Mechanism of cytotoxicity of psychosine

在包括Krabbe病(球状细胞白质营养不良)在内的大多数鞘磷脂疾病中都发现了酶缺陷。然而，这种酶的缺陷如何导致病理还没有明确的定义。我们发现缺失酶的天然底物--神经氨酸(半乳糖鞘氨醇)在球形细胞白质营养不良患者的大脑中积累。由于精神病素是一种有效的毒素，Krabbe病的破坏性病理似乎是由累积的脂质的毒性效应引起的(IGISU&Suzuki，Science 224,753-755)。然而，精神病素的毒性机制尚不清楚。本研究旨在阐明这一点。利用大鼠肝脏线粒体，我们发现精神病毒素对细胞呼吸具有强大的抑制作用(IGISU&Nakamura，BioChem.BiPhys.Res.Comm.137,323-327)。以还原态细胞色素c为底物，(0.1)&lt；mU&gt；M；细胞色素c氧化酶的抑制作用是由于线粒体膜上的酶环境的扰动(如Igisu等人，脂类)所致。纯化后的酶没有抑制作用，而是轻微地激活了酶的活性。相反，当细胞色素c氧化酶与声化的磷脂酰胆碱“重组”时，它抑制了酶的活性。

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.