Labeling of a glucose binding protein in the rabbit intestinal brush border membrane

• Published in: Canadian journal of physiology and pharmacology Vol. 56; no. 5; p. 760
• Main Authors: Lemaire, J, Maestracci, D
• Format: Journal Article
• Language: English
• Published: Canada 01.10.1978
• Subjects: Actins - analysis; Animals; Carrier Proteins - analysis; Cell Membrane - analysis; Electrophoresis, Polyacrylamide Gel; Ethylmaleimide; Glucose - metabolism; Intestinal Mucosa - analysis; Intestinal Mucosa - ultrastructure; Male; Membrane Proteins - analysis; Microvilli - analysis; Phenylmercury Compounds; Rabbits; Sulfhydryl Reagents
• ISSN: 0008-4212; 1205-7541
• DOI: 10.1139/y78-121

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.