Kitano R, Morimoto S
Biochim Biophys Acta. 1975 Nov 10;411(1):113-20. doi: 10.1016/0304-4165(75)90290-1.
The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.
本研究采用不连续蔗糖密度梯度离心法和区带离心法分离犬肾皮质的过氧化物酶体。通过测定参考酶的活性来评估亚细胞颗粒的分离情况，溶酶体的β-甘油磷酸酶、线粒体的琥珀酸脱氢酶、微粒体的葡萄糖-6-磷酸酶以及过氧化物酶体的过氧化氢酶和D-氨基酸氧化酶。通过不连续蔗糖密度梯度离心获得的第1和第2组分（1.6和1.7M蔗糖）中主要观察到D-氨基酸氧化酶和过氧化氢酶的活性。这些组分中含有少量酸性磷酸酶和琥珀酸脱氢酶。在上清液中测定的过氧化氢酶活性相当高，而D-氨基酸氧化酶活性较低。通过区带离心法，在第50组分（1.73M蔗糖）中发现过氧化氢酶和D-氨基酸氧化酶的比活性最高，且无琥珀酸脱氢酶、酸性磷酸酶或葡萄糖-6-磷酸酶活性。这些结果表明，通过区带离心法，犬肾皮质的过氧化物酶体在1.73M蔗糖中与线粒体、溶酶体和微粒体清晰分离。
