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本试验以细鳞鱼仔鱼肠道为材料,通过梯度离心法对刷状缘膜进行了分离纯化。以刷状缘膜酶、碱性磷酸酶、γ-谷氨酰胺转移酶为标志酶,以钠-钾-ATP酶代表底侧膜,琥珀酸脱氢酶指示线粒体。经两个梯度离心处理后,刷状缘膜酶得到了富积纯化。在28000g离心处理中,碱性磷酸酶、γ-谷氨酰胺转移酶富积系数分别超过13倍和10倍,检测不到钠-钾ATP酶,琥珀酸脱氢酶仅为初始匀浆的0.01%;而43000g处理,碱性磷酸酶、γ-谷氨酰胺转移酶富积系数也分别超过13倍和10倍,检测不到钠-钾-ATP酶和琥珀酸脱氢酶。通过刷状缘膜上相关酶类活性测定对分离结果进行了评价。结果显示,以镁为沉淀剂,对肠道匀浆以43000g二次离心可以提高富积系数并降低污染,制作出了适用于营养吸收研究的细鳞鱼仔鱼肠道刷状缘膜囊。
In this experiment, the gut membrane of larva was used as material, and the brush border membrane was separated and purified by gradient centrifugation. Brush margin membrane enzyme, alkaline phosphatase, γ-glutamine transferase as a marker enzyme, sodium-potassium-ATPase represents the bottom membrane, succinate dehydrogenase indicates mitochondria. After two gradient centrifugation, brush edge membrane enzyme enrichment and purification. In the 28000g centrifugation, the accumulation coefficient of alkaline phosphatase and γ-glutamyltransferase were more than 13 times and 10 times respectively, sodium-potassium ATPase was not detected, and succinate dehydrogenase was only 0.01 %, While the accumulation coefficient of 43000g treatment, alkaline phosphatase and γ-glutamyl transferase also exceeded 13 times and 10 times respectively, and sodium-potassium-ATPase and succinate dehydrogenase were not detected. The separation results were evaluated by enzyme activity determination on the brush edge membrane. The results showed that magnesium as a precipitating agent, intestinal homogenate 43000g secondary centrifugation can increase the enrichment coefficient and reduce pollution, made for the study of nutrient absorption of the intestinal fish brushing membrane membrane larvae.