ACETYLCHOLINESTERASE IS ORIENTATED FACING THE CYTOPLASMIC SIDE IN MEMBRANES DERIVED FROM SARCOPLASMIC-RETICULUM

被引:4
作者
CANOVASMUNOZ, MD [1 ]
MUNOZDELGADO, E [1 ]
VIDAL, CJ [1 ]
机构
[1] UNIV MURCIA,FAC BIOL,DEPT BIOQUIM & BIOL MOLEC,E-30071 MURCIA,SPAIN
关键词
ACETYLCHOLINESTERASE; SARCOPLASMIC RETICULUM; EXTERNAL LOCALIZATION;
D O I
10.1016/0167-4838(91)90276-6
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
(1) Microsomal membranes from white rabbit muscle enriched in sarcoplasmic reticulum (SR) were used to investigate the preferential localization of acetylcholinesterase (AChE) in these membranes. (2) Integrity and orientation of the vesicles was assessed by measuring the inulin-inaccessible space of the vesciles and its calcium-loading capacity. (3) Treatment of the membranes with diisopropyl phosphorofluoridate (DEP), an irreversible inhibitor which is free soluble in lipid, produced an almost complete inactivation of AChE. The inhibition was prevented in assays performed with the non-permeant reversible inhibitor BW284c51 (BW). (4) Similar results were obtained if echothiophate iodide (ECHO), an irreversible and poorly permeant inhibitor, instead of DFP was used. (5) Sedimentation profiles of enzyme solubilized with Triton X-100 from membranes inhibited by DFP after protection with BW showed a minor reduction in the relative proprotion of a 4.5 S (G1) form. (6) Treatment of intact or saponin-permeabilized membranes with concanavalin A (ConA) produced enzyme-lectin complexes. In both cases, most of the enzyme was recovered in the sedimented complexes after centrifugation of the Triton-solubilized membranes. (7) Incubation of intact membranes with the antibody AE1 led to the formation of immuno complexes. Sedimentation analyses of the molecular forms of AChE revealed a shift in the sedimentation coefficients, whether the antibody was added before or after solubilization of the enzyme. (8) These results firmly establish an external localization of AChE in SR, most of the protein backbone facing the cytoplasmic side of the membrane.
引用
收藏
页码:259 / 265
页数:7
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