Kunze N, Kleinkauf H, Bauer K
Eur J Biochem. 1986 Nov 3;160(3):605-13. doi: 10.1111/j.1432-1033.1986.tb10081.x.
From rat brain extracts, two carnosine-degrading enzymes have been identified and partially purified by ion-exchange chromatography, hydrophobic interaction chromatography on phenyl-Sepharose CL-4B and gel filtration. These enzymes exhibit distinct differences in their chemical characteristics and substrate specificities. One enzyme, designated carnosinase, preferentially hydrolyzes carnosine and exhibits a low Km value (0.02 mM) towards this substrate. Carnosinase also degrades anserine but not homocarnosine or homoanserine. The other carnosine-degrading enzyme hydrolyzes beta Ala-Arg considerably faster than carnosine and, therefore, has been tentatively designated beta Ala-Arg hydrolase. This enzyme exhibits high Km values with carnosine (Km = 25 mM) and beta Ala-Arg (Km = 2 mM). Homocarnosine and gamma-aminobutyryl-arginine are not degraded by beta Ala-Arg hydrolase. Neither enzyme is inhibited by agents reactive on activated hydroxyl groups, such as diisopropyl fluorophosphate, and also not by a variety of peptidase inhibitors of microbial origin or from other sources. Carnosinase is also not inhibited by bestatin but beta Ala-Arg hydrolase, although not an aminopeptidase, is strongly inhibited by this aminopeptidase inhibitor (IC50 = 50 nM). While carnosinase is strongly inhibited by thiol-reducing agents such as dithioerythritol and 2-mercaptoethanol, beta Ala-Arg hydrolase is stabilized and activated by these substances. Both enzymes are strongly inhibited by metal-chelating agents. Carnosinase, however, is not dependent on exogeneously added metal ions and is strongly inhibited by Mn2+ as well as by heavy metal ions. In contrast, beta Ala-Arg hydrolase requires Mn2+ ions for full enzymatic activity. Based on these differences, selective incubation conditions could be evaluated in order to determine specifically both enzyme activities in crude tissue extracts. In rat, both enzymes are present in all tissues tested, except skeletal muscles, but considerable differences in their relative distribution among different tissues are also observed.
从大鼠脑提取物中，通过离子交换色谱、苯基 - 琼脂糖CL - 4B上的疏水相互作用色谱和凝胶过滤，已鉴定出两种肌肽降解酶并进行了部分纯化。这些酶在化学特性和底物特异性方面表现出明显差异。一种酶，称为肌肽酶，优先水解肌肽，对该底物表现出低Km值（0.02 mM）。肌肽酶也降解鹅肌肽，但不降解高肌肽或高鹅肌肽。另一种肌肽降解酶水解β - 丙氨酰 - 精氨酸的速度比肌肽快得多，因此，暂时被命名为β - 丙氨酰 - 精氨酸水解酶。这种酶对肌肽（Km = 25 mM）和β - 丙氨酰 - 精氨酸（Km = 2 mM）表现出高Km值。高肌肽和γ - 氨基丁酰 - 精氨酸不被β - 丙氨酰 - 精氨酸水解酶降解。这两种酶都不受对活性羟基有反应的试剂（如二异丙基氟磷酸）的抑制，也不受多种微生物来源或其他来源的肽酶抑制剂的抑制。肌肽酶也不受贝抑素抑制，但β - 丙氨酰 - 精氨酸水解酶虽然不是氨肽酶，但被这种氨肽酶抑制剂强烈抑制（IC50 = 50 nM）。虽然肌肽酶被硫醇还原剂（如二硫苏糖醇和2 - 巯基乙醇）强烈抑制，但β - 丙氨酰 - 精氨酸水解酶被这些物质稳定化并激活。两种酶都被金属螯合剂强烈抑制。然而，肌肽酶不依赖于外源添加的金属离子，并且被Mn2 +以及重金属离子强烈抑制。相比之下，β - 丙氨酰 - 精氨酸水解酶需要Mn2 +离子才能具有完全的酶活性。基于这些差异，可以评估选择性孵育条件，以便在粗组织提取物中特异性地测定两种酶的活性。在大鼠中，除骨骼肌外，两种酶存在于所有测试组织中，但在不同组织中的相对分布也观察到相当大的差异。
