Depletion of cultured human fibroblasts of pyridoxal 5'-phosphate: effect on activities of aspartate aminotransferase, alanine aminotransferase, and cystathionine beta-synthase.
Depletion of cultured human fibroblasts of pyridoxal 5'-phosphate: effect on activities of aspartate aminotransferase, alanine aminotransferase, and cystathionine beta-synthase.
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培养的人成纤维细胞中 5-磷酸吡哆醛的消耗：对天冬氨酸转氨酶、丙氨酸转氨酶和胱硫醚 β-合酶活性的影响。
DOI：
10.1016/0003-9861(80)90060-0
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发表时间：
1980
影响因子：
3.9
通讯作者：
Rosenberg,LE
中科院分区：
文献类型：
--
作者：
Lipson,MH;Kraus,JP;Solomon,LR;Rosenberg,LE
人皮肤成纤维细胞在几乎不含吡哆醛的培养液中培养。在这些条件下培养的细胞在几代中生长到融合，没有形态上的退化迹象，也没有两种控制酶-己糖激酶和乳酸脱氢酶的活性变化。在添加吡哆醛的培养液中，这些成纤维细胞的5‘-磷酸吡哆醛含量下降到生长过程中的3%左右。在连续四代中，评估了这种消耗对三种依赖于5‘-磷酸吡哆醛的酶活性的影响。细胞提取物中胱硫氨酸β合成酶和天冬氨酸氨基转移酶的总活性下降到对照的50%，而丙氨酸转氨酶的总活性保持不变。这些酶与辅因子的饱和度也不同。全酶活性占总酶活性的比例下降到15%以下，天冬氨酸氨基转移酶下降到60%，胱硫氨酸β合成酶下降到15%以下。相反，丙氨酸氨基转移酶保持完全饱和的辅因子。当5‘-磷酸吡哆醛耗竭的成纤维细胞在含1 ng/ml吡哆醛的培养液中生长时，5’-磷酸吡哆醛对天冬氨酸氨基转移酶的饱和度达到最大，而要使胱硫氨酸β合成酶达到最大饱和度，则需要加入10 ng/ml的吡哆醛。在培养基中加入100 ng/ml的吡哆醛时，细胞内5‘-磷酸吡哆醛含量最高。有趣的是，在所有的耗竭和再耗竭实验中，吡哆醇激酶的活性保持不变。我们的结论是，在这些条件下培养人成纤维细胞的能力为研究完整的培养细胞和细胞提取物中的脱辅酶-辅酶相互作用提供了一个系统。
Human skin fibroblasts were grown in culture medium containing virtually no pyridoxal. Cells cultured under these conditions grew to confluence for several passages without morphologic signs of degeneration and without changes in activity of two control enzymes, hexokinase and lactate dehydrogenase. The pyridoxal 5′-phosphate content of these fibroblasts fell to about 3% of values obtained during growth in pyridoxal-supplemented medium. The effect of such depletion on the activities of three pyridoxal 5′-phosphate-dependent enzymes was assessed during four consecutive passages. Total activity of cystathionine β-synthase and of aspartate aminotransferase in cell extracts fell to a mean of 50% of control values whereas total activity of alanine aminotransferase remained unchanged. Saturation of these enzymes with cofactor differed as well. The ratio of holoenzyme activity to total enzyme activity fell to less than 15% or predepletion values for cystathionine β-synthase and to 60% for aspartate aminotransferase. In contrast, alanine aminotransferase remained completely saturated with cofactor. Maximal saturation of aspartate amino-transferase with pyridoxal 5′-phosphate was achieved when pyridoxal 5′-phosphate-depleted fibroblasts were grown in medium containing as little as 1 ng/ml of pyridoxal, but addition of 10 ng/ml of pyridoxal was required for maximal saturation of cystathionine β-synthase. Maximal intracellular content of pyridoxal 5′-phosphate was achieved only when 100 ng/ml of pyridoxal was added to the growth medium. Interestingly, the activity of pyridoxine kinase remained constant during all depletion and repletion experiments. We conclude that the ability to grow human fibroblasts under these conditions provides a system for the study of apoenzyme-coenzyme interactions both in intact cultured cells and in cell extracts.