Mechanism of cytotoxicity of psychosine

在包括Krabbe病（球状细胞脑白质营养不良）在内的大多数鞘脂病中发现了酶缺陷。然而，酶缺陷如何导致病理学尚未确定。我们发现，在球状细胞脑白质营养不良患者的脑中，缺失酶的天然底物psychosine（半乳糖基鞘氨醇）积累。由于精神分裂素是一种有效的毒素，Krabbe病的破坏性病理似乎是由积累的脂质的毒性作用引起的（Igisu和Suzuki，Science 224，753 -755）。然而，精神病碱的毒性机制尚不清楚，本研究旨在阐明这一点，使用大鼠肝脏线粒体，我们发现精神病碱可对细胞呼吸产生强有力的抑制作用（Igisu & Nakamura，Biochem.Bioprotein.Res.Commun.137，323 -327）。在含有精神霉素的反应混合物中，精神霉素抑制了磷的吸收和乙酰乙酸的形成，并增加了还原型细胞色素c的含量。以还原型细胞色素c为底物，精神霉素浓度小于5 <mu>M（0.1 <mu>mol/mg线粒体蛋白）时，对细胞色素c氧化酶的抑制率为50%。因此，精神霉素抑制了线粒体中的细胞色素c氧化酶。此外，精神霉素还抑制了线粒体中的细胞色素c氧化酶。加入白蛋白后，抑制作用立即完全消失（终浓度为1%），表明抑制是可逆的。我们发现细胞色素C氧化酶的抑制是由于线粒体膜中酶的环境的扰动（Igisu等，当这种酶被纯化时，psychosine并不抑制而是轻度激活这种酶。相反，当细胞色素c氧化酶与超声处理的磷脂酰胆碱“重组“时，psychosine抑制了这种酶的活性，就像它在线粒体中所做的那样。

Enzymatic defect has been discovered in most sphingolipidoses including Krabbe disease (globoid cell leukodystrophy).However,how the defect of the enzyme causes the pathology has not been defined.We dound accumulation of psychosine(galactosylsphingosine),a natural substrate of the missing enzyme,in the brains affected with globoid cell leukodystrophy. Since psychosine is a potent toxin,the devastating pathology in Krabbe disease appeared to be caused by the toxic effects of the accumulated lipid(Igisu & Suzuki,Science 224,753-755). Nevertheless,the mechnism of toxicity of psychosine has not been known.The present study was undertaken to clarify this.Using mitochondria from rat liver,we found that psychosine can exert a powerfun inhitory effect on cellular respiration(Igisu & Nakamura,Biochem.Biophys.Res.Commun.137,323-327). Pi uptake and acetoacetate formation were suppressed by psychosine and reduced form of cytochrome c increased in the reaction mixture which contained psychosine.Using reducded form of cytochrome c as substrate,less than 5 <mu>M of psychosine(0.1)<mu>mo1es/mg of mitochondrial protein)inhibited cytochrome c oxidase by 50%.Thus,psychosine inhibits cytochrome c oxidase in mitochondria.Besides,the inhibition was completely and immediately abolished upon addition of albumin(final concentration of 1%),indicating that the inhibition is reversible.Furthermore,we found that the inhibition of cytochrome c oxidase is due to the perturbation of the environment of the enzyme in mitochondrial membrane(Igisu et al.,Lipids, in press).When the enzyme was purified,psychosine did not inhibit but mildly activated the enzyme.In contrast,when cytochrome c oxiase was"reconstituted"with sonicated phosphatidylcholine,psychosine suppressed the enzyme activity as it did in mitochondria.