Zolfaghari R, Baker C R, Amirgholami A, Canizaro P C, Behal F J
Department of Surgery, Texas Tech University School of Medicine, Lubbock 79430.
Arch Biochem Biophys. 1987 Oct;258(1):42-50. doi: 10.1016/0003-9861(87)90320-1.
A multicatalytic endopeptidase (ME) with three distinct activities, chymotrypsin-like, cucumisin-like, and trypsin-like, occurred in all rat tissues examined with highest activities in kidney, testes, liver, and spleen; they were assayed with benzyloxycarbonyl-Gly-Gly-Leu-p-nitroanilide (Z-Gly-Gly-Leu-pNA), benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide (Z-Leu-Leu-Glu-2NA), and benzyloxycarbonyl-Gly-Gly-Arg-2-naphthylamide (Z-Gly-Gly-Arg-2NA), respectively. All three activities were recovered from a single protein band on a polyacrylamide gel after electrophoresis of purified human kidney ME. The native enzyme had a Mr of 650,000, and it consisted of about 5,135 amino acid residues. After denaturation and electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels kidney ME dissociated into several low Mr components ranging from 23,000 to 33,000. Kidney ME had a pH optimum of 7.6-8.1 with Z-Gly-Gly-Leu-pNA, 7.3 with Z-Leu-Leu-Glu-2NA, and 9.8 with Z-Gly-Gly-Arg-2NA. SDS enhanced chymotrypsin- and cucumisin-like activities by two to three times whereas trypsin-like activity was not enhanced. The specificity constant (kappa cat/Km) of human kidney ME for Z-Gly-Gly-Leu-pNA was 6.7 X 10(3) M-1 S-1; Z-Gly-Gly-Leu-2NA was not hydrolyzed. The specificity constant for Z-Leu-Leu-Glu-2NA was similar to, and for Z-Gly-Gly-Arg-2NA was one half of that for Z-Gly-Gly-Leu-pNA. ME cleaves only the Phe5-Ser6 bond of bradykinin (BK); however, all three ME activities were inhibited by BK. Strong inhibition of ME by albumin suggests that ME is involved in cleavage of larger polypeptides. Antipain and leupeptin almost completely inactivated the trypsin-like activity whereas they had no significant effect on the other two activities. ME is not a metal-loenzyme nor is the serine residue essential for its activities; however, thiol groups are involved. Na+ and K+ inhibited all ME activities. Trypsin-like activity was more sensitive to divalent cations than the other two.
一种具有三种不同活性（类胰凝乳蛋白酶活性、类黄瓜蛋白酶活性和类胰蛋白酶活性）的多催化内肽酶（ME）存在于所有被检测的大鼠组织中，在肾脏、睾丸、肝脏和脾脏中的活性最高；分别用苄氧羰基 - 甘氨酰 - 甘氨酰 - 亮氨酰 - 对硝基苯胺（Z - Gly - Gly - Leu - pNA）、苄氧羰基 - 亮氨酰 - 亮氨酰 - 谷氨酰 - 2 - 萘酰胺（Z - Leu - Leu - Glu - 2NA）和苄氧羰基 - 甘氨酰 - 甘氨酰 - 精氨酰 - 2 - 萘酰胺（Z - Gly - Gly - Arg - 2NA）对其活性进行测定。纯化的人肾脏ME经聚丙烯酰胺凝胶电泳后，所有这三种活性都从单一蛋白条带中回收。天然酶的分子量为650,000，由约5135个氨基酸残基组成。在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶上变性并电泳后，肾脏ME解离成几个分子量较低的组分，范围从23,000到33,000。肾脏ME对Z - Gly - Gly - Leu - pNA的最适pH为7.6 - 8.1，对Z - Leu - Leu - Glu - 2NA为7.3，对Z - Gly - Gly - Arg - 2NA为9.8。SDS使类胰凝乳蛋白酶和类黄瓜蛋白酶活性增强两到三倍，而类胰蛋白酶活性未增强。人肾脏ME对Z - Gly - Gly - Leu - pNA的特异性常数（kappa cat/Km）为6.7×10³ M⁻¹ S⁻¹；Z - Gly - Gly - Leu - 2NA不被水解。对Z - Leu - Leu - Glu - 2NA的特异性常数与之相似，对Z - Gly - Gly - Arg - 2NA的特异性常数是Z - Gly - Gly - Leu - pNA的一半。ME仅切割缓激肽（BK）的Phe5 - Ser6键；然而，所有三种ME活性都被BK抑制。白蛋白对ME的强烈抑制表明ME参与较大多肽的切割。抗蛋白酶和亮抑蛋白酶几乎完全使类胰蛋白酶活性失活，而对其他两种活性没有显著影响。ME不是金属酶，其活性也不需要丝氨酸残基；然而，巯基参与其中。Na⁺和K⁺抑制所有ME活性。类胰蛋白酶活性比其他两种活性对二价阳离子更敏感。
