Keeling D J, Smith I R, Tipton K F
Naunyn Schmiedebergs Arch Pharmacol. 1984 Jun;326(3):215-21. doi: 10.1007/BF00505321.
A sensitive coupled assay for histidine decarboxylase has been developed. This method involved conversion of [3H]histidine into [3H]histamine by the enzyme sample, with methylation of this product in situ, catalysed by the enzyme histamine N-methyltransferase, to yield [3H]N-tele-methylhistamine. The radioactive product was separated from the substrate by (i) extraction into chloroform, (ii) ion-exchange chromatography and (iii) liquid cation-exchange extraction. The "no tissue" assay blank comprised 0.0007% of the substrate radioactivity. Sample material with a histidine decarboxylase activity of as little as 0.14 fmol/min/ml (measured at 1 microM histidine) gave double the blank value. More than 50 assays could be performed in one day. This assay was used to determine the in vivo changes in mouse brain histidine decarboxylase activity following irreversible inhibition with (+) alpha-fluoromethylhistidine (alpha-FMH). From the time course of recovery of enzyme activity the half-life of histidine decarboxylase in vivo was calculated to be 53 h.
已开发出一种用于组氨酸脱羧酶的灵敏偶联测定法。该方法包括通过酶样品将[³H]组氨酸转化为[³H]组胺，并在组胺N-甲基转移酶的催化下使该产物原位甲基化，生成[³H]N-甲基组胺。通过以下步骤将放射性产物与底物分离：（i）用氯仿萃取；（ii）离子交换色谱法；（iii）液相阳离子交换萃取。“无组织”测定空白占底物放射性的0.0007%。组氨酸脱羧酶活性低至0.14 fmol/分钟/毫升（在1 microM组氨酸下测定）的样品材料产生的数值是空白值的两倍。一天内可进行50多次测定。该测定法用于确定在用（+）α-氟甲基组胺（α-FMH）进行不可逆抑制后小鼠脑组织中组氨酸脱羧酶活性的体内变化。根据酶活性恢复的时间进程，计算出组氨酸脱羧酶在体内的半衰期为53小时。
