Characterization of Binding of Folates and Antifolates to Brush-Border Membrane Vesicles Isolated from Human Kidney

2004 ◽  
Vol 67 (2) ◽  
pp. 453-459 ◽  
Author(s):  
Vijaya L. Damaraju ◽  
Katherine F. Hamilton ◽  
Michelle L. Seth-Smith ◽  
Carol E. Cass ◽  
Michael B. Sawyer
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Human Kidney ◽  
Brush Border Membrane Vesicles

scholarly journals Characterization of an ATP-driven H+ pump in human placental brush-border membrane vesicles

1992 ◽  
Vol 287 (2) ◽  
pp. 423-430 ◽  
Author(s):  
B J Simon ◽  
P Kulanthaivel ◽  
G Burckhardt ◽  
S Ramamoorthy ◽  
F H Leibach ◽  
...  
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Bafilomycin A1 ◽  
Ph Indicator ◽  
Native Form ◽  
Anion Diffusion ◽  
Partial Activity

The presence of an ATP-driven H+ pump as measured by H+ uptake upon addition of ATP was not demonstrable in human placental brush-border membrane vesicles when used in their native form, owing to their right-side-out orientation. However, the presence of the H+ pump in these membranes became evident when the membrane vesicles were transiently exposed to 1% cholate, with subsequent removal of the detergent to re-form the vesicles. Apparently, cholate pretreatment reoriented the H+ pump from an inward-facing configuration to outward-facing. Consequently, H+ uptake in response to externally added ATP was easily demonstrable in these cholate-pretreated vesicles by using the delta pH indicator Acridine Orange. In addition, bafilomycin A1-sensitive ATPase activity was measurable in cholate-pretreated vesicles, but not in native intact vesicles, indicating reorientation of the H+ pump. The reoriented H+ pump was electrogenic because H+ uptake was stimulated by an inside-negative anion-diffusion potential or when the vesicles were voltage-clamped. ATP supported H+ uptake with an apparent Km of 260 microM. ITP and GTP supported the pump activity partially, whereas CTP and UTP did not. Mg2+ and Mn2+ were the most preferred bivalent cations. Co2+ and Zn2+ showed partial activity, whereas Ca2+ and Ba2+ showed little or no activity. The pump was inhibited by nanomolar concentrations of bafilomycin A1 and micromolar concentrations of N-ethylmaleimide, p-chloromercuribenzenesulphonate, NN-dicyclohexylcarbodi-imide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but was relatively insensitive to oligomycin, vanadate and NaN3. The inhibition by N-ethylmaleimide was protectable by ATP. It is concluded that human placental brush-border membranes possess an ATP-driven H+ pump and that, on the basis of its characteristics, it belongs to the class of vacuolar (V-type) H+ pumps.

scholarly journals Characterization of a sodium-dependent concentrative nucleobase-transport system in guinea-pig kidney cortex brush-border membrane vesicles

1994 ◽  
Vol 303 (3) ◽  
pp. 901-905 ◽  
Author(s):  
D A Griffith ◽  
S M Jarvis
Keyword(s):  
Guinea Pig ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Hill Coefficient ◽  
Kidney Cortex ◽  
Pig Kidney ◽  
Na Ions

The characteristics of hypoxanthine transport were examined in purified brush-border membrane vesicles isolated from guinea-pig kidney. Hypoxanthine uptake in the vesicles was specifically stimulated by both Na+ and an inside-negative potential, resulting in a transient accumulation of intravesicular hypoxanthine. Na(+)-dependent hypoxanthine influx was saturable (apparent Km 4.4 +/- 2.1 microM, Vmax. 128 +/- 29 pmol/min per mg of protein at 100 mM NaCl and 22 degrees C). Guanine, thymine, 5-fluorouracil and uracil inhibited hypoxanthine uptake (Ki values 1-30 microM), but adenine and the nucleosides inosine and thymidine were without effect. Guanine competitively inhibited Na(+)-dependent hypoxanthine influx, suggesting that it was a substrate for the active nucleobase transporter in guinea-pig renal membrane vesicles. A sigmoidal dependence between hypoxanthine influx and Na+ concentration was obtained (KNa 13 +/- 2 mM; Hill coefficient, h, 2.13 +/- 0.14), suggesting that at least two Na+ ions are transported per hypoxanthine molecule. This system differs from the Na(+)-nucleobase carrier in cultured LLC-PK1 renal cells, which has a stoichiometric coupling ratio of 1:1. These results represent the first demonstration of an active electrogenic nucleobase carrier in renal apical membrane vesicles.

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