Lipson M H, Kraus J, Rosenberg L E
J Clin Invest. 1980 Aug;66(2):188-93. doi: 10.1172/JCI109843.
Previous attempts to correlate in vivo pyridoxine-responsiveness with in vitro assays of cystathionine beta-synthase activity in synthase-deficient homocystinuric patients have been only partially successful. All such studies, however, have been conducted with extracts of cultured skin fibroblasts grown in medium containing a high concentration (1,000 ng/ml) of pyridoxal. Having recently shown that such growth conditions may obscure important aspects of enzyme-coenzyme interactions by saturating most synthase molecules with their cofactor, pyridoxal 5'-phosphate, we have established conditions for growth of cells in pyridoxal-free medium. Under these conditions, intracellular pyridoxal 5'-phosphate fell by >95%, and saturation of cystathionine beta-synthase apoenzyme with pyridoxal 5'-phosphate decreased from a predepletion value of 70% to <10%. When such depleted cells were grown in media containing pyridoxal concentrations ranging from 0 to 1,000 ng/ml, cellular pyridoxal 5'-phosphate reached a maximum of 30 ng/mg cell protein at a medium pyridoxal concentration of 100 ng/ml. Maximal saturation of aposynthase with coenzyme in control cells was reached at a medium pyridoxal concentration of 10 ng/ml. In contrast, maximal saturation of residual aposynthase in cells from an in vivo responsive patient was achieved at a medium pyridoxal concentration of 25-50 ng/ml, whereas that from cells from an in vivo unresponsive patient was reached at 100 ng/ml. Estimates of the affinity of control and mutant cystathionine beta-synthase for pyridoxal 5'-phosphate in cell extracts supported the differences observed in intact cells. The apparent K(m) of cystathionine beta-synthase for pyridoxal 5'-phosphate in extracts of depleted cells from four in vivo-responsive patients was two to four times that of control. In contrast, the K(m) for pyridoxal 5'-phosphate in two lines from in vivo nonresponsive patients was 16- and 63-fold normal. These results suggest that cystathionine beta-synthase activity in cells from patients containing a mutant enzyme with a moderately reduced affinity for pyridoxal 5'-phosphate can be increased by pyridoxine supplements in vivo, whereas that from patients whose enzyme has a more dramatically reduced affinity for the coenzyme cannot be so modulated because of limits on the capacity of such cells to accumulate and retain pyridoxal 5'-phosphate.
先前尝试将体内吡哆醇反应性与合酶缺陷型同型胱氨酸尿症患者的胱硫醚β-合酶活性体外测定相关联，但仅取得了部分成功。然而，所有这些研究都是用在含有高浓度（1000 ng/ml）吡哆醛的培养基中培养的皮肤成纤维细胞提取物进行的。最近我们发现，这样的生长条件可能会通过用其辅因子磷酸吡哆醛使大多数合酶分子饱和，从而掩盖酶 - 辅酶相互作用的重要方面。因此，我们建立了在无吡哆醛培养基中培养细胞的条件。在这些条件下，细胞内磷酸吡哆醛下降了>95%，胱硫醚β-合酶脱辅基酶被磷酸吡哆醛饱和的程度从预耗尽值的70%降至<10%。当将这种耗尽的细胞在含有0至1000 ng/ml吡哆醛浓度的培养基中培养时，在培养基吡哆醛浓度为100 ng/ml时，细胞内磷酸吡哆醛达到最大值30 ng/mg细胞蛋白。对照细胞中脱辅基合酶与辅酶的最大饱和度在培养基吡哆醛浓度为10 ng/ml时达到。相比之下，体内有反应患者的细胞中残留脱辅基合酶的最大饱和度在培养基吡哆醛浓度为25 - 50 ng/ml时实现，而体内无反应患者的细胞则在100 ng/ml时达到。对细胞提取物中对照和突变型胱硫醚β-合酶对磷酸吡哆醛亲和力的估计支持了在完整细胞中观察到的差异。来自四名体内有反应患者的耗尽细胞提取物中，胱硫醚β-合酶对磷酸吡哆醛的表观K(m)是对照的两到四倍。相比之下，来自两名体内无反应患者的细胞系中，磷酸吡哆醛的K(m)分别是正常水平的16倍和63倍。这些结果表明，对于含有对磷酸吡哆醛亲和力适度降低的突变酶的患者细胞，胱硫醚β-合酶活性在体内可通过补充吡哆醇而增加，而对于其酶对辅酶亲和力显著降低的患者，由于此类细胞积累和保留磷酸吡哆醛的能力有限，其活性无法如此调节。
