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Detection of the membrane protein recognized by the kidney-specific alkylglucoside vector
Y Watanabe1, H Suzuki, K Suzuki
1Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan.
Purpose:
Previously, we suggested that alkylglucoside can be an effective vector for renal-specific drug delivery (Suzuki et al., J. Pharmacol. Exp. Ther, 288:57-61, 1999). The purpose of the present study is to characterize the membrane protein which is recognized by this alkylglucoside.
Methods:
The binding of [125I] tyrosine conjugated with a octylthioglucoside (Glc-S-C8-[125I]Tyr) Glc-S-C8-[125I]Tyr to crude membrane fractions of kidney was determined. In addition, the membrane was cross-linked with this alkylglucoside and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Results:
Glc-S-C8-[125I]Tyr was shown to have a specific binding site on the kidney membrane (Kd = 931 nM and Bmax = 987 pmol/mg protein). Cross-linking of the membrane with Glc-S-C8-[125I]Tyr resulted in the detection of a protein (Mr = 62,000), which was unaffected by reducing agents. The results of this cross-linking study were consistent with previous information on its localization and binding characteristics.
Conclusions:
The kidney membrane protein, to which alkylglucoside binds in a specific manner, has a molecular weight of 62,000. Crosslinking is a useful tool for detecting this novel membrane protein in kidney.