Beier K, Völkl A, Hashimoto T, Fahimi H D
Department of Anatomy and Cell Biology, University of Heidelberg/Federal Republic of Germany.
Eur J Cell Biol. 1988 Aug;46(3):383-93.
Quantitative immunoelectron microscopy in conjunction with quantitative analysis of immunoblots have been used to study the effects of bezafibrate (BF), a peroxisome-proliferating hypolipidemic drug, upon six different enzyme proteins in rat liver peroxisomes (Po). Antibodies against following peroxisomal enzymes: catalase, urate oxidase, alpha-hydroxy acid oxidase, acyl-CoA oxidase, bifunctional enzyme (hydratase-dehydrogenase) and thiolase, were raised in rabbits, and their monospecificities were confirmed by immunoblotting. Female Sprague-Dawley rats were treated for 7 days with 250 mg/kg/day bezafibrate and liver sections were incubated with the appropriate antibodies followed by the protein A-gold complex. The labeling density for each enzyme was estimated by automatic image analysis. In parallel experiments immunoblots prepared from highly purified peroxisome fractions of normal and BF-treated rats were incubated with the same antibodies. The antigens were visualized by an improved protein A-gold method including an anti-protein A step and silver amplification. The immunoblots were also quantitated by an image analyzer. The results revealed a selective induction of beta-oxidation enzymes by bezafibrate with thiolase showing the most increase followed by bifunctional protein and acyl-CoA oxidase. The labeling density for catalase and alpha-hydroxy acid oxidase was reduced, confirming fully the quantitative analysis of immunoblots which in addition revealed reduction of uricase. These observations demonstrate that hypolipidemic drugs induce selectively the beta-oxidation enzymes while other peroxisomal enzymes are reduced. The quantitative immunoelectron microscopy with automatic image analysis provides a versatile, highly sensitive and efficient method for rapid detection of modulations of individual proteins in peroxisomes.
定量免疫电子显微镜结合免疫印迹定量分析已被用于研究苯扎贝特（BF），一种过氧化物酶体增殖性降血脂药物，对大鼠肝脏过氧化物酶体（Po）中六种不同酶蛋白的影响。针对以下过氧化物酶体酶的抗体：过氧化氢酶、尿酸氧化酶、α-羟酸氧化酶、酰基辅酶A氧化酶、双功能酶（水化酶-脱氢酶）和硫解酶，在兔体内产生，其单特异性通过免疫印迹得到证实。雌性Sprague-Dawley大鼠用250mg/kg/天的苯扎贝特治疗7天，肝脏切片与适当的抗体孵育，然后与蛋白A-金复合物孵育。通过自动图像分析估计每种酶的标记密度。在平行实验中，将由正常和BF处理大鼠的高度纯化过氧化物酶体组分制备的免疫印迹与相同的抗体孵育。通过改进的蛋白A-金方法，包括抗蛋白A步骤和银增强，使抗原可视化。免疫印迹也通过图像分析仪进行定量。结果显示苯扎贝特对β-氧化酶有选择性诱导作用，硫解酶增加最多，其次是双功能蛋白和酰基辅酶A氧化酶。过氧化氢酶和α-羟酸氧化酶的标记密度降低，充分证实了免疫印迹的定量分析，该分析还显示尿酸酶减少。这些观察结果表明，降血脂药物选择性诱导β-氧化酶，而其他过氧化物酶体酶减少。具有自动图像分析的定量免疫电子显微镜为快速检测过氧化物酶体中单个蛋白质的调节提供了一种通用、高度灵敏和高效的方法。
