Vanadate inhibition of ATP and p-nitrophenyl phosphate hydrolysis in the fragmented sarcoplasmic reticulum.
The vanadate inhibition of the Ca(2+)-ATPase activity was analysed both in intact sarcoplasmic reticulum vesicles and in the presence of low concentrations of Tween 20, using ATP and p-nitrophenyl phosphate as substrates. The saturation of the internal low-affinity calcium-binding sites protects the enzyme against vanadate inhibition, because: (1) p-nitrophenyl phosphate hydrolysis is not inhibited by vanadate in intact vesicles, but inhibition developed after solubilization with detergents; (2) the vanadate inhibition of the p-nitrophenyl phosphate hydrolysis in solubilized preparations is prevented by free Ca2+ concentrations higher than 10(-3) M and vanadate competes with calcium (10(-5)-10(-3) M); and (3) the vanadate inhibition of ATP hydrolysis is decreased with an increase in vesicular Ca2+ concentration. The presence of magnesium ions is indispensable for the vanadate effect. The vanadate inhibition is non-competitive with respect to Mg-p-nitrophenyl phosphate and uncompetitive with respect to Mg-ATP. However, in the presence of dimethyl sulfoxide, which facilitates phosphorylation of the enzyme, the inhibition is converted to a competitive one with respect to a substrate. The results suggest, that in the process of enzyme operation vanadate interacts with the unliganded E form of Ca(2+)-ATPase, occupying probably an intermediate position between the E2 and E1 forms, with the formation of an E2 Van complex, that imposes the inhibition on the Ca(2+)-ATPase activity.
Vashchenko
V I
VI
Institute of Physiology, Academy of Sciences, Kasakh SSR, Alma-Ata, U.S.S.R.
Utegalieva
R S
RS
Esyrev
O V
OV
D009597:4-Nitrophenylphosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010070:Oxalates / Q000378:metabolism; D019815:Oxalic Acid; D011895:Rana ridibunda; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014638:Vanadates / Q000494:pharmacology*
D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010070:Oxalates; C008644:nitrophenylphosphate; D014638:Vanadates; D000255:Adenosine Triphosphate; D019815:Oxalic Acid; D009597:4-Nitrophenylphosphatase; D000252:Calcium-Transporting ATPases; D008274:Magnesium