Saarem K, Bergseth S, Oftebro H, Pedersen J I
J Biol Chem. 1984 Sep 10;259(17):10936-40.
Vitamin D3 25-hydroxylase activity was measured in subcellular and submitochondrial fractions of human liver. Quantitation of 25-hydroxyvitamin D3 was based on high performance liquid chromatography. Vitamin D3 25-hydroxylase activity was detected in the mitochondrial fraction only. The mitochondrial 25-hydroxylase activity was linear with time up to 60 min and with mitochondrial protein up to 1 mg/ml. An apparent Km value of about 10(-5) M was found. Substrate satuation level was not reached. In the presence of 2.4 X 10(-4) M vitamin D3, the rate of 25-hydroxyvitamin D3 formation was 0.19 nmol X mg of protein-1 X h-1 After fractionation of the mitochondria, 86% of the 25-hydroxylase activity was recovered in the mitoplast fraction. The outer membrane fraction was devoid of activity. It is concluded that human liver contains only one detectable vitamin D3 25-hydroxylase enzyme localized to the mitochondrial inner membrane.
在人肝脏的亚细胞和亚线粒体组分中测定了维生素D3 25-羟化酶活性。25-羟基维生素D3的定量基于高效液相色谱法。仅在线粒体组分中检测到维生素D3 25-羟化酶活性。线粒体25-羟化酶活性在长达60分钟的时间内与时间呈线性关系，在高达1mg/ml的线粒体蛋白浓度下与蛋白浓度呈线性关系。发现表观Km值约为10^(-5)M。未达到底物饱和水平。在存在2.4×10^(-4)M维生素D3的情况下，25-羟基维生素D3的形成速率为0.19nmol×mg蛋白^(-1)×h^(-1)。线粒体分级分离后，86%的25-羟化酶活性在线粒体膜间腔组分中回收。外膜组分无活性。结论是人肝脏仅含有一种可检测到的定位于线粒体内膜的维生素D3 25-羟化酶。
