Separation and purification by two-dimensional gel electrophoresis of a 52–54 kDa bumetanide binding protein from rat liver plasma membranes

Abstract

By affinity labeling with photolabile [ 3H]bumetanide, a 52–54 kDa bumetanide binding protein was identified in the sinusoidal plasma membrane fraction from rat liver. The protein is assumed to represent the carrier for hepatic uptake of loop diuretics. By two-dimensional (2D) gel electrophoresis we have purified this protein from hepatocytes, sinusoidal plasma membranes and subfractions of associated and integral plasma membrane proteins. Amongst more than 20 protein spots, a single integral plasma membrane protein was detected. The apparent pI of this molecule is 6.7. Specific labeling of this protein was not found in the fraction of associated plasma membrane proteins. To detect possible binding of radioactive bumetanide to microsomal cytochrome P450s, photolabeling experiments with integral plasma membrane proteins were performed under nitrogen/carbon monoxide atmosphere and in the presence of piperonyl butoxide. Labeling of the 52–54 kDa protein was not affected by these inhibitors of P450 enzymes. Taken together, these results indicate that the bumetanide binding protein is very likely to be a non-microsomal integral plasma membrane protein.