Irena Klodos

Large-scale preparation of Na,K-ATPase from ox brain

Abstract A method for the isolation of membrane-bound Na,K-ATPase in quantity from brain gray matter is described. The method permits a large amount of enzyme to be obtained rather quickly with about 60% of the original activity of Na,K-ATPase of the tissue being recovered. The enzyme is stable, it has a specific activity of about 200 μmoles ATP split per mg protein per hour. Mg-ATPase comprises about 1% of the total ATPase activity. The enzymatic properties of this Na,K-ATPase do not differ from those in the literature; the turnover number is about 9300 min −1 .

The effect of Mg2+ and chelating agents on intermediary steps of the reaction of Na+, 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. (4) The rate of dephosphorylation after addition of ADP to phospho-enzyme formed without and with EDTA is the same. But as the rate of spontaneous dephosphorylation is lower with EDTA than without, ADP gives a higher increase in the rate of dephosphorylation of phospho-enzyme formed with EDTA than without. (5) The experiments thus show that the reported different sensitivity towards potassium and ADP of phospho-enzyme formed in the presence of a low and high concentration of free magnesium, respectively, is due to the EDTA used to decrease the free magnesium concentration and not to the decrease in the free magnesium as such.

Purification and characterization of (Na+ + K+)-ATPase. VII. Tryptic degradation of the Na-form of the enzyme protein resulting in selective modification of dephosphorylation reactions of the (Na+ + K+)-ATPase

Abstract 1. 1. We have examined the effect of partial tryptic digestion of purified ( Na + + K + )-ATPase in the presence of NaCl on the formation and degradation of the phosphoenzyme formed from ATP. The digestion to a ( Na + + K + )-ATPase activity of 40% barely affects ATP binding and the steady-state level of phosphorylation, but increases the rate of the Na + -ADP-ATP exchange to 150% of control and reduces the K + -phosphatase activity to 15–20% of control (Jorgensen, P.L. (1977) Biochim. Biophys. Acta 466, 97–108). 2. 2. After this tryptic cleavage in the presence of NaCl, the fraction of ADP sensitive phosphoenzyme was 2–3-fold higher than control levels. The presence of KCl depressed the phosphoenzyme level 2–3-fold less than that of the control and the K + -dependent increment of the rate constant for dephosphorylation was less than half that of the control. 3. 3. The results show that partial tryptic digestion of the purified ( Na + + K + )-ATPase in the presence of NaCl selectively affects protein areas involved both in the transformation of the phosphoenzyme from an ADP-sensitive to an ADP-insensitive form and in the stimulation by K + of the dephosphorylation process and the K + -phosphatase activity. The protein areas that are involved in these reactions seem therefore to be close to one another, or identical, and they appear to be spatially separated from the ATP binding area.

(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 + 2.5 mM ADP or 1 mM ATP + 20 mM K+. Similarly to the previously reported results on brain enzyme, both types of dephosphorylation curves have a fast and a slow phase, so that also for kidney enzymes a slow decay of a part of the phosphoenzyme, up to 80% at 1000 mM Na+, after addition of 1 mM ATP + 20 mM K+ is observed. The results obtained with the kidney enzymes seem therefore to reinforce previous doubts about the role played by E1 approximately P(Na3) as intermediate of (Na+ + K+)-ATPase activity. Furthermore, for both kidney enzymes the sum of ADP- and K+-sensitive phosphoenzymes is greater than E tot. In experiments on brain enzyme an estimate of dissociation rate constant for the enzyme-ATP complex, k-1, is obtained. k-1 varies between 1 and 4 s-1 and seems to depend on the ligands present during formation of the complex. The highest values are found for enzyme-ATP complex formed in the presence of Na+ or Tris+. The results confirm the validity of the three-pool model in describing dephosphorylation kinetics of phosphointermediates of Na+-ATPase activity.

Sulfhydryl Groups of Na.K-ATPase: Effects of N-Ethylmaleimide on Phosphorylation from ATP in the Presence of Na+ + Mg2+

Publisher Summary This chapter discusses the effect of N-ethylmaleimide (NEM) on the Na, K-ATPase activity with respect to the molar activity of the modified enzyme. The result of modifying Na, K-ATPase with NEM with different ligands present during the modification is presented in the chapter. In the presence of Na+ alone, Na+ + ATP, or K+ alone, the decrease in Na, K-ATPase activity is almost parallel to the decrease in EP level—the molar activity remains high, 80–90% of the control enzyme. However, with K+ + ATP present in the modification medium, a large amount of EP is retained in spite of a loss of Na, K-ATPase activity. The molar activity is thus decreased markedly, the effect being more pronounced at longer incubation times. The experiments discussed in the chapter suggests that the one SH-group not modified by NEM in the presence of K+ + ATP is essential to phosphorylation, but that the modification of the other 3 SH-groups inactivates the overall enzyme activity. It is also suggested that for the Na-ATPase this inactivation is probably a decreased ability to perform E1P–E2P conformational change, in accordance with the results of Fahn et al.

Fluorescent styryl dyes as probes for Na,K-ATPase reaction. Enzyme source and fluorescence response.

Fluorescent styryl dyes (RH-dyes), when bound to Na,K-ATPase-enriched broken membranes, change their fluorescence in response to some molecular events in the Na,K-ATPase reaction cycle. Although the mechanism of dye response is still controversial, they have recently found increasing application in studies of the reaction mechanism of P-type transport ATPases. It has been suggested’-* that the dyes’ responses to changes in the intramembranous electric field are modified by interactions between these dyes and the Na,K-ATPase protein. It was previously shown’ that the excitation spectra of RH160 depend on the concentration of protein reconstituted into liposomes, and the same is found for absorbance (not shown), an indication of a dye-protein interaction. To evaluate the significance of these interactions we studied RH421 responses to (1) Na+ binding, (2) phosphorylation from either ATP or Pi, or (3) vanadate binding to Na,K-ATPase (FIG. 1). Experiments were performed with membrane-bound Na,KATPase, solubilized enzyme, and enzyme reconstituted into liposomes. Furthermore, we used enzymes from different sources, shark rectal gland and pig kidney, and varied their lipid surroundings in reconstituted preparations. For both enzymes we measured the specific activities at 23°C and 37”C, the phosphorylation level in the presence of 4 mh4 MgCI2, 16 mM NaCI, and 30 pM [Y-~~PI-ATP in 30 mM imidazole (pH 7.5) at 0°C and in the presence of inorganic 32P in the same medium, but without NaC1. Both enzyme preparations and all applied methods were described by Cornelius’ and Fedosova et al.’ Shark enzyme was solubilized in C12E8. The activity of kidney enzyme, solubilized in CHAPS, deteriorated quickly. Therefore, measurements of its catalytic activity and fluorescence measurements were processed in parallel. The results are shown in TABLE 1. The experimental observations can be summarized as follows: 1 . RH421 responses depend on the enzyme source. Formation of several intermediates is reported differently with the two enzymes, for example, formation of the enzyme-vanadate complex is “seen” with the shark enzyme but not with the kidney enzyme (TABLE 1). RH421 bound to proteoliposomes containing kidney enzyme