CYTOPLASMIC TRANSFER OF THE MTDNA NT-8993-T-]G (ATP6) POINT MUTATION ASSOCIATED WITH LEIGH-SYNDROME INTO MTDNA-LESS CELLS DEMONSTRATES COSEGREGATION WITH A DECREASE IN STATE-III RESPIRATION AND ADP/O RATIO
CYTOPLASMIC TRANSFER OF THE MTDNA NT-8993-T-]G (ATP6) POINT MUTATION ASSOCIATED WITH LEIGH-SYNDROME INTO MTDNA-LESS CELLS DEMONSTRATES COSEGREGATION WITH A DECREASE IN STATE-III RESPIRATION AND ADP/O RATIO
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DOI：
10.1073/pnas.91.18.8334
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发表时间：
1994-08-30
影响因子：
11.1
通讯作者：
WALLACE, DC
中科院分区：
文献类型：
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作者：
TROUNCE, I;NEILL, S;WALLACE, DC
在两个Leigh综合征家系中发现了一个与Leigh综合征相关的线粒体DNA编码的ATP 6基因的点突变（在nt 8993处T -> G），该突变降低了ADP刺激的（状态III）呼吸，ADP分子磷酸化与氧原子的比率（ADP/O比率）降低，但不影响2，4-二硝基苯酚（DNP）解偶联呼吸，这表明线粒体H+转运ATP合酶缺陷。从患者和对照类淋巴母细胞系分离的完整线粒体进行了测试，状态III，ADP限制（状态IV），和DNP非偶联呼吸与各种底物。从携带95-100%携带nt 8993 T --> G突变的mtDNA的患者淋巴母细胞分离的线粒体显示状态III呼吸速率比对照低26-50%，而具有正常的DNP解偶联速率。这导致患者线粒体中的状态III/DNP比率为0.52-0.70，而对照中为0.88-0.97。患者线粒体中ADP/O比值也降低了30-40%。使用Percoll梯度将nt 8993突变的患者异质淋巴母细胞去核，并通过电击将胞质与mtDNA缺陷（rho（0））细胞融合。对于nt 8993处的野生型核苷酸（T）同质的胞质杂交体克隆产生与对照胞质杂交体相似的状态III/DNP和ADP/O比率，而对于突变核苷酸（G）同质的胞质杂交体克隆显示状态m呼吸降低24-53%，状态III/DNP比率为0.53-0.64，并且ADP/O比率降低30%。因此，降低的状态III呼吸速率和ADP/O比率与nt 8993处的T -> G突变有关。
A point mutation in the mtDNA-encoded ATP6 gene (T --> G at nt 8993) associated with Leigh syndrome in two pedigrees was found to decrease ADP-stimulated (state III) respiration and the ratio of ADP molecules phosphorylated to oxygen atoms reduced (ADP/O ratio) but did not affect 2,4-dinitrophenol (DNP) uncoupled respiration, suggesting a defective mitochondrial H+-translocating ATP synthase. Intact mitochondria isolated from patient and control lymphoblastoid cell lines were tested for state III, ADP-limited (state IV), and DNP-uncoupled respiration with various substrates. Mitochondria isolated from patient lymphoblasts harboring 95-100% of mtDNAs carrying the nt 8993 T --> G mutation showed state III respiration rates 26-50% lower than controls while having normal DNP-uncoupled rates. This resulted in state III/DNP ratios of 0.52-0.70 in patient mitochondria versus 0.88-0.97 in controls. The ADP/O ratio was also decreased 30-40% in patient mitochondria. Patient lymphoblasts heteroplasmic for the nt 8993 mutation were enucleated by using Percoll gradients and the cytoplasts were fused to mtDNA-deficient (rho(0)) cells by electric shock. Cybrid clones homoplasmic for the wild-type nucleotide (T) at nt 8993 gave state III/DNP and ADP/O ratios similar to those of control cybrids, whereas cybrid clones homoplasmic for the mutant nucleotide (G) showed a 24-53% reduction in state m respiration, a state III/DNP ratio of 0.53-0.64, and a 30% decrease in the ADP/O ratio. Thus, the reduced state III respiration rates and ADP/O ratios are linked to the T --> G mutation at nt 8993.