Labeling of a glucose binding protein in the rabbit intestinal brush border membrane.
Using a double labeling method based on the method of Thomas (Thomas, L. 1973). Isolation of N-ethylmaleimide-labeled phloridzin-sensitive D-glucose binding protein of brush border membrane from rat kidney cortex. Biochim. Biophys. Acta, 291, 454--464), with radioactive N-ethylmaleimide ([3H]NEM and [14C]NEM) in the presence and absence of D-glucose, a protein band which is periodic acid--Schiff staining insensitive and which has a relative mobility (Rm) of 0.55 (corresponding to a molecular weight of 51 000 daltons) as determined by sodium dodecyl sulfate (SDS) electrophoresis was labeled preferentially. When radioactive p-hydroxymercuriphenylsulfonate ([203Hg]PCMBS) is used in the presence and absence of D-glucose, as described by Smith et al. (Smith, M. W., Ferguson, D. R., and Burton, K. A. 1975. Glucose- and phloridzin-protected thiol groups in pig intestinal brush border membranes. Biochem. J. 147, 617--619.), a protein band which has a relative mobility of 0.62 and a corresponding molecular weight of 42 000 daltons was labeled. Control experiments have shown that increasing concentrations of nonradioactive NEM (0.1--5.0 mM) do not substantially modify the electrophoretic pattern of SDS-solubilized brush border membrane. Nonradioactive PCMBS (0.1--10 mM), on the other hand, modifies the electrophoretic pattern and especially causes a change in relative mobility of the 0.55 protein band which migrates after 1 mM PCMBS treatment with a Rm of 0.62. The effect of 1 mM PCMBS can be reversed by adding L-cysteine or dithiothreitol. Actin extracted from rabbit muscle migrates with the same Rm as the 0.55 protein band in our electrophoretic conditions.
D000199:Actins / Q000032:analysis; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D002462:Cell Membrane / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide; D005947:Glucose / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000032:analysis* / Q000648:ultrastructure; D008297:Male; D008565:Membrane Proteins / Q000032:analysis*; D008871:Microvilli / Q000032:analysis*; D010663:Phenylmercury Compounds; D011817:Rabbits; D013439:Sulfhydryl Reagents
D000199:Actins; D002352:Carrier Proteins; D008565:Membrane Proteins; D010663:Phenylmercury Compounds; D013439:Sulfhydryl Reagents; C002910:4-hydroxymercuribenzenesulfonate; D005947:Glucose; D005033:Ethylmaleimide