An efficient method for the isolation and separation of basolateral-membrane and luminal-membrane vesicles from rabbit kidney cortex

Abstract

A procedure for isolation and separation of purified luminal-membrane and basolateral-membrane vesicles from adult and newborn rabbit renal cortex by using Ca2+/Mg2+ precipitation, differential centrifugation and a self-orienting Percoll-gradient centrifugation is described. The purity of the membrane-vesicle suspensions was examined by electron microscopy and by measuring the activity of several marker enzymes. The activity of Na+ + K+-stimulated ATPase in the fraction mainly containing adult rabbit basolateral-membrane vesicles was enriched 16-fold, and the activity of alkaline phosphatase in the fraction mainly containing luminal-membrane vesicles was increased 13-fold, compared with the homogenate. Similar results were obtained with kidneys from newborn rabbits. Uptake studies, with a rapid filtration technique and the spectrophotometric method described in an accompanying paper [Kragh-Hansen, Jørgensen & Sheikh (1982) Biochem. J. 208, 359-368], showed that both adult and newborn rabbit luminal-membrane vesicles, in contrast with the basolateral-membrane preparations, possess an Na+-dependent electrogenic transport system for L-proline. Adult rabbit luminal-membrane vesicles take up citrate and L-malate by Na+-dependent electrogenic processes, whereas adult rabbit basolateral membrane vesicles do not exhibit electrogenic uptake of citrate. By contrast, these vesicles show Na+-dependent electrogenic uptake of L-malate.

Production and Actions of Hydrogen Sulfide, a Novel Gaseous Bioactive Substance, in the Kidneys

“…Basolateral membrane was prepared from the kidney tissue as described previously (Sheikh et al, 1982). HPLC analysis of renal Na ϩ /K ϩ -ATPase activity in the basolateral membranes was performed by detecting the conversion of ATP into ADP as reported previously (Sudo et al, 2000).…”

Demonstration of H+‐ and Na+‐coupled co‐transport of beta‐alanine by luminal membrane vesicles of rabbit proximal tubule.

“…Unless otherwise stated the vesicles were suspended in a solution containing 310 mM-mannitol and 15 mM-HEPES-Tris buffer, pH 7-5, but in a series of experiments luminal membrane vesicles were prepared and suspended in a solution containing 310 mM-mannitol and 15 mm-MES-Tris buffer, pH 5-5. The purity of the membrane vesicle preparation was examined by electron microscopy (Kragh-Hansen et al 1985) and by measuring specific activities of various enzyme markers as previously described (Sheikh, Kragh-Hansen, J0rgensen & R0igaard-Petersen, 1982). The protein concentration in the membrane fractions was determined by the method of Lowry, Rosebrough, Farr & Randall (1951), as modified by Peterson (1977) with serum albumin as a standard.…”

Ba2+‐sensitive K+ channels in luminal‐membrane vesicles from pars convoluta of rabbit proximal tubule

“…The final pellet was washed for 20 min by a solution consisting of 3.75 mM EDTA, 298 mM sucrose and 27 mM Hepes/Tris buffer, pH 7.4, and resuspended in the same medium minus EDTA. The purity of the membrane vesicle preparations was examined by electron microscopy [11] and by measuring specific activities of various enzyme markers as in [12]. The amount of protein was determined as described by Lowry et al [13] and as modified by Peterson [14] and with serum albumin as a standard.…”