Large-scale preparation of sodium-potassium ATPase from kidney outer medulla.

Background: Large amounts of Na,K-ATPase are needed for studies involving protein chemistry. Preparation of Na,K-ATPase from kidney by the widely used, rapid procedure of Jørgensen (Biochim Biophys Acta 356:36-52, 1974; Methods Enzymol 156:29-43, 1988) includes labor-intensive dissection of tissue from the outer medulla and centrifugation into a step gradient of sucrose solution.

Methods: In a large-scale modification presented here, tissue was dissected with a surgical instrument, a rongeur, and centrifugation was simply a five times repeated differential centrifugation.

E2P phosphoforms of Na,K-ATPase. II. Interaction of substrate and cation-binding sites in Pi phosphorylation of Na,K-ATPase.

In this investigation the effects of alkali cations on the transient kinetics of Na,K-ATPase phosphoenzyme formation from either ATP (E2P) or Pi (E'2P) were characterized by chemical quench methods as well as by stopped-flow RH421 fluorescence experiments. By combining the two methods it was possible to characterize the kinetics of Na, K-ATPase from two sources, shark rectal glands and pig kidney. The rate of the spontaneous dephosphorylation of E2P and E'2P was identical with a rate constant of about 1.

E2P phosphoforms of Na,K-ATPase. I. Comparison of phosphointermediates formed from ATP and Pi by their reactivity toward hydroxylamine and vanadate.

The properties of Na,K-ATPase phosphoenzymes formed either from ATP in the presence of Mg2+ and Na+ or from Pi in the absence of alkali cations were investigated by biochemical methods and spectrofluorometry employing the styryl dye RH421. We characterized the phosphoenzyme species by their reaction to N-methyl hydroxylamine, which attacks specifically the protein-phosphate bond. We studied reactions of the phospho- and dephospho-enzymes with vanadate, which is a transition-state analogue of phosphate in this enzyme.

Interpretation of extraordinary kinetics of Na(+)-K(+)-ATPase by a phase change.

We interpret at a molecular level an extraordinary response in the transient kinetics of the phosphointermediate of Na(+)-K(+)-ATPase (I. Klodos, R. L.

Fluorescent styryl dyes as probes for Na,K-ATPase reaction mechanism: significance of the charge of the hydrophilic moiety of RH dyes.

The fluorescence responses of a series of potential-sensitive styryl-based dyes (either zwitterionic RH160, RH421, di-4-ANEPPS, or positively charged RH795, RH414, RH461) to phosphorylation of Na,K-ATPase from ATP or inorganic phosphate, and ouabain binding to phospho- or dephosphoforms, have been characterized and compared in broken membrane preparations of the enzyme. Zwitterionic dyes were more sensitive to molecular events in the Na,K-ATPase reaction cycle than positively charged dyes, but the net charge did not affect the sensitivity of the dyes to a transmembrane electric field. The major part of the response of the zwitterionic dyes to formation of phosphoenzymes was due to a change in the quantum yield of fluorescence.

Kinetic heterogeneity of phosphoenzyme of Na,K-ATPase modeled by unmixed lipid phases. Competence of the phosphointermediate.

Interconversion of phosphoenzyme resistant to K+ and sensitive to ADP (E1P) and phosphoenzyme resistant to ADP and sensitive to K+ (E2P) was studied in bovine brain and dog and pig kidney. The kinetics of dephosphorylation were observed by chasing phosphoenzyme formed from [32P]ATP with unlabeled ATP with or without ADP or K+. Phosphorylation in 0.

The influence of membrane charge on the kinetic properties of Na,K-ATPase.

Lipophilic ions modify the affinity of the cation binding sites of the membrane-bound Na,K-ATPase. We studied the effect of the lipophilic ions tetraphenyl-phosphonium (TPP+) and tetraphenylboron (TPB-) on the binding of Na+ and K+ to the cation site(s) that are exhibited by the enzyme during the catalytic cycle: the high-affinity (inside) Na-binding site, site I, the low-affinity (outside) Na-leaving site, site II, and the high-affinity (outside) K-site, site III. Site I: In the presence of TPP+ (positive charge added to the lipid environment) a higher Na(+)-concentration was needed to obtain phosphorylation of the enzyme, whereas in the presence of TPB- (negative charge added to the lipid environment) phosphorylation was obtained at a lower Na(+)-concentration, but the change in apparent K0.

(Na+ + K+)-ATPase: confirmation of the three-pool model for the phosphointermediates of Na+-ATPase activity. Estimation of the enzyme-ATP dissociation rate constant.

The dephosphorylation kinetics of acid-stable phosphointermediates of (Na+ + K+)-ATPase from ox brain, ox kidney and pig kidney was studied at 0 degree C. Experiments performed on brain enzyme phosphorylated at 0 degree C in the presence of 20-600 mM Na+, 1 mM Mg2+ and 25 microM [gamma-32P]ATP show that irrespectively of the EP-pool composition, which is determined by Na+ concentration, all phosphoenzyme is either ADP- or K+-sensitive. After phosphorylation of kidney enzymes at 0 degree C with 1 mM Mg2+, 25 microM [gamma-32P]ATP and 150-1000 mM Na+ the amounts of ADP- and K+-sensitive phosphoenzymes were determined by addition of 1 mM ATP + 2.

Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+.

To determine the biochemical events of Na+ transport, we studied the interactions of Na+, Tris+, and K+ with the phosphorylated intermediates of Na,K-ATPase from ox brain. The enzyme was phosphorylated by incubation at 0 degrees C with 1 mM Mg2+, 25 microM [32P]ATP, and 20-600 mM Na+ with or without Tris+, and the dephosphorylation kinetics of [32P]EP were studied after addition of (1) 1 mM ATP, (2) 2.5 mM ADP, (3) 1 mM ATP plus 20 mM K+, and (4) 2.

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. III. A minimal model.

A steady-state kinetic investigation of the effect of K+ on the Na+-enzyme activity of the (Na+ + K+)-ATPase in broken membrane preparations is reported. Analysis of the kinetic patterns obtained, together with the results reported in the first two articles of this series permit the following conclusions. 1.

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. II. Kinetic characterization of phosphointermediates.

(1) The kinetics of the phosphorylated enzymic intermediates of (Na+ + K+)-ATPase from ox brain, which are formed by incubation of the enzyme with 25 microM AT32P, 150 mM Na+ and 1 mM Mg2+, have been studied in dephosphorylation experiments at 1 degree C. The dephosphorylation of the 32P-labelled enzyme was initiated by addition of either 1 mM unlabelled ATP, 2.5 mM ADP or 1 mM unlabelled ATP + ADP in concentrations from 25 to 1000 microM.

The effect of chelators on Mg2+, Na+-dependent phosphorylation of (Na+ + K+)-activated ATPase.

1. The effect of free Mg2+, MgEDTA and MgCDTA on the phofphorylation of the (Na+ + K+)-activated ATPase (ATP phosphohydrolase, EC 3.6.

The effect of Mg2+ and chelating agents on intermediary steps of the reaction ofNa+,K+-activated ATPase.

(1) It has been investigated how varying concentrations of free magnesium with and without EDTA influence the properties of the phospho-enzyme formed in the presence of sodium by the (Na+ plus K+)-activated enzyme system. (2) The phospho-enzyme formed in the presence of sodium and a high concentration of free magnesium has the same rate of (a) spontaneous dephosphorylation, (b) dephosphorylation after addition of potassium, and (c) dephosphorylation after addition of ADP, as a phospho-enzyme formed in the presence of sodium and a low concentration of magnesium. (3) With sodium and a given concentration of free magnesium, high or low, EDTA present during formation of the phospho-enzyme leads to a decrease in the rate of (a) spontaneous dephosphorylation, and (b) dephosphorylation after addition of potassium to the phospho-enzyme.