Alonso-Gómez A L, Gancedo B, Alonso-Bedate M, Agapito M T, Delgado M J
Departamento de Biología Animal II (Fisiología Animal), Facultad de Biología, Universidad Complutense, Madrid, Spain.
J Neurochem. 1992 Feb;58(2):587-92. doi: 10.1111/j.1471-4159.1992.tb09759.x.
The kinetics of serotonin N-acetyltransferase (NAT) from the lateral eye of Rana perezi have been characterized. NAT from ocular tissue reached maximal activity at a phosphate buffer concentration of 250 mM and a pH of 6.5. Reaction linearity was highly conserved within the homogenate fraction range tested (0.033-0.33). The time course of ocular NAT reaction showed a high linearity at 25 and 35 degrees C. Km and Vmax estimations for acetyl-CoA at a 10 mM tryptamine concentration were 63.3 microM and 4.42 nmol/h per eye, respectively. Regardless of the acceptor amine (tryptamine or serotonin), the Km was not affected by the acetyl-CoA concentration (50 or 250 microM), whereas the Vmax was significantly increased at a 250 microM acetyl-CoA concentration. Ocular NAT showed a higher affinity for serotonin (Km = 20.7 microM) than for tryptamine (Km = 48-60 microM); Vmax, however, was similar for both substrates. Acetyl-CoA does not protect ocular NAT; in contrast, the use of EGTA (greater than or equal to 4 mM) in the assay is essential to protect the enzyme because NAT in ocular crude homogenate shows rapid inactivation. This result suggests that intracellular calcium levels are involved in the NAT inactivation mechanisms in frog ocular tissue.
泽陆蛙侧眼血清素N - 乙酰转移酶（NAT）的动力学特性已得到表征。眼组织中的NAT在磷酸盐缓冲液浓度为250 mM、pH为6.5时达到最大活性。在所测试的匀浆组分范围内（0.033 - 0.33），反应线性高度保守。眼NAT反应的时间进程在25和35摄氏度时显示出高度线性。在10 mM色胺浓度下，乙酰辅酶A的Km和Vmax估计值分别为63.3 microM和每只眼4.42 nmol/h。无论受体胺是色胺还是血清素，Km均不受乙酰辅酶A浓度（50或250 microM）的影响，而在250 microM乙酰辅酶A浓度下Vmax显著增加。眼NAT对血清素（Km = 20.7 microM）的亲和力高于色胺（Km = 48 - 60 microM）；然而，两种底物的Vmax相似。乙酰辅酶A不能保护眼NAT；相反，在测定中使用EGTA（大于或等于4 mM）对于保护该酶至关重要，因为眼粗匀浆中的NAT显示出快速失活。这一结果表明细胞内钙水平参与了蛙眼组织中NAT的失活机制。
