Kei Nagano

Potassium-ion stimulated p-nitrophenylphosphatase activity occurring in a highly specific adenosine triphosphatase preparation from rabbit brain

Abstract 1. 1. p -Nitrophenylphosphatase activity was detected in a highly specific Na + - and K + -dependent ATPase preparation obtained by NaI treatment of rabbit-brain microsomes. 2. 2. The p -nitrophenylphosphatase activity required K + and was inhibited by Na + . The concentrations of K + and Na + required for half maximal activation and half maximal inhibition were 3.2 and 32 mM, respectively. 3. 3. The p -nitrophenylphosphatase activity was inhibited by ouabain, and this inhibition was not affected by Na + . The concentration of ouabain required for 50% inhibition of p -nitrophenylphosphatase activity was approx. 2.5·10 −5 M, which was 30 times that required for 50% inhibition of ATPase activity. However, the former was decreased to 2.5·10 −6 M by the addition of 40 μM of ATP. 4. 4. In the absence of Na + the preparation was active exclusively with -nitrophenylphosphate. None of the following was hydrolyzed: ATP, AMP, 2′-(3′)-AMP, CMP, IMP, GMP, glucose 6-phosphate, glucose 1-phosphate, β-glycerophosphate or phenylphosphate. 5. 5. The p -nitrophenylphosphatase activity was inhibited by a low concentration of ATP. Inhibition was of the simple non-competitive type with a K i value of 4·10 −5 M. The inhibition of ATPase activity by p -nitrophenylphosphate was also of the non-competitive type, the K i for p -nitrophenylphosphate being 4·10 −2 M. 6. 6. The K m for p -nitrophenylphosphate was 1.5·10 −3 M, and that for ATP, 6·10 −5 M. 7. 7. p -Nitrophenylphosphatase activity was inhibited by 60–80% with 3 mM of any of the following: GTP, UTP, CTP, ADP, AMP, PP i , or P i . 8. 8. ATPase and p -nitrophenylphosphatase were similarly inactivated by either thermal denaturation or trypsin treatment. 9. 9. There was a fairly good correlation between the inhibition curves for p -nitrophenylphosphatase and ATPase in the presence of increasing concentrations of N -ethylmaleimide or diisopropylphosphorofluoridate (DFP). The inhibition of both activities by DFP was markedly enhanced by K + , and the activities were protected against the inhibition by either p -nitrophenyphosphate or ATP. 10. 10. It is suggested that the two activities could be attributed to at least two different active sites which, however, appear to be closely correlated.

Asymmetric distribution of ouabain-sensitive ATPase activity in rat intestinal mucosa

Abstract A careful washed preparation of intestinal brush border membranes from rat contained two-thirds of the sucrase activity of a starting homogenate and 2–4% of the ouabain-sensitive ATPase activity.

Primary structure of the β‐subunit of Torpedo californica (Na+ + K+)‐ATPase deduced from the cDNA sequence

DNA complementary to the Torpedo californica electroplax mRNA coding for the β‐subunit of (Na+ + K+)‐ATPase has been cloned by screening a cDNA library with an oligodeoxyribonucleotide probe. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 305 amino acid residues (including the initiating methionine). The transmembrane topology and the potential N‐glycosylation sites of this polypeptide are discussed.

Separation of two adenosine triphosphatases from erythrocyte membrane

Abstract Post et al. (1960) , Dunham and Glynn (1961) , and Whittam (1962) have presented evidence that ouabain-sensitive adenosine triphosphatase (ATPase) in erythrocytes participates in the active transport of sodium and potassium ion in erythrocytes. In previous papers, our collaborators and some of the present authors ( Nakao et al., 1960a , Nakao et al., 1960b , Nakao et al., 1961 ) reported on the reversible shape transformation of erythrocytes depending upon ATP level inside the cells. These results may indicate the presence of an ATPase which controls shape transformation of erythrocytes. Onishi (1962) suggested the presence of actin-like and myosin-like proteins in erythrocytes in his preliminary experiments. Furthermore, evidence that ouabain does inhibit the ATPase activity partially but not completely ( Dunham and Glynn 1961 ), strongly suggests the presence of a different ATPase (i.e. ouabain-insensitive ATPase) other than the ouabain-sensitive ATPase. However, it has not been proved whether the ouabain-sensitive and ouabain-insensitive components of the ATPase are two separate enzymes or not ( Dunham and Glynn 1961 ). In the present paper, separation of two ATPases from erythrocyte membranes and some of their properties will be presented.

Cloning and analysis of the 5′-flanking region of rat Na+/K+-ATPase α1 subunit gene.

Abstract We cloned a 13.3 kilobase (kb) fragment of genomic DNA spanning at least the first two exons of the rat Na+/K+-ATPase α1 subunit gene (NKAA1) and 1.5 kb of the 5′-flanking region. S1 nuclease mapping analysis of the 5′ end of the Na+/K+-ATPase mRNA indicated that the transcription initiation site was located 262 base pairs (bp) upstream of the translation initiation codon. The transcription initiation site of the Na+/K+-ATPase α1 subunit gene was identical among six tissues of adult rat (kidney, brain, heart, thyroid, liver and lung). A TATA-box-like sequence (at position −32), two Sp1 factor binding sequences (−137, −56), an active transcription factor consensus binding sequence (−71) and two glucocorticoid-responsive element half consensus sequences (−750, −481) were found in the 5′-flanking region. The sequence of the first exon and the 5′-flanking region of the rat NKAA1 was 63% homologous to that of the horse equivalent. Maximum homology (82%) between the two genes was observed in the region from 361 bp upstream of the translation initiation site to the 3′ end of the first exon. The TATA-like box, Sp1 binding site and the active transcriptional factor (ATF) consensus site in this region were conserved in both rat and horse.

On the possible role of the phosphorylated intermediate in the reaction mechanism of (Na+−K+)-ATPase

Abstract 1. Phosphorylation and dephosphorylation of (Na + −K + )-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) by [γ- 32 P]ATP was examined using highly specific NaI-treated preparations from pig-brain microsomes. 2. The phosphorylated enzyme consisted of an essentially single chemical species. The incorporated phosphate was relatively stable in an acidic medium (pH 1–2), and was easily released from the enzyme by treatment with hydroxylamine at a neutral pH. 3. The chemical nature of the phosphate bond was similar under the following alternative conditions: (a) whether the labeling was stopped by HClO 4 or dodecyl sulfate; (b) whether K + was present or not in addition to Na + ; or (c) whether the labeling was performed at 37° or 0°. 4. Longer incubation of the enzyme preparation with [γ- 32 P]ATP resulted in another form of incorporation which was hydroxylamine-resistant. 5. The high specificity of the ATP-hydrolyzing reaction of (Na + −K + )-ATPase to Na + and K + was partially lost at 0°, while the specific response of the phosphorylated enzyme to Na + and K + was still maintained. 6. Based on these results, possible relationships of the phosphorylated (Na + −K + )-ATPase to the ATP-hydrolyzing reaction of the enzyme are discussed.

Molecular weight of Na, K-ATPase approximated by the radiation inactivation method.

Abstract The Na, K-ATPase has not yet been purified as a single molecular species, mainly because of its insolubility. Nevertheless, an approximate molecular weight of the dried enzyme can be obtained by the radiation inactivation technique introduced by Hutchinson and Pollard (1961) . Using this procedure Kepner and Macey (1966) reported a value of about one million as the molecular weight for the ATPase of freeze-dried human erythrocyte ghosts. Their preparation contained both the ouabain sensitive and insensitive ATPase. A reinvestigation of this problem seemed worthwhile in that our preparation of Na, K-ATPase from brain microsomes is highly specific, essentially free of the ouabain-insensitive enzyme ( Nakao et al. , 1965 ). This preparation showed that the phosphorylated protein, obtained in a reaction with ATP 32 requiring both Na and Mg ions, consisted of a single chemical species as indicated by the time course of hydrolysis and by its sensitivity towards hydroxylamine. In this report we have estimated the target molecular weight of the enzyme involved in the phosphorylation reaction with ATP and in the overall hydrolysis of ATP.

Protective effect of Na+ and K+ against inactivation of (Na+ + K+-ATPase by high concentrations of 2-mercaptoethanol at high temperatures

Purified dog kidney (Na+ + K+)-ATPase (EC 3.6.1.3) was inactivated with high concentrations of 2-mercaptoethanol at 50-55 degrees C. The inactivation was prevented by NaCl or KCl, with KCl being more effective than NaCl (the former ion being about one order more efficient under a typical set of experimental conditions). A disulfide bond in the beta-subunit of the enzyme protein was prevented from reductive cleavage by NaCl or KCl in accordance with protection of the enzyme activity. Choline chloride did not exert a significant protective effect over a similar concentration range. (Na+ + K+)-ATPase was also inactivated with high concentrations of 2-mercaptoethanol in the presence of low concentrations of dodecyl sulfate. This inactivation was also prevented by NaCl or KCl, with the latter being again more efficient than the former. These results indicate that Na+ and K+ bound to their respective ion-binding sites on the alpha-subunit exert a protective effect on a disulfide bond on the beta-subunit. This suggests some sort of interaction between the alpha- and the beta-subunits.

The inhibition of Na,K-activated adenosinetriphosphatase by a large molecule derivative of p-chloromercuribenzoic acid at the outer surface of human red cell

Abstract A large mercurial compound was newly synthesized from Aminoethyldextran (mean MW 250,000) and p-chloromercuribenzoic acid with dicyclohexylcarbodiimide. The reagent inhibited Na, K-activated ATPase of the red cell membrane when applied to intact cells, but not the ouabain-insensitive-ATPase, whereas both were inhibited when it was applied to the membrane preparations. The active transport of rubidium in intact cells was inhibited, but was not the passive transport. The reagent may be a useful tool for studying the polarity and orientation of Na,K-ATPase and other proteins in the cell membranes.

Approximation of molecular weight of (Na+-K+)-ATPase

Abstract 1. 1. A (Na+-K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparation from pig brain microsomes was disrupted into smaller particles by sonication in the presence of a detergent. A portion of the dissected protein was active, completely inhibited by ouabain, and remained in the supernatant after centrifugation at 100 000 × g. A portion of the supernatant activity was retarded over a wide range in gel filtration through a Sepharose 4B column. Fractions of retarded activity were eluted at the corresponding positions in rechromatography through the same column. 2. 2. The partial specific volume of the eluted active protein was determined as 0.85 by means of a sort of equilibrium centrifugation in sucrose media with several different densities. 3. 3. Sucrose gradient centrifugation was performed using 2 fractions of the eluted active protein and their molecular sizes were estimated. 4. 4. Agreement was obtained regarding the smallest molecular size of active (Na+-K+)-ATPase protein estimated by various methods. The values also roughly coincided with the estimated molecular size in the target bombardment experiments reported previously by ourselves. 5. 5. The possibility of application of the techniques adopted here to other unpurified protein preparations was discussed.