Sastry B V, Janson V E
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee.
J Ocul Pharmacol. 1994 Spring;10(1):203-15. doi: 10.1089/jop.1994.10.203.
Choline acetyltransferase catalyzes the synthesis of acetylcholine from choline and acetylcoenzyme A (ACoA) in both nervous and non-nervous tissues. Carnitine acetyltransferase occurs in several tissues and transfers acetyl groups from ACoA to carnitine forming acetylcarnitine and exhibits weak choline acetyltransferase activity. Several haloacetylcholines and haloacetylcarnitines were synthesized to develop selective inhibitors of choline acetyltransferase and carnitine acetyltransferase. Acetylcholine is a transmitter for some presynaptic neurons and/or amacrine cells in retina. Selective inhibitors of choline acetyltransferase and carnitine acetyltransferase were used in the evaluation of choline acetyltransferase and carnitine acetyltransferase activities in the rat retina. Choline acetyltransferase and carnitine acetyltransferase activities were assayed by transferring of [14C]acetyl group from [14C]ACoA to choline or carnitine and estimating [14C]-acetylcholine or [14C]acetylcarnitine. This study gave the following results: (a) Bromoacetylcholine (BrACh) was a selective inhibitor of purified choline acetyltransferase (I50, 2.2 microM); (b) (R)-bromoacetylcarnitine [(R)-BrACa] was more potent for inhibiting purified carnitine acetyltransferase (I50, 4 microM) than purified choline acetyltransferase (I50, 46 microM); (c) Rat retinal sonicate gave choline acetyltransferase activity of 98 +/- 6 nmol of ACh formed/mg/10 min. When the carnitine acetyltransferase was completely inhibited by (R)-BrACa, the activity for choline acetyltransferase decreased to 47 +/- 1 nmol, and this decrease was possibly due to the formation of some [14C]acetylcholine by carnitine acetyltransferase. The net retinal choline acetyltransferase activity was 51 nmol acetylcholine/mg protein/10 min; (d) Rat retinal sonicate contained carnitine acetyltransferase activity of 102 +/- 7 nmol acetylcarnitine formed/mg protein/10 min. This was not altered by inhibition of choline acetyltransferase with BrACh. This means that choline acetyltransferase did not use carnitine as a substrate. Choline acetyltransferase and carnitine acetyltransferase activities did not change after dialysis of retinal sonicates at 4 degrees C for 24 hrs. These observations suggest that BrACh and (R)-BrACa are useful for assessing the correct values for choline acetyltransferase and carnitine acetyltransferase activities in retinal tissues.
胆碱乙酰转移酶在神经组织和非神经组织中催化由胆碱和乙酰辅酶A（ACoA）合成乙酰胆碱。肉碱乙酰转移酶存在于多种组织中，它将ACoA上的乙酰基转移至肉碱形成乙酰肉碱，并表现出较弱的胆碱乙酰转移酶活性。合成了几种卤代乙酰胆碱和卤代乙酰肉碱，以开发胆碱乙酰转移酶和肉碱乙酰转移酶的选择性抑制剂。乙酰胆碱是视网膜中一些突触前神经元和/或无长突细胞的递质。胆碱乙酰转移酶和肉碱乙酰转移酶的选择性抑制剂被用于评估大鼠视网膜中胆碱乙酰转移酶和肉碱乙酰转移酶的活性。通过将[14C]乙酰基从[14C]ACoA转移至胆碱或肉碱，并测定[14C] - 乙酰胆碱或[14C]乙酰肉碱来检测胆碱乙酰转移酶和肉碱乙酰转移酶的活性。本研究得出以下结果：（a）溴乙酰胆碱（BrACh）是纯化胆碱乙酰转移酶的选择性抑制剂（半数抑制浓度I50，2.2 microM）；（b）（R）-溴乙酰肉碱[（R）-BrACa]对纯化肉碱乙酰转移酶的抑制作用（I50，4 microM）比对纯化胆碱乙酰转移酶的抑制作用更强（I50，46 microM）；（c）大鼠视网膜超声匀浆的胆碱乙酰转移酶活性为98±6 nmol乙酰胆碱形成量/mg/10分钟。当肉碱乙酰转移酶被（R）-BrACa完全抑制时，胆碱乙酰转移酶活性降至47±1 nmol，这种下降可能是由于肉碱乙酰转移酶形成了一些[14C]乙酰胆碱。视网膜胆碱乙酰转移酶的净活性为51 nmol乙酰胆碱/mg蛋白质/10分钟；（d）大鼠视网膜超声匀浆的肉碱乙酰转移酶活性为102±7 nmol乙酰肉碱形成量/mg蛋白质/10分钟。用BrACh抑制胆碱乙酰转移酶对此活性无影响。这意味着胆碱乙酰转移酶不将肉碱用作底物。在4℃对视网膜超声匀浆进行24小时透析后，胆碱乙酰转移酶和肉碱乙酰转移酶的活性未发生变化。这些观察结果表明，BrACh和（R）-BrACa可用于评估视网膜组织中胆碱乙酰转移酶和肉碱乙酰转移酶活性的正确值。
