Kapsa R M, Quigley A F, Han T F, Jean-Francois M J, Vaughan P, Byrne E
Melbourne Neuromuscular Research Centre and Department of Clinical CSIRO Division of Molecular Science, Parkville Laboratory, Parkville, Victoria 3052, Australia.
Nucleic Acids Res. 1998 Oct 1;26(19):4365-73. doi: 10.1093/nar/26.19.4365.
Progressive age-related oxidative phosphorylation (OxPhos) decline is well known in human tissues. Depletion of mitochondrial DNA (mtDNA) causes OxPhos defects in patients with myopathic syndromes and deficient mtDNA replication has been observed in cells cultured from patients with mitochondrial disease. Patients undergoing treatment for AIDS develop OxPhos defects via mtDNA depletion resulting from inhibition of mtDNA polymerase gamma (Polgamma) by 2'-deoxy 3'-azido thymidine. These findings by others give rise to a possible link between mtDNA replication and bioenergetic decline in disease and during ageing. We have designed an in vitro assay for Polgamma function in small tissue samples to explore this possible link. Platelet homogenate Polgamma showed an activity with a K m of 150 microM (dTTP), a V max of 11.8 pmol/min/mg, inhibited (41% inhibition; 50 microM) by ethidium bromide. Determination of several storage characteristics showed that platelets were a convenient source of Polgamma for assay. Polgamma activity in 45 subjects did not coincide with significant age-related decline (P<0.002; P) observed in cytochrome oxidase (CytOx) activity or with citrate synthase activity. Of the activities studied, the only significant age-wise variation was a 24% CytOx deficiency in elderly (>50; n = 19) compared to young (<51; n = 24) individuals (P<0.01; t). These results suggest a maintenance of total cellular mtDNA Polgamma processive levels during ageing, largely independent of total cellular bioenergetic status or mitochondrial number/density. The processive component of Polgamma is therefore unlikely to make a major contribution to age-related bioenergetic activity decline. This does not, however, preclude the possibility that transient periods of inhibition at crucial points of the cell cycle or development may augment existing intracellular deficiencies. The assay described here greatly facilitates study of Polgamma activity in patients with conditions involving mtDNA depletion or rearrangement.
与年龄相关的氧化磷酸化（OxPhos）进行性下降在人体组织中是众所周知的。线粒体DNA（mtDNA）的耗竭会导致患有肌病综合征的患者出现氧化磷酸化缺陷，并且在从线粒体疾病患者培养的细胞中观察到mtDNA复制不足。接受艾滋病治疗的患者会因2'-脱氧3'-叠氮胸苷抑制mtDNA聚合酶γ（Polgamma）而导致mtDNA耗竭，进而出现氧化磷酸化缺陷。其他人的这些发现揭示了mtDNA复制与疾病及衰老过程中生物能量下降之间可能存在的联系。我们设计了一种用于检测小组织样本中Polgamma功能的体外试验，以探究这种可能的联系。血小板匀浆中的Polgamma表现出一种活性，其米氏常数（K m）为150微摩尔（dTTP），最大反应速度（V max）为11.8皮摩尔/分钟/毫克，会受到溴化乙锭的抑制（抑制率为41%；50微摩尔）。对几种储存特性的测定表明，血小板是用于检测Polgamma的便利来源。45名受试者的Polgamma活性与细胞色素氧化酶（CytOx）活性中观察到的与年龄相关的显著下降（P<0.002；P）或柠檬酸合酶活性均不相符。在所研究的活性中，唯一显著的年龄差异是老年（>50岁；n = 19）个体相较于年轻（<51岁；n = 24）个体存在24%的CytOx缺陷（P<0.01；t）。这些结果表明，在衰老过程中细胞总mtDNA Polgamma的持续水平得以维持，很大程度上独立于细胞总生物能量状态或线粒体数量/密度。因此，Polgamma的持续成分不太可能对与年龄相关的生物能量活性下降产生主要影响。然而，这并不排除在细胞周期或发育的关键点出现短暂抑制期可能会加剧现有细胞内缺陷的可能性。本文所述的检测方法极大地便利了对涉及mtDNA耗竭或重排病症患者的Polgamma活性的研究。
