Separation of the intracellular secretory compartment of rat liver and isolated rat hepatocytes in a single step using self-generating gradients of iodixanol

被引:23
作者
Plonne, D
Cartwright, I
Linss, W
Dargel, R
Graham, JM
Higgins, JA [1 ]
机构
[1] Univ Sheffield, Dept Mol Biol & Biotechnol, Sheffield S10 2TN, S Yorkshire, England
[2] Univ Jena, Inst Pathobiochem, D-6900 Jena, Germany
[3] Univ Jena, Inst Anat, D-6900 Jena, Germany
[4] Liverpool John Moores Univ, Peter Jost Enterprise Ctr, Liverpool L3 3AF, Merseyside, England
基金
英国生物技术与生命科学研究理事会;
关键词
D O I
10.1006/abio.1999.4311
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A novel method is described for the separation on a single gradient of the major intracellular organelles of the secretory pathway, the Golgi, the smooth endoplasmic reticulum, and the rough endoplasmic reticulum. Total microsomes were prepared from rat; liver by differential centrifugation and resuspended in 20% iodixanol. The microsomal suspension was then layered. between a 30% iodixanol cushion and a layer of 15% iodixanol and centrifuged in a vertical rotor for 2 h, The microsomes distributed in four visible bands, The gradients were collected by upward displacement and were characterized (i) by determination of UDP galactose-galactosyltransferase (Golgi marker) NADPH-cytochrome c reductase (endoplasmic reticulum marker) and RNA (rough endoplasmic reticulum marker); (ii) by immunoblotting for TGN38 (trans-Golgi marker) and GS28 (cis-Golgi marker) and for protein disulfide isomerase (endoplasmic reticulum lumenal marker); (iii) by determination of the lipid composition; and (iv) by electron microscopy. The results suggest that the top band (density 1.045-1.090 g/ml), which contains 68% of the galactosyltransferase activity, consists of vesicles derived from the Golgi. The second broad band in the middle of the tube (density 1.130-1.160 g/ml), which contains 54% of the NADPH-cytochrome c reductase activity, consists mainly of vesicles derived from the smooth endoplasmic reticulum, overlapped at the top by a small band of Golgi-derived lamellae. The two bands at the bottom of the tube (density 1.130-1.160 and density 1.180-1.220 g/ml) appear to contain two subfractions of vesicles derived from the rough endoplasmic reticulum. (C) 1999 Academic Press.
引用
收藏
页码:88 / 96
页数:9
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