哺乳动物25-羟基维生素D3 24R-和1α-羟化酶的测定。
Measurement of mammalian 25-hydroxyvitamin D3 24R-and 1 alpha-hydroxylase.
作者信息
Tanaka Y, DeLuca H F
出版信息
Proc Natl Acad Sci U S A. 1981 Jan;78(1):196-9. doi: 10.1073/pnas.78.1.196.
An in vitro assay of mammalian 25-hydroxyvitamin D3 1 alpha- and 24R-hydroxylases in kidney has been developed. It had been suggested that 25-hydroxyvitamin D binding protein present in mammalian blood and tissues inhibits the enzyme activities in cell-free preparations by binding the substrate 25-hydroxyvitamin D3 more strongly than the hydroxylases bind it. This inhibitory effect is overcome by the addition of substantial amounts of unlabeled 25-hydroxyvitamin D3 to saturate the binding sites of this protein. The resulting metabolites produced in vitro by rat kidney homogenates were isolated and firmly identified by ultraviolet absorption spectrometry and mass spectrometry as 1,25-dihydroxyvitamin D3 and (24R)-24,25-dihydroxyvitamin D3. Maximal 1 alpha-hydroxylation of 25-hydroxyvitamin D3 could be demonstrated in kidney homogenates prepared from vitamin D-deficient rats. Thyroparathyroidectomy of these rats resulted in total suppression of the 1 alpha-hydroxylase. Homogenates of kidney from rats given vitamin D showed little or no 1 alpha-hydroxylase and substantial 24R-hydroxylase activity. Thyroparathyroidectomy of these rts markedly increased the 24R-hydroxylase activity.
已经开发出一种用于检测哺乳动物肾脏中25-羟基维生素D3 1α-羟化酶和24R-羟化酶的体外分析方法。有人提出，哺乳动物血液和组织中存在的25-羟基维生素D结合蛋白通过比羟化酶更紧密地结合底物25-羟基维生素D3来抑制无细胞制剂中的酶活性。通过添加大量未标记的25-羟基维生素D3以饱和该蛋白的结合位点，可以克服这种抑制作用。大鼠肾脏匀浆体外产生的代谢产物通过紫外吸收光谱法和质谱法进行分离并确认为1,25-二羟基维生素D3和（24R）-24,25-二羟基维生素D3。在由维生素D缺乏的大鼠制备的肾脏匀浆中可以证明25-羟基维生素D3的最大1α-羟化作用。这些大鼠的甲状腺甲状旁腺切除术导致1α-羟化酶完全被抑制。给予维生素D的大鼠的肾脏匀浆显示几乎没有或没有1α-羟化酶活性，但有大量的24R-羟化酶活性。这些大鼠的甲状腺甲状旁腺切除术显著增加了24R-羟化酶活性。
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