Gibson K M, Sweetman L, Nyhan W L, Page T M, Greene C, Cann H M
Clin Chim Acta. 1982 Dec 9;126(2):171-81. doi: 10.1016/0009-8981(82)90033-x.
A new assay has been developed for 3-hydroxy-3-methylglutaryl-CoA lyase, the final enzyme in the leucine degradative pathway. The assay was performed by incubating lysates of fibroblasts with glutaryl-3-14C-3-hydroxy-3-methyl-glutaryl coenzyme A. The products were analysed by high performance liquid chromatography with continuous liquid scintillation counting. This provided simultaneous identification and quantification of one of the enzymatic products, [3-14C]acetoacetic acid. The mean 3-hydroxy-3-methylglutaryl-CoA lyase activity in fibroblasts from five controls was 732 +/- 81 (SD) pmol/min X mg protein. Using this assay, we have studied skin fibroblasts cultured from a patient with 3-hydroxy-3-methylglutaric aciduria and found 3% of normal 3-hydroxy-3-methylglutaryl-CoA lyase activity. The activities in skin fibroblasts cultured from the parents were 46 and 53% of control activity which is consistent with heterozygocity. Kinetic studies of 3-hydroxy-3-methylglutaryl-CoA lyase in skin fibroblasts cultured from two normal subjects yielded Km values of 14.4 and 18.8 mumol/l for 3-hydroxy-3-methylglutaryl-CoA.
已开发出一种针对3-羟基-3-甲基戊二酰辅酶A裂解酶的新检测方法，该酶是亮氨酸降解途径中的最后一种酶。检测方法是将成纤维细胞裂解物与戊二酰-3-¹⁴C-3-羟基-3-甲基戊二酰辅酶A一起孵育。通过高效液相色谱结合连续液体闪烁计数法对产物进行分析。这实现了对一种酶促产物[3-¹⁴C]乙酰乙酸的同时鉴定和定量。来自五个对照的成纤维细胞中3-羟基-3-甲基戊二酰辅酶A裂解酶的平均活性为732±81（标准差）pmol/分钟×毫克蛋白。使用该检测方法，我们研究了一名3-羟基-3-甲基戊二酸尿症患者培养的皮肤成纤维细胞，发现其3-羟基-3-甲基戊二酰辅酶A裂解酶活性为正常水平的3%。从该患者父母培养的皮肤成纤维细胞中的活性分别为对照活性的46%和53%，这与杂合性相符。对两名正常受试者培养的皮肤成纤维细胞中3-羟基-3-甲基戊二酰辅酶A裂解酶的动力学研究得出，3-羟基-3-甲基戊二酰辅酶A的Km值分别为14.4和18.8μmol/L。
