Lee R S, Ford H C
Department of Pathology, Wellington School of Medicine, New Zealand.
J Biol Chem. 1988 Oct 15;263(29):14878-83.
An enzyme with FAD pyrophosphatase activity was extracted from human placental syncytiotrophoblast microvilli and purified to near-homogeneity. The enzyme has been identified as 5'-nucleotidase by several criteria. Throughout purification, parallel increases in the specific activities of FAD pyrophosphatase and AMP phosphatase were observed. The enzyme was a glycoprotein with a subunit molecular weight of 74,000. EDTA treatment resulted in a marked decline in both activities, and restoration of FAD pyrophosphatase activity but not 5'-nucleotidase activity was accomplished by the addition of Co2+ or, to a lesser extent, Mn2+. The substrate specificity of the 5'-nucleotidase activity that we observed agreed closely with the results of others. The pyrophosphatase activity was relatively specific for FAD. ADP, ATP, NAD(H), and FMN were not hydrolyzed, and ADP strongly inhibited both activities. For FAD pyrophosphatase activity, a Km of 1.2 x 10(-5) M and a Vmax of 1.1 mumol/min/mg protein were determined in assays performed in the presence of Co2+. In the absence of added Co2+, the Vmax declined but the Km was unchanged. For 5'-nucleotidase (AMP as substrate) the Km was 4.1 x 10(-5) M and the Vmax 109 mumol/min/mg protein. Hydrolysis of FMN to riboflavin was observed in partially purified detergent extracts of microvilli that contained alkaline phosphatase activity and lacked FAD pyrophosphatase and 5'-nucleotidase activity. The presence of both FAD pyrophosphatase and FMN phosphatase activities in syncytiotrophoblast microvilli supports the view that the placental uptake of vitamin B2 involves the hydrolysis of FAD and FMN to riboflavin which is then absorbed, a sequence postulated for intestinal absorption and liver uptake.
从人胎盘合体滋养层微绒毛中提取出一种具有黄素腺嘌呤二核苷酸（FAD）焦磷酸酶活性的酶，并将其纯化至接近均一状态。通过多项标准已将该酶鉴定为5'-核苷酸酶。在整个纯化过程中，观察到FAD焦磷酸酶和AMP磷酸酶的比活性平行增加。该酶是一种糖蛋白，亚基分子量为74,000。用乙二胺四乙酸（EDTA）处理会导致两种活性显著下降，添加Co2+（在较小程度上添加Mn2+）可恢复FAD焦磷酸酶活性，但不能恢复5'-核苷酸酶活性。我们观察到的5'-核苷酸酶活性的底物特异性与其他人的结果密切相符。焦磷酸酶活性对FAD相对特异。二磷酸腺苷（ADP）、三磷酸腺苷（ATP）、烟酰胺腺嘌呤二核苷酸（NAD(H)）和黄素单核苷酸（FMN）不被水解，并且ADP强烈抑制这两种活性。对于FAD焦磷酸酶活性，在存在Co2+的测定中，确定其米氏常数（Km）为1.2×10^(-5) M，最大反应速度（Vmax）为1.1 μmol/分钟/毫克蛋白。在不添加Co2+的情况下，Vmax下降但Km不变。对于5'-核苷酸酶（以AMP为底物），Km为4.1×10^(-5) M，Vmax为109 μmol/分钟/毫克蛋白。在含有碱性磷酸酶活性且缺乏FAD焦磷酸酶和5'-核苷酸酶活性的微绒毛部分纯化去污剂提取物中，观察到FMN水解为核黄素。合体滋养层微绒毛中同时存在FAD焦磷酸酶和FMN磷酸酶活性支持了这样一种观点，即胎盘对维生素B2的摄取涉及FAD和FMN水解为核黄素，然后被吸收，这一过程与肠道吸收和肝脏摄取所假定的序列相同。
