Fonda M L
Anal Biochem. 1986 May 15;155(1):14-22. doi: 10.1016/0003-2697(86)90217-4.
The alternate procedures used in the tyrosine apodecarboxylase assays for pyridoxal 5'-phosphate were evaluated to determine optimal conditions. Two preparations of tyrosine apodecarboxylase from Streptococcus faecalis were used: a cell suspension and a partially purified cell-free form. The activity of the decarboxylase was measured in two different assays using [14C]tyrosine or [3H]tyrosine as substrate. The presence of serum proteins caused greater inhibition of the assay for serum pyridoxal phosphate using [14C]tyrosine as substrate than the assay with [3H]tyrosine. In contrast, addition of deproteinized serum extract did not appear to inhibit either assay. The rate of reconstitution of the apodecarboxylase in the cell suspension was at least four times slower than that of the cell-free enzyme. The rate of reconstitution of the cell-free enzyme was faster in acetate than in citrate buffer. Inorganic sulfate or phosphate, at normal plasma concentrations, did not alter either the reconstitution rate of tyrosine decarboxylase or the final activity obtained in the assays using either substrate. The tyrosine apodecarboxylase assay for pyridoxal phosphate can be optimized by using deproteinized sera or plasma and incubating the cell-free apoenzyme with the coenzyme in acetate buffer for a time sufficient to obtain maximum reconstitution.
对用于测定磷酸吡哆醛的酪氨酸脱羧酶分析中的替代程序进行了评估，以确定最佳条件。使用了两种来自粪链球菌的酪氨酸脱羧酶制剂：细胞悬液和部分纯化的无细胞形式。脱羧酶的活性在两种不同的分析中进行测量，使用[14C]酪氨酸或[3H]酪氨酸作为底物。血清蛋白的存在对以[14C]酪氨酸为底物的血清磷酸吡哆醛分析的抑制作用比对以[3H]酪氨酸为底物的分析更大。相比之下，添加脱蛋白的血清提取物似乎不会抑制任何一种分析。细胞悬液中脱羧酶的重构速率比无细胞酶至少慢四倍。无细胞酶在乙酸盐缓冲液中的重构速率比在柠檬酸盐缓冲液中更快。在正常血浆浓度下，无机硫酸盐或磷酸盐不会改变酪氨酸脱羧酶的重构速率或使用任何一种底物进行的分析中获得的最终活性。通过使用脱蛋白的血清或血浆，并将无细胞脱辅酶与辅酶在乙酸盐缓冲液中孵育足够长的时间以获得最大重构，可以优化用于磷酸吡哆醛的酪氨酸脱羧酶分析。
