Morgan E W, Yan B, Greenway D, Petersen D R, Parkinson A
Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City 66160-7417.
Arch Biochem Biophys. 1994 Dec;315(2):495-512. doi: 10.1006/abbi.1994.1531.
The enzymatic hydrolysis of para-nitrophenylacetate by rat liver microsomes is predominantly catalyzed by two esterases: one with high affinity (Km approximately 25 microM) and one with low affinity (Km approximately 400 microM) for the substrate. Two kinetically distinct esterases were similarly detected in liver microsomes from mouse, hamster, guinea pig, rabbit, cat, cynomolgus monkey, and human, but only the high-affinity enzyme was detectable in dog liver microsomes. The tissue distribution of these kinetically distinct esterases was examined in rats. High-affinity (Km 20-35 microM esterase activity toward para-nitrophenylacetate was detected in testis, lung, prostate, and pancreas. The activity in testicular microsomes was comparable to that in liver microsomes. Low-affinity (Km 200-700 microM) esterase activity was detected in kidney, small intestine, lung, spleen, heart, and brain. The activity in kidney microsomes was comparable to that in liver microsomes. The high-affinity esterase in testicular and liver microsomes was highly sensitive to the inhibitory effects of phenylmethylsulfonyl fluoride (PMSF), whereas the low-affinity esterase in kidney and liver microsomes was relatively resistant. These results suggested that rat liver microsomes contain two esterases with high activity toward para-nitrophenylacetate, a PMSF-sensitive esterase with high substrate affinity, and a PMSF-insensitive esterase with low substrate affinity. In support of the hypothesis, we have purified and characterized two esterases, designated hydrolases A and B, which appear be the only abundant enzymes in rat liver microsome that rapidly hydrolyze para-nitrophenylacetate. Hydrolase A hydrolyzed para-nitrophenylacetate with high affinity (Km approximately 25 microM), and was inhibited by extremely low concentrations of PMSF (IC50 approximately 100 nM). In contrast, hydrolase B hydrolyzed para-nitrophenylacetate with low affinity (Km approximately 400 microM) and was inhibited only by relatively high concentrations of PMSF (IC50 approximately 100 microM Paraoxon, the active metabolite of parathion, and cresylbenzodioxaphosphorin oxide, the active metabolite tri-ortho-tolylphosphate, completely inhibited the hydrolysis of pra-nitrophenylacetate by rat liver microsomes and by hydrolases A and B, whereas the sulfhydryl agent, para-chloromercurobenzoate, was not inhibition. These results suggest that hydrolases A and B are both serine esterases. The N-terminal amino acid sequence of hydrolases A and B were similar but distinct (23 the first 30 amino acid residues were identical), indicating that these two esterases are isozymes.(ABSTRACT TRUNCATED AT 400 WORDS)
一种对底物具有高亲和力（Km约为25微摩尔），另一种对底物具有低亲和力（Km约为400微摩尔）。在小鼠、仓鼠、豚鼠、兔子、猫、食蟹猴和人类的肝脏微粒体中同样检测到两种动力学特性不同的酯酶，但在狗的肝脏微粒体中仅能检测到高亲和力酶。在大鼠中研究了这些动力学特性不同的酯酶的组织分布。在睾丸、肺、前列腺和胰腺中检测到对乙酸对硝基苯酯具有高亲和力（Km为20 - 35微摩尔）的酯酶活性。睾丸微粒体中的活性与肝脏微粒体中的相当。在肾脏、小肠、肺、脾脏、心脏和大脑中检测到低亲和力（Km为200 - 700微摩尔）的酯酶活性。肾脏微粒体中的活性与肝脏微粒体中的相当。睾丸和肝脏微粒体中的高亲和力酯酶对苯甲基磺酰氟（PMSF）的抑制作用高度敏感，而肾脏和肝脏微粒体中的低亲和力酯酶相对耐受。这些结果表明，大鼠肝脏微粒体含有两种对乙酸对硝基苯酯具有高活性的酯酶，一种是对底物具有高亲和力的PMSF敏感酯酶，另一种是对底物具有低亲和力的PMSF不敏感酯酶。为支持该假设，我们已纯化并鉴定了两种酯酶，命名为水解酶A和B，它们似乎是大鼠肝脏微粒体中仅有的能快速水解乙酸对硝基苯酯的丰富酶。水解酶A以高亲和力（Km约为25微摩尔）水解乙酸对硝基苯酯，并被极低浓度的PMSF（IC50约为100纳摩尔）抑制。相反，水解酶B以低亲和力（Km约为400微摩尔）水解乙酸对硝基苯酯，仅被相对高浓度的PMSF（IC50约为100微摩尔）抑制。对硫磷的活性代谢产物对氧磷和三邻甲苯基磷酸酯的活性代谢产物甲酚苯并二氧磷杂环己烷完全抑制大鼠肝脏微粒体以及水解酶A和B对乙酸对硝基苯酯的水解，而巯基试剂对氯汞苯甲酸则无抑制作用。这些结果表明水解酶A和B都是丝氨酸酯酶。水解酶A和B的N端氨基酸序列相似但不同（前30个氨基酸残基中有23个相同），表明这两种酯酶是同工酶。（摘要截短为400字）
