Differential characteristics and regulation of arylamine and arylalkylamine N-acetyltransferases in the frog retina (Rana perezi).
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)
Alonso-Gómez
A L
AL
Department Biología Animal II (Fisiología Animal), Facultad de Biología, Universidad Complutense, Madrid, Spain.
Valenciano
A I
AI
Alonso-Bedate
M
M
Delgado
M J
MJ
D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D009924:Organ Culture Techniques; D011898:Ranidae; D012160:Retina / Q000201:enzymology*; D012701:Serotonin / Q000494:pharmacology
D012701:Serotonin; D001191:Arylamine N-Acetyltransferase