenal trypsin-like enzyme (mATLE) is 31 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the enzyme is extremely basic, binding to carboxymethyl-Sephadex at pH 8.5. The pH optimum of mATLE using t-butoxycarbonyl-Glu-Lys-Lys-aminomethylcoumarin as a substrate is 8.5-8.7, and its Km value for this substrate is 2.2 mM. mATLE activity was inhibited by soybean trypsin inhibitor, lima bean trypsin inhibitor, and aprotinin but not by metal chelators or thiol-directed reagents. Sequencing of cleavage products released from Peptide B revealed that the enzyme preferentially cleaves between and following the paired basic residues at positions 23 and 24 of Peptide B (thus generating [Met-enkephalin]-Arg-Phe and Arg-[Met-enkephalin]-Arg-Phe). Dynorphin A was cleaved following a single lysine at position 11 but not at the paired arginine site. Our results suggest that mATLE is a trypsin-like serine protease with the specificity appropriate to that of a proenkephalin processing enzyme.
一种假定的脑啡肽原加工酶，其活性针对碱性残基，从洗涤过的牛肾上腺髓质嗜铬颗粒膜中纯化了2000多倍。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，这种膜结合的肾上腺胰蛋白酶样酶（mATLE）的分子量为31 kDa，该酶具有极强的碱性，在pH 8.5时与羧甲基-葡聚糖凝胶结合。以叔丁氧羰基-谷氨酸-赖氨酸-赖氨酸-氨基甲基香豆素为底物时，mATLE的最适pH为8.5 - 8.7，其对该底物的Km值为2.2 mM。mATLE活性受到大豆胰蛋白酶抑制剂、利马豆胰蛋白酶抑制剂和抑肽酶的抑制，但不受金属螯合剂或硫醇导向试剂的抑制。对从肽B释放的裂解产物进行测序表明，该酶优先在肽B的第23和24位成对碱性残基之间及之后进行切割（从而产生[甲硫氨酸-脑啡肽]-精氨酸-苯丙氨酸和精氨酸-[甲硫氨酸-脑啡肽]-精氨酸-苯丙氨酸）。强啡肽A在第11位的单个赖氨酸之后被切割，但在成对的精氨酸位点未被切割。我们的结果表明，mATLE是一种胰蛋白酶样丝氨酸蛋白酶，其特异性与脑啡肽原加工酶的特异性相符。
