Fowler B, Kraus J, Packman S, Rosenberg L E
J Clin Invest. 1978 Mar;61(3):645-53. doi: 10.1172/JCI108976.
We have compared in vivo pyridoxine responsiveness with in vitro cystathionine beta-synthase activity in extracts of confluent fibroblasts from 14 synthase-deficient patients. Enzyme activity was measured with and without addition of its cofactor, pyridoxal-5'-phosphate, using a radioisotopic assay which detects as little as 0.25% of control activity. Six of seven lines from responsive patients had measurable activity without the added cofactor (0.6-15% of mean control). Two of these lines showed a five- and sevenfold stimulation of cystathionine beta-synthase activity with added pyridoxal-5'-phosphate; in the other four, the cofactor addition increased activity only modestly, as in controls. Two of seven lines from nonresponsive patients had measurable activity (each 3% of mean control) which increased two- and fivefold with the added cofactor. Cystathionine beta-synthase activity was undetectable in one line from a responsive patient and in five lines from nonresponsive ones. To characterize control and mutant synthase further, dissociation constants for pyridoxal-5'-phosphate were estimated and thermostability (54 degrees C) was studied in two control and five mutant lines. In one mutant, both parameters were normal; in the others, the affinity for the cofactor was reduced 3-to 11-fold and thermostability was much impaired. We conclude that at least three general classes of cystathionine beta-synthase mutants exist: those with no residual activity; those with reduced activity and normal affinity for pyridoxal-5' phosphate; and those with reduced activity and a reduced affinity for the cofactor. Pyridoxine responsiveness in vivo cannot be correlated simply with the presence or absence of residual synthase activity in vitro or with stimulation of in vitro enzyme activity by cofactor.
我们比较了14名合胞体成纤维细胞提取物中胱硫醚β-合酶活性的体外情况与14名合酶缺乏患者体内的维生素B6反应性。使用一种放射性同位素测定法，在添加和不添加其辅因子磷酸吡哆醛的情况下测量酶活性，该测定法可检测低至对照活性0.25%的酶活性。来自反应性患者的7个细胞系中有6个在未添加辅因子的情况下具有可测量的活性（为平均对照的0.6 - 15%）。其中2个细胞系在添加磷酸吡哆醛后胱硫醚β-合酶活性有5倍和7倍的刺激；在另外4个细胞系中，添加辅因子后活性仅适度增加，与对照情况相同。来自无反应性患者的7个细胞系中有2个具有可测量的活性（各为平均对照的3%），添加辅因子后活性增加了2倍和5倍。在来自一名反应性患者的一个细胞系以及来自无反应性患者的5个细胞系中未检测到胱硫醚β-合酶活性。为了进一步表征对照和突变合酶，估计了磷酸吡哆醛的解离常数，并在2个对照细胞系和5个突变细胞系中研究了热稳定性（54℃）。在一个突变体中，这两个参数均正常；在其他突变体中，对辅因子的亲和力降低了3至11倍，热稳定性严重受损。我们得出结论，至少存在三类胱硫醚β-合酶突变体：无残余活性的；活性降低但对磷酸吡哆醛亲和力正常的；以及活性降低且对辅因子亲和力降低的。体内维生素B6反应性不能简单地与体外残余合酶活性的有无或辅因子对体外酶活性的刺激相关联。
