Reversible inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid of the plasma membrane (Ca(2+)+Mg2+)ATPase from kidney proximal tubules.
Calcium accumulation by purified vesicles derived from basolateral membranes of kidney proximal tubules was reversibly inhibited by micromolar concentrations of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of anion transport. The inhibitory effect of this compound on Ca2+ uptake cannot be attributed solely to the inhibition of anion transport: (Ca(2+)+Mg2+)ATPase and ATP-dependent Ca2+ transport, respectively. The rate constant of EGTA-induced Ca2+ efflux from preloaded vesicles was not affected by DIDS, indicating that this compound does not increase the permeability of the membrane vesicles to Ca2+. In the presence of DIDS, the effects of the physiological ligands Ca2+, Mg2+, and ATP on (Ca(2+)+Mg2+)ATPase activity were modified. The Ca2+ concentration that inhibited (Ca(2+)+Mg2+)ATPase activity in the low-affinity range decreased from 91 to 40 microM, but DIDS had no effect on the Km for Ca2+ in the high-affinity, stimulatory range. Free Mg2+ activated (Ca(2+)+Mg2+)ATPase activity at a low Ca2+ concentration, and DIDS impaired this stimulation in a noncompetitive fashion. The inhibition by DIDS was eliminated when the free ATP concentration of the medium was raised from 0.3 to 8 mM, possibly due to an increase in the turnover of the enzyme caused by free ATP accelerating the E2----E1 transition, and leading to a decrease in the proportion of E2 forms under steady-state conditions. Alkaline pH totally abolished the inhibition of the (Ca(2+)+Mg2+)ATPase activity by DIDS, with a half-maximal effect at pH 8.3.(ABSTRACT TRUNCATED AT 250 WORDS)
Guilherme
A
A
Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil.
Meyer-Fernandes
J R
JR
Vieyra
A
A
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors*; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002463:Cell Membrane Permeability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000201:enzymology*; D008051:Lipid Bilayers / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D011487:Protein Conformation; D012756:Sheep
D008051:Lipid Bilayers; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium