Sedimentation and immunological analyses of GLUT4 and α2‐Na,K‐ATPase subunit‐containing vesicles from rat skeletal muscle: evidence for segregation

FEBS Letters

Aledo, J.Carlos¹  Hundal, Harinder S.¹ 

[1] Department of Anatomy and Physiology, The University of Dundee, Dundee, DD1 4HN, Scotland, UK

关键词: Membrane;    Transport;    Na pump;    Insulin;   

DOI  :  10.1016/0014-5793(95)01282-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

In skeletal muscle insulin induces the translocation of both the GLUT4 glucose transporter and the α2 subunit of the Na,K-ATPase from an intracellular membrane (IM) compartment to the plasma membrane (PM). Fractionation studies of rat skeletal muscle using a discontinuous sucrose gradient have indicated that the insulin-induced loss of both proteins occurs from a fraction containing intracellular membranes (IM) of common density. This raises the possibility that both proteins may be colocalized in a single intracellular compartment or are present in separate membrane vesicles that are of similar buoyant density. In this study we report that membrane vesicles from the insulin-responsive IM fraction can in fact be separated on the basis of differences in their sedimentation velocities; immunoblot analyses of fractions collected from a sucrose velocity gradient revealed the presence of two separate peaks for GLUT4 and the α2 subunit of the Na,K-ATPase. One of these peaks representing a fast sedimenting population of vesicles (with a sedimentation coefficient of 2697 ± 57 S) reacted against antibodies to the α2 subunit of the Na,K-ATPase, whereas, the second peak contained a population of much slower sedimenting vesicles (with a sedimentation coefficient of 209 ± 4 S) were practically devoid of the α2-subunit. By contrast, the slow sedimenting vesicles were enriched by ∼32-fold in GLUT4 relative to the starting IM fraction when the fractional protein content was taken into account. Immunoprecipitation of GLUT4-containing vesicles from the insulin-sensitive IM fraction revealed that no immunoreactivity towards either the α2 or the β1 subunits of the Na,K-ATPase could be observed, signifying that the insulin-responsive subunits of the Na,K-ATPase and GLUT4 were present in different membrane vesicles and that it was unlikely, therefore, that the insulin-induced redistribution of these proteins to the PM occurs from a common intracellular pool.

【 授权许可】

Unknown