Mantle D, Lauffart B, Gibson A
Neurochemistry Department, Regional Neurological Centre, Newcastle General Hospital, Newcastle upon Tyne, UK.
Clin Chim Acta. 1991 Feb 28;197(1):35-45. doi: 10.1016/0009-8981(91)90346-e.
The purification and characterization of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle are described. The characteristics of leucyl aminopeptidase were as follows: optimum activity was at pH 9.5 in the presence of 5 mmol/l Mg2+ or 0.5 mmol Mn2+. No activation of enzyme activity was obtained following addition of other divalent cations or sulphhydryl reagents. Only the leucyl-AMC and methionyl-AMC derivatives were appreciably hydrolysed. The mol mass was estimated as 280 kDa. Approx. 50% inhibition of activity was obtained following addition of p-hydroxymercuriphenyl sulphonate (10 mumol/l), N-ethyl maleimide (2 mmol/l), o-phenanthroline (5 mmol/l), bacitracin (1 mmol/l), amastatin (1 microgram/ml) and bestatin (0.1 mumol/l); no inhibition of activity was obtained in the presence of phenylmethanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 microgram/ml) or pepstatin (100 microgram/ml). The following oligopeptides were hydrolysed by the enzyme: luliberin 7-10, proctolin and [Leu5]enkephalin; oligopeptides not appreciably hydrolysed included neurotensin, angiotensin-I, substance-P and bradykinin. Pyroglutamyl aminopeptidase had the following characteristics: optimum activity was at pH 8.5 in the presence of 1 mmol/l dithiothreitol (an absolute requirement for maintenance of enzyme activity). Maximum activity was obtained in the absence of divalent cations. Only the pyroglutamyl-AMC derivative was appreciably hydrolysed. The mol mass of this enzyme was estimated as 22 kDa. Approximately 50% inhibition of activity was obtained on addition of phenanthroline (4 mmol/l) and antipain (7 microgram/ml); no inhibition of activity was obtained following addition of phenyl methanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 microgram/ml) or pepstatin (100 microgram/ml). Only oligopeptides with a pyroglutamyl N-terminal residue (thyroliberin, neurotensin, and luliberin) were hydrolysed by the enzyme.
本文描述了从人体骨骼肌中纯化和鉴定亮氨酰氨肽酶和焦谷氨酰氨肽酶的过程。亮氨酰氨肽酶的特性如下：在存在5 mmol/L Mg2+或0.5 mmol Mn2+的情况下，最适活性pH为9.5。添加其他二价阳离子或巯基试剂后，酶活性未得到激活。只有亮氨酰-AMC和甲硫氨酰-AMC衍生物能被明显水解。估计其分子量为280 kDa。添加对羟基汞苯磺酸盐（10 μmol/L）、N-乙基马来酰亚胺（2 mmol/L）、邻菲啰啉（5 mmol/L）、杆菌肽（1 mmol/L）、氨肽酶抑制剂（1 μg/ml）和苯丁抑制素（0.1 μmol/L）后，活性约被抑制50%；在存在苯甲磺酰氟（1 mmol/L）、 lima豆胰蛋白酶抑制剂（100 μg/ml）或胃蛋白酶抑制剂（100 μg/ml）的情况下，酶活性未被抑制。该酶能水解以下寡肽：促黄体素释放激素7-10、促肠肌肽和[亮氨酸5]脑啡肽；未被明显水解的寡肽包括神经降压素、血管紧张素-I、P物质和缓激肽。焦谷氨酰氨肽酶具有以下特性：在存在1 mmol/L二硫苏糖醇（维持酶活性的绝对必要条件）的情况下，最适活性pH为8.5。在不存在二价阳离子的情况下可获得最大活性。只有焦谷氨酰-AMC衍生物能被明显水解。该酶的分子量估计为22 kDa。添加邻菲啰啉（4 mmol/L）和抑肽酶（7 μg/ml）后，活性约被抑制50%；添加苯甲磺酰氟（1 mmol/L）、 lima豆胰蛋白酶抑制剂（100 μg/ml）或胃蛋白酶抑制剂（100 μg/ml）后，酶活性未被抑制。该酶只能水解具有焦谷氨酰N端残基的寡肽（促甲状腺素释放激素、神经降压素和促黄体素释放激素）。
