Alonso-Gómez A L, Valenciano A I, Alonso-Bedate M, Delgado M J
Department Biología Animal II (Fisiología Animal), Facultad de Biología, Universidad Complutense, Madrid, Spain.
Neurochem Int. 1995 Mar;26(3):223-31. doi: 10.1016/0197-0186(94)00131-d.
Arylamine N-acetyltransferase activity (A-NAT: E.C.2.3.1.5) from Rana perezi retina was studied using p-phenetidine as specific substrate. Enzyme characteristics and regulation were compared with respect to the arylalkylamine N-acetyltransferase (AA-NAT: E.C.2.3.1.87) from the same tissue. A-NAT activity is distributed in both neural retina and choroid-pigmented epithelium complex, showing a 10-fold higher specific activity in neural retina. In contrast, AA-NAT activity is restricted to neural retina. Subcellular localization in neural retina indicated that both enzymatic activities are in the supernatant fraction (39,000 g, 20 min). p-Phenetidine acetylation was linear as a function of the neural retina amount in the assay (1/16 to 1 retina), and it is insensitive to phosphate buffer pH in the range 6.5-8.4. A-NAT kinetic showed a hyperbolic shape for both cosubstrates. Kinetic constants were KM = 11.2 microM, Vmax = 0.49 nmol/h/mg prot. for p-phenetidine (50 microM acetyl-CoA), and KM = 113.4 microM, Vmax = 3.1 nmol/h/mg prot. for acetyl-CoA (5 mM p-phenetidine). The additivity test for both enzymatic activities in retina homogenates demonstrated that both acceptor amines do not compete for the catalytic sites. Serotonin addition in the assay modifies differentially the kinetic characteristics of both enzymes. Serotonin acted as a strong mixed inhibitor, mainly competitive in nature (competitive Ki = 18.1 microM; non-competitive Ki = 1.9 mM) for AA-NAT. However, it acted as a weak inhibitor with respect to A-NAT, mainly non-competitive, (competitive Ki = 5.7 mM; non-competitive Ki = 8.7 mM).(ABSTRACT TRUNCATED AT 250 WORDS)
以对乙氧基苯胺作为特异性底物，研究了泽蛙视网膜的芳胺N - 乙酰基转移酶活性（A - NAT：E.C.2.3.1.5）。将该酶的特性和调控与同一组织中的芳烷基胺N - 乙酰基转移酶（AA - NAT：E.C.2.3.1.87）进行了比较。A - NAT活性分布于神经视网膜和脉络膜色素上皮复合体中，在神经视网膜中的比活性高10倍。相比之下，AA - NAT活性仅局限于神经视网膜。神经视网膜中的亚细胞定位表明，两种酶活性均存在于上清液部分（39,000 g，20分钟）。在测定中，对乙氧基苯胺的乙酰化与神经视网膜量呈线性关系（1/16至1个视网膜），并且在6.5 - 8.4的磷酸盐缓冲液pH范围内不敏感。A - NAT动力学对两种共底物均呈双曲线形状。动力学常数为：对乙氧基苯胺（50μM乙酰辅酶A）时，KM = 11.2μM，Vmax = 0.49 nmol/h/mg蛋白；乙酰辅酶A（5 mM对乙氧基苯胺）时，KM = 113.4μM，Vmax = 3.1 nmol/h/mg蛋白。视网膜匀浆中两种酶活性的加和试验表明，两种受体胺不会竞争催化位点。测定中添加血清素会对两种酶的动力学特性产生不同的影响。血清素对AA - NAT表现为强混合型抑制剂，主要为竞争性（竞争性Ki = 18.1μM；非竞争性Ki = 1.9 mM）。然而，它对A - NAT表现为弱抑制剂，主要为非竞争性（竞争性Ki = 5.7 mM；非竞争性Ki = 8.7 mM）。（摘要截短于250字）
