Purification and reconstitution of Na+/D-glucose cotransport carriers from guinea pig small intestine.
Sodium/D-glucose cotransport carriers solubilized from intestinal brush border membranes were purified and incorporated into liposomes made of soybean phospholipids, and transport properties of the reconstituted system were studied. The brush border membrane vesicles prepared from guinea pig small intestine were first treated with deoxycholate and papain in order to remove unnecessary membrane proteins, and then the remaining membrane proteins were solubilized with Triton X-100. The solubilized proteins were fractionated by-gel-filtration according to molecular size and the fractions containing proteins of molecular weight of around 340 K daltons were further separated by chromatofocusing according to isoelectric point. The Na+ gradient-dependent overshoot uptake of D-glucose was seen when the proteins which were finally eluted in the pH range of 5.0-5.5 were incorporated into the liposomes. The proteins purified and incorporated into liposomes could be visualized on sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis as a single band of 160,000 dalton glycoprotein. The proteoliposomes constructed with the purified proteins revealed about 20-fold higher accumulation of D-glucose as compared with those constructed with unpurified membrane protein extracts. Kinetically, however, the reconstituted system revealed somewhat different characteristics from those of the native brush border membrane vesicles, i.e. retarded time course of overshooting uptake and an S-shaped relationship between 1-min uptake of glucose (the quasi-initial rate of glucose uptake) and glucose concentration. The reciprocal of the uptake rate was linearly proportional to the reciprocal of the square of glucose concentration and the estimated Hill coefficient was about 2.
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000032:analysis; D005947:Glucose / Q000378:metabolism; D006168:Guinea Pigs; D007421:Intestine, Small / Q000032:analysis*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D009004:Monosaccharide Transport Proteins
D002352:Carrier Proteins; D008081:Liposomes; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D005947:Glucose