A HIGH-YIELD PREPARATION FOR RAT-KIDNEY BRUSH-BORDER MEMBRANES - DIFFERENT BEHAVIOR OF LYSOSOMAL MARKERS
A HIGH-YIELD PREPARATION FOR RAT-KIDNEY BRUSH-BORDER MEMBRANES - DIFFERENT BEHAVIOR OF LYSOSOMAL MARKERS
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DOI：
10.1016/0005-2736(81)90243-1
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
MURER, H
中科院分区：
文献类型：
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作者：
BIBER, J;STIEGER, B;MURER, H
大鼠肾皮质切片用Polytron匀浆，在等渗介质中加入5 mmol/L EGTA[乙二醇双(β-氨基乙醚)-N，N‘’-四乙酸]。用氯化镁(12 mm o l/L)沉淀2次，经差速离心法纯化刷状缘膜。刷状边缘标记酶碱性磷酸酶和氨基肽酶M富含17.0.+-。5.3倍和16.7倍+-。分别是3.7倍。用这种方法获得了高得率的刷状边缘膜(48.3.+-.碱性磷酸酶7.9%；47.0.+-。氨基肽酶M占9.5%)。酸性磷酸酶浓缩5倍，而其他溶酶体酶(氨基葡萄糖苷酶、葡萄糖苷酶、组织蛋白酶D)浓缩0.2倍。采用自由流动电泳法和蔗糖密度梯度离心法可将酸性磷酸酶活性从碱性磷酸酶和亮氨酸氨基肽酶中分离出来。与埃弗斯等人的钙法制备的囊泡相比，目前描述的镁/EGTA法制备的囊泡具有更好的传输性能。(1978)，而用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法没有观察到蛋白质图谱的差异。
Rat kidney cortex slices were homogenized with a polytron in an isoosmotic medium containing 5 mmol/l EGTA [ethyleneglycol bis(.beta.-aminoethyl ether)-N,N''-tetraacetic acid]. By 2 precipitations with MgCl2 (12 mmol/l) and differential centrifugation, brush border membranes were purified. The brush border marker enzymes alkaline phosphatase and aminopeptidase M were enriched 17.0 .+-. 5.3-fold and 16.7 .+-. 3.7-fold, respectively. By this method a high yield of brush border membranes was obtained (48.3 .+-. 7.9% for alkaline phosphatase; 47.0 .+-. 9.5% for aminopeptidase M). The acid phosphatase was enriched 5-fold, whereas other lysosomal enzymes (glucosaminidase, glucuronidase, cathepsin D) were enriched 0.2-fold. Acid phosphatase activity could not be washed out, but could be separated from alkaline phosphatase and leucine aminopeptidase by means of free flow electrophoresis and sucrose density gradient centrifugation. Vesicles prepared by the presently described Mg/EGTA-method show better transport properties, compared to vesicles prepared by the Ca method of Evers et al. (1978), whereas by SDS[sodium dodecyl sulfate]-polyacrylamide gel electrophoresis no differences in the protein patterns were observed.