ATP-driven proton transport in vesicles from the kidney cortex

Abstract

Publisher Summary: This chapter describes an improved preparation of endocytotic vesicles from rat renal cortex. This method yields vesicles that are more enriched in proton pump activity. The preparation of endocytotic vesicles takes about 6 hr and does not require an ultracentrifuge. Therefore, endocytotic vesicles from kidney can serve as an excellent model to study the characteristics of the vacuolar proton pump (H+-ATPase). This chapter describes the way of tissue homogenization is of critical importance for the success of the preparation. Although the proton pump (H+- adenosine tri-phosphate (ATP)-ase) in isolated endocytotic vesicles is strongly inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), N-Ethylmaleimide (NEM), and diethylstilbestrol (DES), neither of these inhibitors can be used as a specific marker for the pump. Experiments showing the distribution of inhibitor-sensitive ATPases indicate that much higher activities of DCCD-, NEM-, and DES-sensitive ATPases are present in fractions of the Percoll gradient that do not contain endocytotic vesicles with an ATP-driven proton pump