Hermann Bader

(K+)-dependent acyl phosphatase as part of the (na+ + K+)-dependent ATPase of cell membranes.

Summary 1. A (K + )-dependent acetyl phosphatase (acyl phosphate phosphohydrolase, EC 3.6.1.7) activity of a (Na + + K + )-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparation from cell membranes of guinea-pig kidney cortex was compared with the (Na + (Na + + K + )-dependent ATPase activity. 2. Of a variety of phosphate compounds tested only acetyl phosphate and carbamyl phosphate showed a (K + )-dependent hydrolysis which was comparable with the (Na + + K + )-dependent hydrolysis of ATP. 3. A close relationship was shown between the (K + )-dependent hydrolysis of acetyl phosphate and the (K + )-dependent part of the hydrolysis of ATP by the (Na + + K + )-dependent ATPase preparations. Both activities showed the following characteristics: K + could be replaced by Rb + Cs + , NH 4 + or Li + but not by Na + . The K m s for Mg 2+ , K + , Rb + , NH 4 + and Li + were similar. The optimal pH was 7.8 for both. Both activities were inhibited by ouabain, Ca 2+ , Hg 2+ , and F − with similar K i s, They had similar activation energies. ATP and acetyl phosphate were each competitive inhibitors of the other as substrate. 4. These close relationships suggest that the (K + )-dependent acetyl phosphatase activity arises from the (Na + + K + )-dependent ATPase system and that an acyl phosphate may be a functional intermediate in the system.

Comparison of sources of a phosphorylated intermediate in transport ATPase

Abstract 1. 1. The relationship between the enzymatic activity and the phosphorylated intermediate of the (Na + + K + )-dependent ATPase (EC 3.1.6.3) from 11 species and 6 tissues was tested. 2. 2. The range in specific activity of the ATPase was more than 400-fold, yet the range in the ratio of the (Na + + K + )-ATPase activity to the phosphorylated intermediate was only 2-fold, and the range in the ratio of the (K + )-acylphosphatase (EC 3.6.1.7) activity of the (Na + + K + )-ATPase activity was also only 2-fold. 3. 3. The (Na + + K + )-ATPase of all sources tested required Na + for phosphorylation and its dephosphorylation was greatly accelerated in the presence of K + . 4. 4. The turnover of the phosphorylated enzyme at 0° in the absence of K + , estimated by washout of 32 P, showed a range of 3-fold. 5. 5. The phosphorylated enzymes yielded identical 32 P-labeled peptides after digestion with pepsin (EC 3.4.4.1) or pronase and electrophoresis. The 32 P-labeled peptides of the (Na + + K + )-ATPase of all sources tested were sensitive to hydroxylamine. 6. 6. The results indicate that the mechanism of the (Na + + K + )-dependent ATPase is likely to be similar in most animal species and tissues.

Inhibition of human erythrocyte Ca++-transport ATPase by phenothiazines and butyrophenones

Abstract Antipsychotic agents such as the phenothiazines-trifluoperazine, fluphenazine and chlorpromazine- and the butyrophenones-penfluridol, fluspirilene and pimozide — which were shown to interact with calmodulin in a Ca++-dependent fashion, competitively inhibit Ca++-transport ATPase of human erythrocyte membranes. Activation of the ATPase by calmodulin is half-maximally antagonized by these agents in the concentration range of 2.6–22 μM whereas inhibition of the basal ATPase (in the absence of calmodulin) is achieved at about 10-fold higher concentrations.

Isolation and characterization of a phosphorylated intermediate in the (Na+ + K+) system-dependent ATPase*

Summary 1. A phosphorylated intermediate material in the (Na + + K + )-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) transport system was solubilized by peptic digestion and.subjected to paper electrophoresis. 2. Brief digestion released one fragment which moved toward the cathode. Prolonged digestion converted this fragment into two subfragments. 3. The subfragments and the parent material were characterized as probably being acyl phosphates by their lability in strong acid and at neutral and alkaline pH, by their ease of methanolysis or ethanolysis, by their lability in the presence of hydroxylamine or molybdate, and by their response to bivalent ions. 4. The mol. wt. of the subfragments was estimated to be about 2200 by gel filtration. 5. The subfragments could be oxidized with performic acid or acetylated with acetic anhydride. 6. The subfragments were not soluble in ether.

Acetyl phosphate as a substitute for ATP in (Na− + K+)-dependent ATPase

Abstract 1. 1. In the presence of Mg 2+ and Na + , acetyl phosphate replaced ATP as an agent for phosphorylating Na + - and K + -dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3). 2. 2. The phospho-enzymes produced by both substrates were similar in the following respects: (a) electrophoretic mobility and chemical reactivity of phosphopeptides released by proteolytic digestion of the denatured enzyme, (b) the quantity of phospho-enzyme, (c) the concentration of Na + for half-maximal phosphorylation, (d) a requirement for Mg 2+ , (e) the half-life at o°. Furthermore, each substrate inhibited phosphorylation by the other, and the inhibitor ouabain enhanced the inhibitory effect of acetyl phosphate. 3. 3. In the presence of Mg 2+ and K + , acetyl phosphate, unlike ATP, also phosphorylated the enzyme. The quantity of phospho-enzyme was less and its turnover was faster. K + appeared to accelerate both phosphorylation and dephosphorylation. 4. 4. The same active site may be an intermediate in both the Na + - and K + -dependent ATPase activity and the K + -dependent acetyl phosphatase activity of this enzyme system in functionally different conformations of an active center.

Gradient elution of phosphatides from silicic acid column

Abstract Previous methods for the separation of phosphatides using silicic acid chromatography have been modified. A continuous concave-upward gradient of methanol in chloroform has been employed. The separation was highly, reproducible with respect to quantity and distribution of lipid phosphorus and was free of factitious secondary peaks.

Further characterization of a phosphorylated intermediate in (Na+ + K+)-dependent ATPase

Abstract 1. A radioactive acyl phosphate intermediate in the (Na+ + K+)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) of guinea-pig kidney was treated with proteolytic enzymes. The soluble products were separated by paper electrophoresis at pH 2.1. 2. Differential digestion with pepsin (EC 3.4.4.1) yielded a sequence of six fragments. Their molecular weights ranged from about 10000 to about 2200 as estimated by gel filtration. 3. All fragments were positively charged and contained at least one sulfhydryl group and no disulfide bond. 4. Hydrolysis of the intermediate or the peptic fragments with pronase gave a positively charged fragment with a molecular weight of 380. This fragment contained no sulfhydryl group. At least part of the positive charge was not that of histidine or lysine. The pronase fragment was not arginyl phosphate. 5. The results provide further evidence that the acyl phosphate group is attached to a protein containing one or more sulfhydryl and basic groups.

Effects of Vinca Alkaloids on Calmodulin-Dependent Ca2+-Transport ATPase

SummaryThe Vinca alkaloids vinblastine, vindesine and vincristine were found to be inhibitors of the calmodulin-dependent fraction of red blood cell Ca2+-transport ATPase with IC50-values of 35, 100 and 220 μM, respectively. However, in the concentration range of 1–10 uM all three substances inhibit 10–15 % of the calmodulin-dependent ATPase activity. The concentration of vinblastine for 50 % inhibition of the calmodulin-dependent fraction of the Ca2+transport into inside-out vesicles is very similar to that found for the Ca2+-ATPase. It was shown that the inhibitory potency of vinblastine can be reduced by increasing calmodulin concentrations, suggesting a competitive mechanism. With respect to other ATPases the action of vinblastine on Ca2+-transport ATPase seems to be specific since Mg2+-ATPase and (Na+ + K+)-ATPase are hardly affected up to 300 μM.