Toshiko Nakao

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.

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.

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.

Microdetermination of protein not affected by the presence of various buffers, sucrose, ATP, and eluates from polysaccharide derivatives

Abstract Paper states standard and microdetermination of protein that is not affected by the presence of various substances including buffers, sucrose, ATP, eluates from columns of polysaccharide derivatives, etc. The method was originally based on Kiharas method (4), although the basic principle is somewhat different. The protein was stained with amido black under defined condition, filtered, and washed through a small membrane filter, and the filter was extracted with SDS-methanol. The color intensity was measured. The recovery of proteins was almost 100%. From 2 to 100 μg of protein could be successfully determined on a standard scale and from 1 to 20 μg on a microscale.

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.

Partial purification and characterization of sodium- and potassium-dependent adenosine triphosphatase from rat-heart muscle

Abstract 1. 1. The cardiac ATPase preparation obtained by deoxycholate treatment according to S kou was found to contain a considerable amount of Na + - and K + -independent as well as Na + - and K + -dependent ATPase activity. The former activity was partially inibited by Na + . 2. 2. The cardiac Na + - and K + -dependent ATPase was partially purified from rat-heart muscle, by treatment with 2 M NaI, and a highly specific Na + - and K + - dependent ATPase was obtained. 3. 3. Rat cardiac Na + - and K + -dependent ATPase obtained by treatment with 2 M NaI was compared with that obtained by the deoxycholate procedure. The two enzymes were essentially the same in their pattern of activation by Na + and K + in the presence of Mg 2+ . 4. 4. The characteristics of the cardiac Na + - and K + -dependent ATPase were studied mainly using the NaI-treated enzyme. The observed properties were similar to those of Na + - and K + -dependent ATPase preparations from other tissues. 5. 5. The ouabain-sensitive portion in the total ATPase activity of the preparation was liable to be overstimated when deoxycholate-treated enzyme preparations (especially fresh ones) were used, on account of contamination by Na + -inhibited activity. 6. 6. An apparent increase in the ratio of Na + - and K + -dependent ATPase to independent ATPase activity was observed in deoxycholate-treated enzyme preparations during storage at 5°. It is suggested that the apparent increase was not the result of activation of Na + - and K + -dependent ATPase activity but rather due to the relatively more rapid inactivation of Na + - and K + -independent activity.

Purification and characterization of ribonuclease from rabbit reticulocytes

Abstract A method is described for the purification of ribonmuclease (EC 2.7.7.16) from rabbit reticulocytes by heart treatment at pH 2.5 followed by chromatography on a carboxymethylcellulose column. The enzymei was purified 5000-fold, and was free from both acid and alkaline phosphatases (EC 3.I.3.2 and EC 3.I.3.I, respectively), non-specific phosphodiesterase (EC 3.I.4.I) and deoxyribonuclease (EC 3.I.4.5). The enzyme had a pH optimum at 5.8. It was heat and acid stable and could be stored at —20° for I year without loss of activity. The products of hydrolysis of yeast ribonucleic acid by the enzyme were found to contain both 2′- and 3′-purine and pyrimide nucleotides,t he ratio of ratio of adenylic to cytidylic acid being approximately I:I. An incomplete digest of yeast ribonucleic acid by the enzyme was separated on a diethylaminoethyl-cellulose column and both mono- and oligonucleotide fractions were identified. Nucleoside 2′- and 3′-cyclic phosphates were hydrolyzed to their corresponding non-cyclic mononucleotides by the enzyme. A possible physiological role of the enzyme is discussed on the basis of the following experiments: (I) degradation of rabbit reticulocyte ribosome by the enzyme; (2) chronological difference in its activity in rabbit reticulocytes; and (3) intracellular localization of the enzyme.