Reconstitution of D-glucose transport catalyzed by a protein fraction from human erythrocytes in sonicated liposomes.

Abstract

A protein fraction was obtained from human erythrocyte ghosts by solubilization with Triton X-100 or octylglucoside. Triton X-100 was removed from the protein by Bio-Beads SM-2 and octylglucoside, by diafiltration. The solubilized protein fraction catalyzed D-glucose uptake when reconstituted in sonicated liposomes. The min at 370, and sedimented at 78,000 X g for 30 min. The pellet was washed once with 100 ml of 0.1 mM EDTA, treated with 100 ml of 0.5 M NaCl, 5 mM Tris-H2SO4 (pH 7.4) for 20 min at 40, washed once with 50 ml of the same salt solution and twice with 50 ml of 10 mM Tris-H2SO4 (pH 7.4), and stored at -70°(5 mg of protein per ml in 10 mM Tris, H2SO4). We confirmed the previous observation that EDTA and NaCl treatment removed several proteins from the ghosts and converted the ghosts into small vesicles (25). Solubilization with detergents Solubilization with Triton X-100. Triton X-100 was treated with SnCl2 to remove peroxides (26,27). Most of the peroxides (more than 85%) were removed, as estimated by the ferrous thiocyanate method (28). Sodium-chloride-treated vesicles (40 mg of protein in 20 ml) were incubated with 0.5% of purified Triton X-100 in 10 mM Tris-H2SO4 (pH 7.4) for 20 min at 4°. After centrifugation at 100,000 X g for 60 min, the supernatant was treated with Bio-Beads SM-2 overnight (6 g wet weight of beads per 20 ml) at 40 (29), divided into small fractions, and stored at -70°.Solubilization with Octylglucoside. Sodium-chloridetreated vesicles (14 mg of protein in 7 ml) were incubated with 30 mM octylglucoside in 10 mM Tris-H2SO4 (pH 7.4) for 30 min at 4°. After centrifugation at 100,000 X g for 60 min, the supernatant was subjected to diafiltration (XM-50; cut-off molecular weight, 50,000) against 10 mM TrisH2SO4 (pH 7.4) for 6 hr to overnight at 40. The protein fraction was divided into small fractions and stored at -70°. Reconstitution of D-glucose uptake system Acetone-washed soybean phospholipids (16) in a deoxygenated buffer (150 mM KC1, 10 mM Tris.HCl, pH 7.4) were sonicated in a test tube for 15-30 min at 20-400 with a sonicator (80 W, 80 kHz, Generator model G80-80-1, Tank model T80-80-1-R$, Laboratory Supplies, Hicksville, N.Y.) at 25-40 mg dry weight of lipid per ml. The solubilized membrane fraction was added to the sonicated liposomes, 15-20 mg/ml of phospholipids and 0.5-1.0 mg/ml of protein (total volume, 0.5-1.5 ml). The mixture was flushed with N2 gas and sonicated further for 1-3 min at 30-35' (30). The reconstitution was not specific for the soybean phospholipids; a mixture of egg phosphatidylcholine and soybean phosphatidylethanolamine could be used in place of the soybean phospholipids. Uptake of radioactive sugars A portion (85-120 gl)

Functional reassembly of membrane proteins in planar lipid bilayers

“…The receptor complex was eluted with carbamylcholine and then reconstituted into vesicles by cholate dialysis. Reconstitution by cholate dilution or by freeze-thaw sonication (Kasahara & Hinkle, 1976) were also used but they gave lower sodium influx values than the cholate dialysis procedure (Huganir et al 1979). The carbamylcholinesensitive sodium influx of reconstituted vesicles was inhibited by abungarotoxin and by the local anaesthetic procaine.…”

Peroxisomal ATP Import Is Essential for Seedling Development inArabidopsis thaliana

“…The yeast membrane fractions or E. coli inclusion bodies were reconstituted into liposome suspension by the freeze-thaw procedure (Kasahara and Hinkle, 1976). The liposomes had been prepared from acetone-washed L-a-phosphatidylcholine (PC) by sonication (5 min on ice, Branson Sonicator 250, output 2, duty cycle 30%) to a final concentration of 2% (w/v) in 100 mM Tricine-KOH, pH 7.8, 20 mM potassium gluconate, and 30 mM AMP (unless stated otherwise).…”

Properties of the Peribacteroid Membrane ATPase of Pea Root Nodules and Its Effect on the Nitrogenase Activity

“…Co-reconstitution of the bacteriorhodopsin with peribacteroid membrane or with cytoplasmic membrane from bacteroids was achieved by a two-step procedure. In the first step the purple membrane was sonicated with phospholipids as described earlier (Kasahara and Hinkle, 1976). The peribacteroid membrane or cytoplasmic membrane was fused with the purple membrane-phospholipid vesicles in a second step by two freeze-thaw-sonication cycles (Racker, 1979).…”