Sodium, potassium-requiring adenosinetriphosphatase activity I. Purification and properties

520

BIOCHIMICA ET BIOPHYSICA ACTA

BBA

65072

SODIUM, P O T A S S I U M - R E Q U I R I N G ADENOSINETRIPHOSPHATASE ACTIVITY I. PURIFICATION AND PROPERTIES" R E N Z O R E N D I AND M A R I E L O U I S E U H R * *

The Departments of Pathology and Biochemistry, University of Colorado, Medical School, Denver, Colo. (U.S.A.) (Received M a r c h i 7 t h , 1964)

SUMMARY

Purification of the ATPase activity stimulated by Na + and K + from calf kidney resulted in a preparation which is completely dependent upon the addition of these monovalent cations. Na+, K+-requiring ATPase activity hydrolyzes only the terminal phosphate group of ATP. The enzymatic activity is specific for ATP and does not hydrolyze other nucleoside triphosphates. The enzymatic activity is inhibited by nucleoside diphosphates and is stimulated by tile addition of phosphoenolpyruvic acid and pyruvate kinase. For optimal activity, the enzymatic activity requires also the presence of Mg-°÷ or Mns+. Fe s+ or Cos+, but not Cas+ or Ba 2+, can partially replace Mgs+. However, all divalent cations, with the exception of Mn2+, proved inhibitory when added together with Mg2+. The ATPase activity is not affected by inhibitors of the mitochondrial ATPase. It is inhibited by various mercurial compounds and by phloridzin. The inhibitory power of this latter compound is related to the MgC1z and KC1 concentration. INTRODUCTION

The presence of an ATPase activity which is activated by the addition of sodium and potassium ions has been detected in brain I-8, red blood cellsS,l°, livern, Is, heart Is, kidney8,14,15, intestine ss, and other tissuesl~, Is. Evidence has been reported indicating that this enzymatic activity is closely related to the active transport of monovalent cationsS,S,lO,17,18.

It was desirableto establishwhether this activated ATPase is a separate activity, and whether the ATPase observed in the absence of monovalent cations is a contaminant enzyme(s). Uxing calf kidney it was possible to purify the monovaJent cation activated ATPase approx, fifty-fold. Concomitantly, the non-activated ATPase activity Was removed. The purified activity differsin various respects from the beefA b b r e v i a t i o n : PCMB, p - c h l o r o m e r c u r i b e n z o i c acid. * Aided b y g r a n t s f r o m t h e U n i t e d S t a t e s Public H e a l t h Service No. C-598I (C2) a n d No. AM o731i. ** F u l b r i g h t Fellow f r o m t h e U n i v e r s i t y of Q u e e n s l a n d , Brisbane, Australia.

Biochim. Biophys. Acta. 89 (1964) 52o-531

Na +, K+-REQUIRINGATPase ACTIVITY

521

heart mitochondria ATPase TM, myosin ATPase 2° and the non-activated ATPase(s) of the calf-kidney homogenate. Such differences are: a) substrate specificity, b) bivalent cation requirements, and c) effect of inhibitors. On the basis of these observations the monovalent cation activated enzyme has been called Na +, K+-requiring ATPase (ATP phosphohydrolase (Mg2+, Na +, K + requiring, EC 3.6.1.3). The effect of some inhibitors has also been studied. Since it has been suggested that the biochemical target for mercurial diuretics is the ATPase ls,21, these compounds were tested on the purified enzyme. Mercurials, both diuretics and nondiuretics, e.g. PCMB, inhibit the activity at low concentrations. Further inhibitors studied were phloridzin and its analogues. It has been shown by LARISet al.22, 2s that in red blood cells, phloridzin inhibits ATPase activity. The purified kidney enzyme is also inhibited by phloridzin, phloretin and 4'-methylphloretin. This effect is increased at high concentrations of MgC1v however at low K + concentrations this group of compounds activates the ATPase. A preliminary report on some aspects of this problem has been presented~4.

EXPERIMENTS Materials

Calf kidney was obtained from the slaughter house and kept frozen before use. Pentadecafluoreoctanoic acid was purchased from Eastman Kodak and neutralized with Tris buffer. ATP-Na was converted into free acid by passage through an Amberlite I2O (H + form) column, then neutralized with Tris. Phosphoenolpyruvic acid, pyruvate kinase (EC 2.7.I.4O) and phloridzin were purchased from California Biochemical Corporation. ATP, ADP, UTP, CTP, GTP, and IDP were obtained from Schwarz Chemical Company. ITP was purchased from Pabst Laboratories. Phloretin was purchased from Mann Research Lab. The phloretin analogues were kindly provided by the firm Leo, Halsingborg, Sweden. 4-Amino-6-chlorobenzene-I,3-disulphonmethylamideand 6-chloro-7-methylsulphonamide-3,4-dihydro-2-N-methyl-I,Z,4-benzothiadiazine-I,I-dioxidewere kindly provided by Dr. M. A. PARENrI of the Inst. Carlo Erba, Milano, Italy, 6-C1-7sulfamyl-I,Z,4-benzothiadiazine-I,I-dioxide was kindly provided by Dr. E. ALPERT of Merck, Sharp and Dohme, West Point, Pa. 6-Ethoxy-z-benzothiazolesulfonamide and 6-ethoxy-2-mercaptobenzothiaz~le were kindly provided by Dr. P. W. O'CONNELL of the Upjohn Company, Kalamazoo, Michigan. 2-EN-(z-hydroxy-3-hydroxymercuripropyl)-carbamyl]-phenoxyacetic acid (hereafter called Mercurial II) and 2-3-(carboxymethylthiomercuri)-2-methoxypropylcarbamyl phenoxyacetate (hereafter called Mercurial III) were kindly provided by Dr. R. O. CLINTONof the SterlingWinthrop Research Inst., Rensselaer, N.Y. Hydrochlorothiazide was kindly provided by Dr. A. J. PLUMMERof Ciba Pharmaceutical Products Inc., Summit, N.J. Hydroflumethiazide and 4-amino-6-trifluoromethyl-m-benzenedlsulfonamide were kindly provided by Dr. H. L. DICKISO~r of Bristol Lab., Syracuse, N.Y. Mersalyl was provided by Dr. A. SCRIBNERof Winthrop Lab., New York, N.Y. Mercuhydrin and Neohydrin were kindly provided by Dr. H. L. FRIEDMANof Lakeside Lab., Milwaukee, Wisconsin. Aldactone was kindly provided by Dr. W. W. JENKINS of Searle Company, Chicago, Ill. Biochim. Biophys. Acta, 89 (1964) 52o-531

522

R. RENDI, M. L. UHR

Analytical methods el was determined by the method of LOHMANN AND JENDROSSEK 25, scaled down to a final volume of 2. 7 ml. Protein was determined b y the biuret method in the presence of deoxycholate ~e. ATP, ADP, and AMP were determined b y chromatography on a Dowex-I formate form z~.

Definition of unit and specific activity A unit of activity is defined as t h a t amount of enzyme which catalyzes the release of I / , m o l e Pt per min. Specific activity is expressed as units per mg of protein.

Partial purification of Na +, K+-requiring ATPase activity from calf kidney All operations were carried out at 4 ° . Step I - Homogenate. 50 g of calf kidney were homogenized with 15o ml of 0.25 M sucrose in a Waring Blendor for 9 ° sec. This suspension was filtered through gauze and the filter washed with 50 ml of 0.25 M sucrose. Step I I -Sucrose-residue. The suspension of Step I was centrifuged for 5 min at 3500 x g. The pellet was suspended with 75 ml of 0.25 M sucrose, filtered again through gauze and the suspension centrifuged for 5 min at 3500 x g. The pellet was resuspended in o.25 M sucrose. Step I I I - Salt-residue. The suspension of Step I I was centrifuged for IO min at 3500 x g. The pellet was extracted with IOO ml of I M NaC1, 0.02 M Tris buffer (pH 7.6). The suspension was stirred for 20 min, then centrifuged for io min at 8500 x g. The pellet was resuspended in 0.25 M sucrose, 0.02 M Tris buffer (pH 7.6) and brought to 50 ml. Step I V - Deoxycholate-residue. The salt-residue fraction was centrifuged and suspended in i M NaC1 containing 5" lO-3 M T r i s - E D T A and 0.05 M Tris buffer (pH 7.6). The suspension was stirred vigorously for IO min, and then deoxycholate was added to a final concentration of 0.2%. The partially clarified material was centrifuged for IO min at 7700 x g. The supernatant and "fluffy layer" were decanted off, and centrifuged for 60 min at 78 ooo x g in a Model L Spinco preparative ultracentrifuge. ~ e pellet was suspended in 0.o5 M Tris buffer (pH 7.6) containing lO -4 M EDTA, argl dialyzed overnight against the same solution. RESULTS

Purification of the ATPase When 15o/*moles of NaC1 and 5 /,moles of KC1 were added to homogenates prepared from calf kidney an increase in the hydrolysis of A T P of about 20-30% is observed. This effect is linear with increasing concentrations of the homogenate fraction. The activity produced b y the monovalent cations is also linear with time. When low concentrations of strophanthidin (5" lO-5 M) were added, ATPase activity was reduced to values equivalent to those observed in the absence of monovalent cations. Data regarding the partial purification of the Na+, K+-requiring ATPase activity

Biochim. Biophys. Acta, 89 (1964) 52o-531

.Na +, K+-REQUIRINGATPase ACTIVITY

523

TABLE I PURIFICATION OF THE NA +, K+-RE~UIRING A T P a s e ACTIVITY FROM CALF KIDNEY

Test system: IOO #*moles of Tris buffer (pH 7.65), 2.5/,moles of MgCIs, 2.5/~moles of A T P and various a m o u n t s o f the different fractions in a final volume of i ml. W h e n needed 15 °/,moles of NaCI and 5/~moles of KCI are added. Incubation for 5 rain at 37 °. Background A TPase rag total

Fraction

#votein

Total unit

Na +, K +-requiring A TPase

Specific activity

Total unit

Ca)

I. 2. 3. 4.

Homogenate Sucrose-residue Salt-residue Detergent-residue

i i 500 3 3 °0 i 7oo 165

Specific activity

b

(b)

680 19° 13o o.8

0.059 0.068 o.o76 o.oo 5

a

i5i

O.Ol 3 0.068 0.096 0.520

225

163 86

0.22 1.18 1.25 lO 7

from calf kidney are presented in Table I. In the present paper the ATPase activity which is observed in the absence of added monovalent cations will be called background ATPase activity, while the increase in activity upon the addition of monovalent cations will be called Na +, K+-requiring ATPase activity. A considerable purification of the Na +, K+-requiring ATPase was obtained with a recovery of 40-50% of the units. The background ATPase during the steps of purification was reduced to less than 1% of the total ATPase activity. TABLE

II

STOICHIOMETRY OF THE A T P a s e REACTION Test system : 2oo/,moles of Tris buffer (pH 7.65), 5/~moles of ATP, 5/~moles of MgCll and 24o/~g protein of detergent-residue fraction prepared from calf kidney in 2 ml were incubated for 5 min at 37 °. The reaction was terminated by centrifuging off the enzyme. Products were determined as described in the experimental section, and expressed in total/,moles. Initial

Final

A dditious

I. N o n e 2. W i t h 1o/*moles of KC1 and 3oo/,moles of NaC1

ATP

ATP

ADP

AMP

P,

4.6

4.5

0.05

o

0.04

4.6

3.8

0.72

o

0.80

Characteristics of the reaction The data presented in Table II demonstrate that only the terminal phosphate group of ATP is cleaved by the Na +, K+-requiring ATPase activity. Although the salt-residue fraction contains enzyme(s) capable of hydrolyzing several nucleotide triphosphates, Na + and K + stimulated only the hydrolysis of ATP (Table III). With the more purified deoxycholate-residue fraction, again one finds specificity for ATP. With crude preparations, proportionality was observed with time and increasing amounts of enzyme preparation. On the other hand, the deoxycholate-residue fraction showed a poor proportionality with time, and almost no proportionality with increasing amounts of the enzymatic preparation. Upon the addition of phosphoenolpyruvic acid and of puryvate kinase, proportionality was reestablished, and up to B i o c h i m . B i o p h y s . A c t a , 89 (1964) 52o-531

524

R. RENDI, M. L. UHR TABLE III SUBSTRATE

S P E C I F I C I T Y F O R T H E Na +, K + - R E Q U I R I N G ATease A C T I V I T Y A N D F O R T H E B A C K G R O U N D ATPase(s)

Test system as in Table I with 8oo pg protein of salt-residue fraction or 200 pg protein of detergentresidue fraction prepared from calf kidney. Incubation for 5 man at 37 % pmolesP~ released Sail-residue fraction Without N a +, K +

ATP UTP ITP CTP GTP

N a +, K +requiring

0.28 0.28 0.24 o.I2 o.I6

0.40 o o 0.o 4 o

Deoxycholatv-residue fr~ion WitA~ut N a +, K +

N a +, K+requiring

0.005 o o o o

0.47 o o o o

A D P

--

--

o

o

IDP

--

--

o

o

A M P

o.28

o

o

o

PP

o.12

o

o

o

t h r e e - f o l d i n c r e a s e i n a c t i v i t y w a s f o u n d (see Fig. I). T k i s s u g g e s t s t h a t t h e N a +, K + - r e q u i r i n g A T P a s e a c t i v i t y is i n h i b i t e d b y A D P , w h i c h is f o r m e d d u r i n g t h e reaction, and that the addition of phosphoenolpyruvic acid and pyruvate kinase regenerates ATP from ADP. Inhibition could be obtained with all the nucleoside d i p h o s p h a t e s , b u t A D P a n d I D P w e r e t h e m o s t effective. The data in Table IV demonstrate that chloride ion can be replaced by either nitrate or sulphate ions without affecting the enzymatic activity, and that K+ can be

2.C

3o .=_ E O 04 a. -

1.C

o

iO

1C)0 150 200

~g PROTEIN

Fig. I. Relationship between A T P hydrolysis a n d the concentration of the Na +, K+-requiring ATPase in the presence of an ATP-generatlng system. A - - - - • , with a n ATP-generating system; O - - - O, with only ATP. Abscissa: enzyme concentration; ordinate: total Pl released. Test system: IOO/~moles of Tris buffer (pH 7.65), 2. 5 pmoles of MgCll, 2.5/,moles of ATP, 15o/,moles of NaCI, 5/*moles of KC1 and various amounts of detergent-residue fraction prepared from calf kidney in a final volume of i ml. W h e n noted, 4 pmoles of phosphoenolpyruvate and 4 ° ~ug of p y r u v a t e kinase were added. After 20 IELin at 37 ° the reaction was terminated by- the addition of perchloric acid.

Biochim. Biophys. Acta, 89 (1964) 52o-53I

N a +, K + - R E Q U I R I N G

ATPase ACTIVITY

525

T A B L E IV E F F E C T OF MONOVALENT CATIONS AND OF ANIONS ON T H E ~ a + , K + - E E ~ U I R I N G A T P a s e A C T I V I T Y

T e s t s y s t e m as in T a b l e I w i t h ~8o/*g p r o t e i n o f d e t e r g e n t - r e s i d u e fraction f r o m calf k i d n e y a n d w i t h o u t a n A T P - g e n e r a t i n g s y s t e m . I n c u b a t i o n for 5 m i n a t 37 % Expt. z

I. 2. 3. 4.

With With With With

5 pmoles 5/,moles 5/,moles 5/,moles

of of of of

umoles P~ released

KC1 KC1 a n d 15o p m o l e s of NaC1 KC1 a n d 1 5 o / , m o l e s of N a N O , KC1 a n d 7 5 / , m o l e s of NasSO 4

o 0.50 o.49 o.49

Expt. I I

I. 2. 3. 4. 5. 6. 7. 8. 9. IO. 11. 12.

NO a d d i t i o n W i t h I5O p m o l e s o f NaC1 W i t h 1 5 o / , m o l e s of NaC1 a n d 5 / , m o l e s of RbC1 W i t h 15 ° p m o l e s of NaC1 a n d 5 / , m o l e s o f NH4C1 W i t h 15o ~umoles of NaC1 a n d 5/*moles LiC1 W i t h 15 ° jumoles of NaC1 a n d 5 p m o l e s of CsC1 W i t h 15o/*moles of NaC1 a n d 5 / , m o l e s of KC1 W i t h 5 # m o l e s of KC1 a n d 1 5 o / , m o l e s of RbC1 W i t h 5 / , m o l e s of KC1 a n d 15o p m o l e s of NH4C1 W i t h 5 p m o l e s of KC1 a n d 1 5 o / , m o l e s of CsC1 W i t h 5/*moles of KC1 a n d 15o/*moles of LiC1 W i t h 5 p m o l e s of KC1

o o 0.24 o.3 o o.26 o.18 o.31 o o o o o

replaced b y Rb+, NH,+, Li+, and Cs+, while no monovalent cation can replace Na +. Fig. 2 shows that Mg 2+ can be replaced b y Mn 2+ and to a lesser degree b y Fe z+ and Co*+. Both Ba *+ and Ca *+ are unable to replace Mg *+. These bivalent cations are inhibitory when added to a complete system containing Mg *+. Their effectiveness as inhibitors is: Ca*+>BaS+>Fe*+>Co 2+. No significant inhibition was found with Mn 2+. The data in Table V show that the background ATPase(s) are stimulated equally well b y all bivalent cations tested. 0.7 0.6 ~0.5 c:

E

0.41

?2 ~'0.3 o

EO.2 0.1 1.25 2.5

5 10 -a M M e "

I 10

Fig. 2. Effect of b i v a l e n t c a t i o n s o n t h e N a +, K + - r e q u i r i n g A T P a s e a c t i v i t y . [ 7 - - - - [ ] , with MnSO4; { ~ - - - - O , w i t h MgC12; A - - - - A , w i t h FeSO4; 0 - - - - 0 , w i t h Co(NOt)2; m - - - - I , w i t h BaClt, A - - - - A , w i t h CaC1 v A b s c i s s a : c o n c e n t r a t i o n o f b i v a l e n t c a t i o n s ; o r d i n a t e : t o t a l Pi released. T e s t s y s t e m as in Fig. I w i t h 2oo p g p r o t e i n of d e t e r g e n t - r e s i d u e f r a c t i o n p r e p a r e d f r o m calf k i d n e y a n d w i t h o u t a n A T P - g e n e r a t i n g s y s t e m . I n c u b a t i o n for 5 rain a t 37 °. B i o c h i m . B i o p h y s . A c t a , 89 (1964) 52o-531

5:26

R. RENDI, M. L. UHR TABLE V BIVALENT CATION SPECIFICITY OF N a +, K+-RE~UIRING A T P a s e ACTIVITY AND OF THE BACKGROUND ATPase(s) PREPARED FROM CALF KIDNEY

T e s t s y s t e m as in T a b l e I w i t h 35 ° m g p r o t e i n of s a l t - r e s i d u e f r a c t i o n f r o m c a l f k i d n e y . I n c u b a t i o n for 5 m i n a t 37 °. l,noles P~ released Additions

i. 2. 34. 5. 6. 7"

None 2. 5 • IO -a M 2.5 "IO-S M 2.5" lO -8 M 2.5" lO-8 M 2.5" IO -s M 2.5 " lO -8 M

MgCI 2 CaC12 BaC18 FeSO, Co(NOB) ~ MnSO 4

Without Na+, K+

Na ÷. K + requiring

o o. 18 o.13 o.14 o.17 o.16 o.18

o 0.2o o o o.13 o.1o o.23

Effect of inhibitors The reaction is inhibited by various cardiac glycosides. Strophanthidin, ouabain, digitoxigenin and bufotalin showed similar inhibitory effects while digitoxin was IO times less potent. The effect of these inhibitors is not a simple detergent effect since deoxycholate, a natural detergent similar in chemical structure to the cardiac glycosides, is IOO times less potent. Protamine inhibits the enzymatic activity at concentrations as low as 50/,g/ml. Also the background ATPaze is inhibited at similar concentrations of protamine. It was noted that the addition of protamine produced the precipitation of the enzymatic preparations. Histone shows similar effects but at higher concentrations. Neither spermine nor spermidine (5 mM) had an effect on the

0 0 02 ~. 02 "~ 0.~ 0.~

0.2 0.'

•

~

~

6.

8

10"" M PHLORfDZIhl Fig. 3. The i n h i b i t i o n of t h e N a +, K + - r e q u i r i n g A T P a s e a c t i v i t y c a u s e d b y p h l o r i d z i n i n t h e p r e s e n c e of t w o different c o n c e n t r a t i o n s of MgC1v A b s c i s s a : m o l a r c o n c e n t r a t i o n of p h l o r i d z i n ; o r d i n a t e : t o t a l Pi released. ( 3 - - - - © , w i t h 5 " lO-a M MgCll; D - - - - D , w i t h 5 "xo-S M MgCI v T e s t s y s t e m : as in Fig. 1 w i t h 2oo/~g p r o t e i n of d e t e r g e n t - r e s i d u e f r a c t i o n p r e p a r e d f r o m c a l f k i d n e y a n d w i t h o u t a n A T P - g e n e r a t i n g s y s t e m . I n c u b a t i o n for 5 rain a t 37 °. B i o c h i m . B i o p h y s . A c t a , 89 (I964) 52o--531

N a +, K + - R E Q U I R I N G

ATPase ACTIVITY

TABLE EFFECT

OF INHIBITORS

ON

527

VI

A Y W a s e ACTIVITY OF CALF-KIDNEY PREPARATIONS

T e s t s y s t e m : i o o / ~ m o l e s o f T r i s b u f f e r p H 7 . 6 5 , 2 . 5 / * m o l e s o f A T P , 2 . 5 / ~ m o l e s o f MgCI~ a n d 2oo/zg protein of detergent-residue from calf kidney were added in a final volume of i ml incubated f o r 5 r a i n a t 37 °. #*moles P, released Expt.

Additions

Na +, K +requiring

None i o -8 M s o d i u m a z i d e 6 . lO -4 M b i l i r u b i n 2 • IO -2 M g u a n i d i n e 1 . 5 " IO - s M d i n i t r o p h e n o l

0.78 0.80 0.78 0.72 0.70

None 2 - IO -6 M 4" Io-4 M 2 - lO -5 M 4" lO-4

0.67 o.15 o.14

None 5 I°-S 5 lO-6 5 IO-6 5 I°-S 5 lO-6 3 IO-e 6 . lO -6 lO -4 M lO -2 M lO -2 M

strophanthidin phloridzin strophanthidin M phloridzin

and o.14

M PCMB M mersalyl M Mercurial II M Mercurial III M HgCI~ M phenylmercuric acetate M phenylmercuric acetate orthoiodobenzoic acid iodoacetic acid iodoacetamide

0.64 0.20 o.19 o.21 o.58 o.o5 o.3o 0.25 o.42 o.21 0.42

enzymatic activity. The enzymatic activity was not affected by the following steroids when tested at a concentration of 5o#g/ml: aldactone, progesterone, deoxycorticosterone, corticosterone, cortisone, testosterone, estradiol, estriol and estrone. In an effort to show that this ATPase activity is different from the mitochondrial ATPase, different compounds affecting these enzymes were tested on the Na +, K +TABLE

VII

LACK OF INHIBITION BY PHLORIDZIN, STROPHANTHIDZIN AND BY MERSALYL ON MITOCHONDRIAL A T P a s e Test system as in Table I with monovalent cations and 30o/~g protein of rat-liver mitochondria. I n c u b a t i o n f o r 5 m i n a t 37 °. l, moles P~ released Additions

1. 2. 3. 4. 5.

None 8 . lO -4 5 " 1° - 5 2 . lO -5 2 • io -s

M M M M

phloridzin strophanthidin mersalyl PCMB

Witho~ D N P

wi~h 5"xo -4 M DNP

1.o 9 1.o8 i.io I .o9 o.80

1.49 1.51 1.47 1.45 I.IO

Biochim.

Biophys.

A c t a , 89 (1964) 5 2 o - 5 3 x

528

rll

R. RENDI, M. L. UHR

0.3

Q.

i

0.2

0

E

0.1

i

I

I

I

2

4

5

1 0 3 M KCl

Fig. 4. T h e effect of t h e c o n c e n t r a t i o n of KC1 on N a +, K + - r e q u i r i n g A T P a s e a c t i v i t y in t h e presence of phloridzin. Abscissa: m o l a r c o n c e n t r a t i o n of KC1, o r d i n a t e : t o t a l Pl released. O - - - - O , w i t h o u t phloridzin; A - - - - A , w i t h i o - 4 M phtoridzin. Conditions as in Fig. I w i t h I 2 o / , g p r o t e i n o f d e t e r g e n t - r e s i d u e fraction p r e p a r e d f r o m calf k i d n e y a n d w i t h o u t an A T P - g e n e r a t i n g s y s t e m . I n c u b a t i o n for 5 m i n a t 37 °.

requiring ATPase (Table VI). It can be seen that inhibitors of the mitochondrial ATPase, like sodium azide, bilirubin, guanidine, have no effect on the calf-kidney Na +, K+-requiring ATPase activity, while high concentrations of DNP (1.5" lO -3 M) have a slight inhibitory effect. As shown in Expt. 3 of Table VI different sulphydryl reagents and mercurial diuretics inhibit the ATPase activity. None of the other diuretics, enumerated in the experimental section and tested at the concentration of I mg/ml, proved inhibitory. Table VI also shows that the Na +, K+-requiring ATPase activity but not the background ATPase(s) is inhibited by low concentrations of phloridzin. The effect of phloridzin on the enzyme is rather complex, the inhibitory power of this compound

0.~

B

i0.2 0.1

F ~'

~'

~

10 .4 M PHLORIDZIN

Fig. 5. T h e effect of phloridzin o n t h e N a +, K + - r e q u i r i n g A T F a s e in t h e presence of h i g h a n d low c o n c e n t r a t i o n s o f KC1. Abscissa: phloridzin c o n c e n t r a t i o n ; o r d i n a t e : t o t a l PI released. C ) - - - - C ) , w i t h 2. lO -4 M KC1; & - - - - A , w i t h 5" IO-S M KC1. C o n d i t i o n as in Fig. i w i t h i8o/~g p r o t e i n of d e t e r g e n t - r e s i d u e fraction p r e p a r e d f r o m calf k i d n e y w i t h o u t a n A T P - g e n e r a t i n g s y s t e m . I n c u b a t i o n for 5 rain a t 37 °.

Biochim. Biophys. Acta, 89 (1964) 52o-531

Na +, K+-REQUIRING ATPase ACTIVITY

529

being affected b y the concentration of MgC12 and of KC1. Greater inhibitions were o b t a i n e d in the presence of higher concentrations of MgC12 (see Fig. 3). When the amount of KC1 is varied in the presence of a constant amount of phloridzin (Fig. 4), one has an initial enhancement of the reaction followed by a low inhibition and finally after the KC1 concentration reaches lO -3 M the inhibitory effect of phloridzin becomes more pronounced. That phloridzin in part stimulates the ATPase activity at low KC1 concentrations is seen in Fig. 5. Of the various phloridzin analogues only phloretin and 4'-methylphloretin showed inhibitory effects similar to that of phloridzin. 2,6-dimethylphloretin, 2,4,4'-trimethylphioretin, 2,4,6-trimethylphloretin, 2,4',6trimethylphloretin, 2'-methylphloretin, 2,3-dimethylphloretin, and 4,4'-dimethylphloretin had no effect at concentrations between lO-4 and 4" IO-4 M. Neither phloridzin nor strophanthidin had any effect on the ATPase activity of rat-liver mitochondria (Table VII). DISCUSSION

The aim of the work presented in this paper is to determine whether the stimulation of the ATPase activity by monovalent cations is due to an enzymatic system different from other "background" ATPase(s). The data reported in Table I show that this is in fact the case. Upon purification of the monovalent cation activated ATPase it was possible to remove the other ATPase(s) present in the calf-kidney homogenate. Two other sets of data differentiate the monovalent cation requiring ATPase from the background ATPase(s). First, the monovalent cation stimulated activity is elicited only in the presence of Mg2+, Mn 2+, Co2+ and Fe ~+ and not in the presence of Ca z+ and Ba ~+, while the background ATPase(s) are activated by all six bivalent cations. Secondly, ATP is the only nucleoside triphosphate which is hydrolyzed by the monovalent cation requiring activity, while all of the five nucleoside triphosphates are hydrolyzed by the background activity. The nucleotide specificity reported here strengthens the idea that this activity corresponds to the Na +, K+-sensitive portion of the ATPase of different tissues. POST et al. 9 reported that in human erythrocytes, ATP, but not I T P is hydrolyzed. With a similar system, HOlCFMAN28 reported that ATP, but not ITP, UTP, or GTP, is hydrolyzed by the strophanthidin-sensitive ATPase of ghosts from human erythrocytes. However, SKou 2 reported that I T P is hydrolyzed by the monovalent cationsensitive ATPase prepared from crab-brain microsomes at about 5o% of the rate of ATP. JARNEFELT29 found that all five nucleotides were hydrolyzed by a Na+, K +stimulated activity from rabbit-brain microsomes. These differences in nucleotide specificity may be due either to species differences or to the presence of nucleoside diphosphokinase in the preparations used by SKOU AND JARNEFELT. The influence of the monovalent cations on the reaction also indicates a close relationship between this ATPase activity and monovalent cation-sensitive ATPases described by other investigators~,4, 9. POST et al. 9 showed that NH4+ can replace K+ for the ATPase of ghosts prepared from human erythrocytes. SKOU2 showed with crab brain and with mammalian preparations that NH4+, Rb+, Cs +, Li+ can replace K+, while none of the monovalent cations can replace Na +. Inhibition of the monovalent cation-sensitive ATPase b y Ca 2+ was reported by other investigators~-4,1°, 29. The various similarities between the calf-kidney Na +, K+-requiring ATPase activity studied here and the characteristics of the monovalent-cation stimulated Biochim. Biophys. Acta, 89 (1964) 52o-531

53 °

R. RENDI, M. L. UHR

activities studied by others is suggestive evidence of a probable similarity of the enzymatic systems. Nevertheless, this does not imply that other types of ATPases are involved in monovalent cation transport. The Na+-activated ATPase from the electric organ of the Electrophorus is such an example 8°. In the more purified system, the possibility that ADP was an inhibitor of the reaction was suggested by the finding that no proportionality existed between the ATPase activity and either time or enzyme concentration. Two experimental findings support this hypothesis : I) the rate of the reaction is inhibited by several nucleoside diphosphates including ADP, and 2) the rate of the reaction is stimulated by the addition of phosphoenolpyruvic acid and pyruvate kinase which convert nucleoside diphosphates formed during the reaction to the corresponding nucleoside triphosphates. The addition of an ATP-generating system also stimulated Initochondrial ATPaseZg,sz. The inhibition by ADP of the Na+, K+-requiring ATPase activity reported here may explain some observations on the ability of ATP and argininephosphates to restore the K÷-sensitive efflux of Na + in giant-squid axon poisoned with DNP (ref. 32). The K+-sensitive efflux of Na+ is closely related to the Na+, K+-requiring ATPase activity 2,°,z°. In a giant-squid axon the addition of DNP inhibits this efflux of Na+, and upon microinjections of ATP and argininephosphate, the K+-sensitive efflux can be reestablished. Various data suggest that the effect of argininephosphate is to maintain a high ATP/ADP ratio. If the giant-squid axon contains a Na +, K+requiring ATPase activity similar to the one described here, an obvious explanation of the need for a high ATP/ADP ratio for the K÷-sensitive Na+ efflux would be that the enzymatic activity itself is inhibited if this ratio is decreased. Inhibition of the enzymatic activity by sulphydryl reagents and mercurial diuretics has been reported also by SKOU88, TAYLOR~, and by LANI~O~ AND NORRIS15. The possibility that the physiological mechanism of action of mercurial diuretics is on the Na +, K+-requiring ATPase activity does not seem tenable at the present moment. In fact PCMB which has been shown to inhibit the ATPase activity zS,zz, is not a diuretic, and it prevents the diuretic effects of mersalyl and other mercurial diuretics 3.. As in the case of mercurial diuretics the inhibition of the ATPase by phloridzin in the present system may not be of physiological significance. It is well known that phloridzin is an inhibitor of the active transport of some rnonosaccharides in the intestine and kidney 85. CRANE et al. as and CSAKY et a l Y have shown that strophanthidin and ouabain, which are inhibitors of active ion transport, inhibit sugar active transport in hamster and frog intestine respectively. The inhibition of the ATPase by phloridzin is different from the one exhibited by cardiac glycosides, since K +, as shown here, enhances the inhibitory power of phlofidzin. This is contrary to the competitive inhibition observed between strophanthidin and K + (refs. 9, 14). This difference between the two inhibitors agrees with the model suggested by CRANE8s for sugar active transport, in which two different sites are postulated for the inhibitory action of phloridzin and strophanthidin. On the other hand, differences between the effect of phloridzin on the ATPase and on sugar transport are evident. Thus, phloretin inhibits the ATPase and does not affect the transport of sugar in the hamster intestine 39. Phloridzin, on the other hand, is known to inhibit the facilitated diffusion of glucose in the red blood cell4°, although in this system no evidence for involvement of active ion transport has been shown. Biochim. Biophys. Acta, 89 (1964) 52o-531

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Biochim. Biophys. Acta, 89 (I964) 52o-531