An improved method of reconstitution of adipocyte glucose transport activity

✍ Scribed by Frances W. Robinson; Teresa L. Blevins; Kazuo Suzuki; Tetsuro Kono

• Publisher: Elsevier Science
• Year: 1982
• Tongue: English
• Weight: 827 KB
• Volume: 122
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(82)90244-5

✦ Synopsis

The glucose transport activity of rat epididymal fat cells was reconstituted into egg lecithin liposomes with a high degree of reproducibility. The activity was solubilized with 20 mM sodium cholate in Buffer B (10 mM Tris-HCl, pH 7.5). After elimination of small molecules by gel filtration, the transport activity was incorporated into egg lecithin liposomes (Sigma, Type IX-E, homogeneously dispersed into Buffer B) by sonication (5 s), freezing (-70°C) thawing, and a second sonication (5 s). The sonication was done in a 16.8-mm polystyrene test tube (Sarstedt, 55-468) placed in a cup horn (from Heat Systems Ultrasonics) connected to a Branson's sonicator (W-185) at setting No. 3 (70 W of output). The optimum sample size was 80 ~1, and the optimum clearance between the test tube and the sonicator horn was 2-3 mm. The concentration of egg lecithin at the reconstitution step was 25 mg/ml, and that of the microsomal protein was approximately O-3-0.5 mg/ml. The glucose transport activity of reconstituted liposomes was assayed by incubating the latter with a mixture of D-["H]glucose and L-[ %]glucose. The incubation was terminated by the addition of HgCl*, and the reaction mixture was filtered with a Millipore filter (GSWP). The difference in the rates of uptake of D-glUCOSe and L-glucose was regarded as representing the carrier-mediated glucose transport activity. The results of the assay indicated that the glucose transport activity could be reconstituted in a highly reproducible manner. The reconstituted activity was proportional, within a limit of experimental error, to the amount of protein used for reconstitution and was almost completely blocked by cytochalasin B, phloretin, or HgCl,. However, a small amount of Dglucose was found to bind with the egg lecithin preparation.