EFFECT OF REACTIVE OXYGEN SPECIES ON LYSOSOMAL MEMBRANE INTEGRITY - A STUDY ON A LYSOSOMAL FRACTION
EFFECT OF REACTIVE OXYGEN SPECIES ON LYSOSOMAL MEMBRANE INTEGRITY - A STUDY ON A LYSOSOMAL FRACTION
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DOI：
10.1007/bf02915141
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发表时间：
1993-12-01
期刊：
影响因子：
--
通讯作者：
SVENSSON, I
中科院分区：
文献类型：
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作者：
ZDOLSEK, JM;SVENSSON, I
利用溶酶体富集的大鼠肝匀浆的“轻线粒体”部分，测定了活性氧物种过氧化氢、超氧阴离子自由基和羟基自由基的影响。溶酶体内pH的变化和溶酶体标记酶N-乙酰-氨基葡萄糖苷酶的释放被用作溶酶体膜完整性变化的指标。用四甲基偶氮唑盐-硫代巴比妥酸法测定其脂质过氧化作用。次黄嘌呤/黄嘌呤氧化酶产生的超氧阴离子自由基和大剂量的过氧化氢(0.5-1.5 mM)均不能引起溶酶体损伤。然而，如果Fe(III)ADP被包括在超氧阴离子自由基产生系统中，则溶酶体膜被检测到损伤，表现为溶酶体膜pH的升高和N-乙酰氨基葡萄糖苷酶的释放，但仅在大约7min的滞后阶段之后。另一方面，脂质过氧化则是逐渐进行的。过氧化氢处理的溶酶体表现出类似的剂量依赖性变化，尽管只有在同时加入Fe(III)ADP和还原氨基酸半胱氨酸的情况下。然而，在后一种系统中，溶酶体膜稳定性的改变发生得更快，显示出仅2分钟的滞后期。脂质过氧化在10min内趋于平稳，且进展较快，无滞后期。结果表明，超氧阴离子自由基和过氧化氢本身都不会对溶酶体造成损害。然而，有效的Fe(II)形式的催化活性铁允许产生强大的氧化物种--可能是通过Fenton反应形成的羟基--的反应发生，导致溶酶体膜的过氧化，导致质子梯度的消散和酶含量的泄漏。
Using a lysosome-enriched ''light mitochondrial'' fraction of a rat liver homogenate, the effects of the reactive oxygen species hydrogen peroxide, superoxide- and hydroxyl radicals were determined. Alterations in the intralysosomal pH and the release of a lysosomal marker enzyme, N-acetyl-glucosaminidase, were used as indicators of changes in the lysosomal membrane integrity. Lipid peroxidation of the fraction was assayed by TBARS measurement. Neither superoxide radicals, generated by hypoxanthine/xanthine oxidase, nor a bolus dose of hydrogen peroxide (0.5-1.5 mM) induced any lysosomal damage. If, however, Fe(III)ADP was included in the superoxide radical-generating system, lysosomal membrane damage was detected, both as an increase in lysosomal pH and as a release of N-acetyl-glucosaminidase, but only after a lag phase of about 7 min. Lipid peroxidation, on the other hand, proceeded gradually. Lysosomes treated with hydrogen peroxide displayed similar dose-dependent alterations, albeit only if both Fe(III)ADP and the reducing amino acid cysteine were added. In the latter system, however, alterations of the lysosomal membrane stability occurred more rapidly, showing a lag phase of only 2 min. Lipid peroxidation, which proceeded faster and displayed no lag phase, levelled out within 10 min. The results indicate that neither superoxide radicals nor hydrogen peroxide are by themselves damaging to lysosomes. Available catalytically active iron in Fe(II) form, however, allows reactions yielding powerful oxidative species - probably hydroxyl radicals formed via Fenton reactions - to take place inducing peroxidation of the lysosomal membranes resulting in dissipation of the proton-gradient and leakage of their enzyme contents.