Mechanism of cytotoxicity of psychosine

酶缺陷在大多数鞘脂病中被发现，包括Krabbe病（球状细胞白质营养不良）。然而，酶的缺陷是如何引起病理的尚未明确。我们发现精神碱（半乳糖半胱氨酸）的积累，一种缺失酶的天然底物，在受球状细胞白质营养不良影响的大脑中。由于精神素是一种强效毒素，甲蟹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 & Suzuki,Science 224,753-755）。然而，精神素的毒性机制尚不清楚。本研究是为了澄清这一点。利用大鼠肝脏的线粒体，我们发现精神素可以对细胞呼吸发挥强大的抑制作用（Igisu & Nakamura, biochemistry . biophysics . res . community .137,323-327）。精神碱抑制了Pi的摄取和乙酰乙酸的形成，在含有精神碱的反应混合物中细胞色素c的还原形式增加。以还原形式的细胞色素c为底物，低于5 < μ >M的精神碱（0.1 < μ >mo1es/mg线粒体蛋白）对细胞色素c氧化酶的抑制作用为50%。因此，精神素抑制线粒体中的细胞色素c氧化酶。此外，加入白蛋白（终浓度为1%）后，抑制完全立即消失，表明抑制是可逆的。此外，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受到干扰（Igisu等人，Lipids, in press）。当酶被纯化时，精神碱对酶没有抑制作用，只是轻度激活。相反，当细胞色素c氧化酶与超声磷脂酰胆碱“重组”时，精神碱抑制了酶的活性，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.