Study of the differential modifications of ( [formula omitted])-ATPase and its partial reactions by dimethylsulfoxide

Abstract

1. 1. Dimethylsulfoxide inhibits ( Na + + K +)-activated ATPase but increases [ 14C]ADP binding to the enzyme and stimulates the associated K +-dependent p- nitrophenylphosphatase . 2. 2. Enhancement of [ 14C]ADP binding occurs by a decrease of the dissociation constant from K D = 0.95 μ M to 0.09 μM in the presence of 30% dimethylsulfoxide. 5 mM Na + enhances ADP affinity in the presence and absence of dimethylsulfoxide about 3 fold. However, the decrease of the ADP affinity in the presence of K + is abolished by increasing concentrations of dimethylsulfoxide. Two ADP-binding sites, a high-affinity ( K D = 0.17 μ M ) and a low-affinity site ( K D = 0.40 μ M , appeared in the presence of 30% dimethylsulfoxide and 0.2 mM Mg 2+ or Ca 2+. Mg 2+ and Ca 2+ decreased the affinity of the enzyme for ADP in the presence and absence of dimethylsulfoxide. 3. 3. Dimethylsulfoxide decreased the rate of Na + -dependent phosphorylation of an EDTA-washed enzyme (E 1 conformation) and reduced the hydrolysis of the [ 32P]phosphoprotein. However, dimethylsulfoxide enhanced the Na +-dependent phosphorylation of the Rb +-bearing dephosphoenzyme ( Rb + · E 2 ). 4. 4. Dimethylsulfoxide increased the dissociation constant of the ouabain-receptor complex when ouabain binding was studied via the Na +-dependent pathway. 5. 5. The results suggest that dimethylsulfoxide acts on several sites: It favors the E 1 conformational state of the enzyme by an increased affinity for nucleotide triphosphates and diphosphates, by reducing the affinity of K + for the enzyme in the presence of nucleotides and possibly by an expulsion of K + from the K +-dephospho-enzyme. Inhibition of ( Na + + K +)-ATPase may occur by a decreased interaction of the phosphokinase and phosphatase sites with the phosphorylacceptor site.