Vol. 174, No. 2, 1991 January
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
31, 1991
Pages
THE EFFECT OF MICROMOLAR Na+/K+-ATPase
Ca2 + ON THE ACTMTIES
969-974
OF THE DIFFERENT
ISOZYMES IN THE RAT MYOMETRIUM
Agnes Turi, Janos Somogyi and Nandor Mullner
Inst. Biochem. I., Semmelweis Univ. Sch. Med. POB 260 Budapest, H1444 Hungary Received
November
26,
1990
In the present work we show the existence of two Nat/K+-ATPase isozymes in rat myometrial microsomes and suggest that they have different Ca*’ sensitivities. The catalytic subunits (alphal, alpha2) of Na+/K+-ATPase were labelled by fluoresceinisothiocyanate and separated by SDS gel electrophoresis. The two isozyme Ca*+sensitivities were studied by comparing the kinetics of Ca*+, strophantidin, ouabain and N-ethylmaleimide inhibitions. Our results indicate that the activity of the high ouabainsensitive part (alpha2 type) of Na+/K+-ATPase enzyme could only be inhibited by micromolar Ca +. Furthermore, treatment of the rmcrosomal preparation wtth 1mM Nethylmaleimide selectively inactivated the high Ca*+ sensitive isoform of myometrial Na /K+-ATPase. B 1991~~~~~~~~press, I”~.
Recently in several tissues different isozymes of Na+ /K+ -ATPase are expressed (l-3). By application of a high-resolution SDS-gel electrophoretic system the separation of slower and faster migrating catalytic subunits of Na+ /K+ -ATPase was possible (1). The faster migrating subunit is homogenous alphal, but the slower migrating alpha proved to be heterogenods in different species containing alpha2 and alpha3 isoforms (4). The deduced amino acid sequence of the three isoforms showed 85% homology (5). The isozymes differ in several substantial properties. There is a significant difference between the Na+ /K+ -ATPase isozymes in their sensitivities to cardiac glicosides (1,6-7). Furthermore they have different susceptibility to several sulfhydryl reagents and proteases (WV
Two different alpha subunit mRNAs (a1 and aI1) have been detected in the myometrium (9). However the isozymes have been examined so far neither by comparing their gel electrophoretic mobility nor functionally. Na+ /K+ -ATPase activity of the myometrial microsomal or plasmamembrane fraction could be detected only after detergent treatment of the preparations (10). Previously we found the myometrial Na+ /K+ -ATPase activity inhibited about 50% in the presence of 35uM Ca2+, while the complete loss of enzyme activity occured only at loo-fold concentration (11).
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
In the present work we examined wether the different Nat /Kt -ATPase isoforms exist in the myometrium which could explain the observed biphasic inhibiton of Ca2+ on the enzyme activity. METHODS
AND MATERIALS
Preparation of microsomal fractions: Rat myometrium was homogenized in 0.25M sacharose, 30mM histidine-HCl (pH 7.2) containing medium, and the microsomal fraction was isolated by differential centrifugation of the homogenate at 12.00Oxg for 20 min. then lOO.OOOxgfor 60 min. Rat brain and kidney microsomal fractions were isolated on the same manner. The so called “Ca2+ insensitive” preparation was obtained by SDS-treatment of m ometrial microsomes at 0.35:l.O detergent/protein ratio as described by Jorgensen r 12). The solubilized portion was removed by centrifugation at lOO.OOOxgfor 60 min. We obtained the “Ca2+ sensitive” preparations by treating the myometrial microsomal fraction with DMSO at a final concentration of 40% (v/v) for 10 min at 25’C temperature. After DMSO-treatment 0.5mg SDS was added to l.Omg protein and the preparation was centrifuged as above. Purified Nat/KC-ATPase was isolated from pig kidne according to Jorgensen (12). Determination of Nat/K+-ATPase activity: The Nat /yK+-ATPase activity was measured as it was described earlier (10). Protein was determined by the Lowry method (13). For measuring the effect of Ca2+ at micromolar range a Ca2+-EGTA buffer system was used. The final concentrations of free Ca2’ in the Na+/K+- ATPase assay medium were adjusted using a Ca2+ ionselective electrode (Radelkis, Hungary). The measurements of free Ca2+ were calibrated by the method of Tsien et al (14). N-ethylmaleimide treatment: 3mg of microsomal protein of Ca2+ sensitive preparation was treated with 1mM N-ethylmaleimide in 3ml 30mM imidazole-HCl at pH 7.28, at 25’C temperature for 30 min, then centrifuged at lOO.OOOxgfor 60 min, washed and resuspended in the homogenization buffer. Labelling the catalytic subunits of Nat/K+-ATPase by fluorescein isyothiocyanate (FITC): lOtlug of miometrial microsomal preparation and 20ug of purified kidney Na+/K+-ATPase were labelled with FITC to a final concentration of 1OuM in the absence and presence of 10mM ATP as described by Schmitt and McDonough (15). The labelled samples were dissolved in Laemli sample buffer (16) and applied to 7.5% SDS polyacrylamide el. The resolution of alpha subunits was achieved using Laemli SDS-PAGE system as mo cfified by Sackett et al (17). The labelled gels were transilluminated by ultraviolet lamp and photographed through an ultraviolet filter. The negative images (which provided dark signals on a clear background) were scanned with LKB leser densitometer. Materials: DMSO was purchased from Fluka, EGTA was obtained from Serva. Bovin serum albumin and fluorescein isothiocyanate were Sigma products. N-ethylmaleimide was produced by Boehringer-Mannheim. The chemicals not listed above were from Reanal (Hungary) and were of analytical purity. RESULTS Ca*’ was ineffective on the Nat /K+-ATPase activity of the SDS-treated (“Ca2+ insensitive”) preparation at a concentration less than 1OOuM (Fig. 1). However, the enzyme activity of the DMSO t SDS-treated (“Ca2’ -sensitive”) preparation could be inhibited by 50% at a free Ca2+ concentration of 2-5uM. Moreover we observed that the complete inhibition could be accomplished only at milimolar concentration of Ca *+. Based on these results we supposed that the myometrial Na+/K+-ATPase comprises two enzyme fractions which can be influenced by low and high concentrations of Ca2+, respectively. FITC specificly binds to the catalytic subunits of the Nat /K+-ATPase in the presence of 1OOmM Nat at pH 9.2 (18). This observation could be exploited to the identification of 970
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
0.7
BIOPHYSICAL
I
RESEARCH
I
COMMUNICATIONS
1 iB
A 08
m 8 ; r
0.7
0.6 x
2 d
06
* 05
02
OL bz-3 28
29 Y-poshon
Firmre 1 The effect of Ca2+ on the Nat/K+-ATPase DMSO t SDS treated microsomal preparations (0).
30 immb------a
activity
31
1.
05 28 Y-start
-28
29 Y-stlx
* 30 =31
of the SDS- (x) and
lmg of microsomalprotein was treated with 0.35mgSDSfor 10mm at 25 OCin lml volume.The sameamountof protein waspreincubatedwith 40% (v/v) DMSO for 10 min at room temperatureand then was treated with 0.65mgSDS as above described.After the treatments the samplej weie centrifuged the pellets were resuspended m the hogemzatronbuffer and the Na /K ATPase activities were determined.The values expressedas percentageof Na+/K+ATPase activity measuredin the presenceof 1 mM EGTA are meansof 6 experiments (S.A. < 10%) The specificactivities of Nat /K+-ATPase in the SDS- and DMSO +SDS treated preparationswere 13.8and 17.1,respectively. Figure 2. Labelling of Nat /Kt -ATPase catalytic subunits with fluorescein-isothiocyanate. Myometrial microsomefraction (A) and kidney Nat/K+-ATPase (B) containing60 and 1Ougprotein, respectivelywere labelledwith FITC as describedin the Methods. The (*) indicatesthose sampleswhich were preincubatedwith 1OmMATT to demonstratethe competition betweenATP and FITC. The sampleswere applied to a 7.5% (w/v) SDS polyacrylamidegel (pH 9.2) transilluminatedand photographed.The negative imagesof FITC labelled proteins were scannedby LKB leser densitometer. Only 90-120kD of region of the gel is shownas it is indicated by the molecularweight standards(97.4 and 116.0kD).
alpha subunit isoforms even in a crude plasma membrane preparation as it was suggested by Schmitt et al. (15). Fig. 2 shows the FITC-labelled catalytic subunits of Nat /Kt ATPase of the myometrial DMSO t SDS-treated preparation compared with the purified kidney enzyme. The presented densitogram demonstrates the FITC-labelled gels in the area of alpha subunits. Kidney Nat /Kt -ATPase contained only
one type of catalytic
subunit (alphal), however, in the myometrium a slower and a faster migrating alpha isoform could be distinguished. The lighter component was comparable to the alpha subunit of kidney enzyme, and the other labelled protein with higher apparent molecular weight was suggested to be alpha2.
FITC labelling of the catalytic subunits was
significantly reduced in the presence of 10mM ATP in consequenceto their competition. From kinetic analysis of cardiac glycoside inhibition of the enzyme activity the existence of Nat /Kt
-ATPase isozymes can be concluded. Within a given speciesa direct correlation
was found between the strophantidin or ouabain affinity of Nat /Kt -ATPase and the combination of isoenzyme types (1,6,19). Fig 3. shows the inhibition of strophantidin on the Nat /Kt -ATPase activity of rat brain, kidney and myometrial microsomal preparation. Myometrial Nat /K+-ATPase
activity showeda biphasic inhibition curve in the function of
strophantidin concentration asit was found in brain preparation too, but not in kidney. Rat brain is known to contain different catalytic subunits (alpha2-3 and alphal) with high and low strophantidin sensitivity, while in the kidney Nat /Kt -ATPase alpha subunit is 971
31
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Old 168
IO-7 106 strophantidr
165
10-L 163 (M i
ouatmn
iM)
Firmre 3. The effect of strophantidin on the Na+/K+-ATPase activity of rat brain (o), kidney (x) and myometrium (0). The enzyme activity was determined in the DMSO+SDS
treated preparation obtained from brain, kidney medulla and miometrial microsome fraction as it was described in the Methods. (The values are means of 5 experiments +/S.A). Fi_eure4 The effect of Ca2+ on the ouabain inhibition of myometrial Na+/K+-ATPase Ouabain’inhibition of Na+/K+-ATF’ase wasdetermined in the SDS-treated ( anel a) and +SDS-treated (panel b) preparations in the presence of 1mM E8 TA (x) and (0). The difference between ouabain sensitivity in the absence and presence of by the dotted line in panel b. Data represent means of 3-4 determinations (S.A. < 10%). homogen (alphal) and the enzyme activity could be inhibited only at a high concentration of the cardiac glycosides. Biphasic inhibition of myometrial Na+/K+-ATPase activity by strophantidin also demonstrated the existence of different isozymes. Ouabain at low concentrations like strophantidin selectively inhibits the activity of Na+/K+-ATPase isoform containing alpha2.3 catalytic subunit. Furthermore isozymes can be distinguished by their different sensitivities to sulfhydryl reagents as Nethylmaleimide (1,8). We used these properties to obtain indirect evidences that Ca2+ sensitivities of the two isozzymes also differ from each other. Fig 4b. demonstrates inhibitory effect of ouabain on the myometrial Nat /K+-ATPase activity in the presence of 10mM EGTA or 5uM Ca2+. Ca2+ reduced the enzyme activity of DMSOt SDStreated microsomes and higher concentrations of ouabain (K05 = 10-4M) were needed to inhibit the residual Nat/K+-ATPase activity than in the presence of EGTA (K05 = 1.6~10-~). The plotted curve shows the difference between enzyme activity measured in the presence and absence of Ca2’ at increasing concentrations of the inhibitor representing ouabain inhibition of the Ca2+ sensitive enzyme fraction &5=5x10-6M). Ouabain at a concentration of 5~10-~M inhibited about 80% the Ca2+ activity. sensitive but only 20% the residual Ca2+ insensitive part of the Na+/K+-ATPase Fig 4a. illustrates that ouabain sensitivity of the Na+/K+-ATPase in the SDS-treated (“Ca2 + insensitive”) preparation was not influenced by micromolar Ca 2t. To confirm the above results the inverses of these experiment were also carried out. (Fig. Sa-b). Compared to the control samples (OmM ouabain) it can be seen, that ouabain at a concentration of 5~10-~M abolished the Ca2+ sensitivity of the myometrial Nat /Kt -ATPase in the DMSOt SDS treated microsomes [b 1. On the Nat/K+ -ATPase activity of SDS-treated preparation Ca2 + was inneffective in the presence of ouabain as well as in the control [a 1. 972
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
0'
06
05
COMMUNICATIONS
1
3
5
Ca*+
/ pM1
7
Fieu+re 3 Effect of ouabain on the Cazt sensitivity of myometrial Nat /Kt -ATPase. Na /K -ATPase activity of SDS-treated (panel a) and the DMSO+ YS-treated ( b) reparation was measuredin+ the absence.(x) and presence of 5x10- M ouabain dif Perent concentrations of Ca . The dotted lutes express the difference between the sensitivity of the total and the partially inhibited Nai /Kt -ATPase activity. Each are means of 3-4 determinations (S.A. < 10%). Fizure 6. Effect of Nje+tbylmaleimide on the Cazt sensitivity of Nat/K+-ATPase. sensitive” (DMSO + SDS-treated) preparation with 1mM NTreatment of the “Ca ethylmaleimide (NEM) was performed as described in the Methods. Na+/K+-ATPase acttvi of the control (x) and the NEM-treated sample (0) was measured. Data represent one o“f tree identical experiments.
Significant
differences
were observed in the sensitivity
of rat brain and kidney Nat /Kt
-
to inhibition by sulfhydryl reagents as N-ethylmaleimide (NEM). Fig 6. ATPase demonstrates that a milde NEM treatment which is described to selectively block alpha2 type isoform (l,S), influenced the Ca2+ sensitivity of Nat /K+ -ATPase in the myometrial DMSO t SDS-treated
preparation.
The NEM-treatment
reduced the Nat /Kt
-ATPase
activity and abolished Ca2+ sensitivity of the enzyme too. DISCUSSION Nat /Kt
-ATPase isozymes having different type of catalytic subunits (alphal, alpha2 and In many species and tissues alpha1 alpha3) differ from each other in several properties. appears less sensitive to cardiac glycosides than the other two. This isozyme is more protected against proteases (1). Alpha2 has more reactive sulfhydryl groups which were tested by measuring incorporation of the labelled N-ethylmaleimide and sensitivity of the enzyme activity against
the sulphydryl
reagent treatment
(1,8). An antimetabolic
agent
pyrithiamin selectively blocked the activity of alpha2 isozyme type (20). Changes in the lipid environment also have different effects on the activity of the isoforms (21). Tissue distribution of the Na+/K+-ATPase
isoforms shows wide variations. mRNA
hibridization studies concerning to myometrial Nat /K+-ATPase presumed the existence of alpha1 and alpha2 isoforms and our results supported this. Two distinct FITC labelled proteins were demonstrated by SDS gel electrophoresis in the area of catalytic subunit of Na+ /K+ -ATPase. These bands proved to be sensitive to preincubation with ATP showing the competition of these two substratesfor the binding site. Inhibiton of enzyme activity by strophantidin showed a biphasic sensitivity confirming the existence of different isozymes. 973
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Our further results showed that myometrial Na+/K’ -ATPase activity could be partially inhibited by micromolar Ca 2t and the high Ca2’ sensitivity is characteristic to the isozyme containing alpha2 catalytic subunit. Recently several results indicate that an alternate regulation of the different isoforms of Nat /Kt -ATPase can be possible in the tissues. Authors suggest that the slower migrating (alpa2-3) types hormonally-sensitive versions of the enzyme (22-23). We suppose that alfa2 isoform of myometrial Na+/K+-ATPase beeing sensitive to the alteration of intracellular Ca2 + level could participate in the hormonal regulation of uterus contractility.
REFERENCES Sweadner,K.J. (1979) J. Biol. Chem. 260,9016-9022. Siegel,G.J., Desmond,T. and Ernst,S.S. (1986) J. Biol. Chem. 261, 13768-13776. Lytton,J. (1985) J. Biol. Chem. 260, 10075-10080. Hara,Y., Nikamoto,A., Kojima,T., Matsumoto,A. and Nakao,N. (1988) FEBS Letters 238,27-305. p7;ge;en,P.L.and Collins,J.H. (1986) Biochim. Biophys. Acta 860, Feige,G:, Leuter T. and dePover,A. (1988) The NaC,Kt-ATPase (Skou,J.C., Norby,J.G., Maunsbach,A.B. and Esmann,M. eds.) pp. 377-384. Lelievre,L.G., Charlemagne,D., Mouas,C. and Swynghedauw,B. (1986) Biochem. Pharmacol. 35,3443-3453. Matsuda,T., Iwatw,H.and Cooper,J.R. (1985) Biochim.Biophys. Acta. 817, 17-24. Herrera,V.L.M., Emanuel,J.R.,Ruiz-Opozo,N., Levenson,R.and Nadal- Ginard,B. (1987) J. Cell. Biol. 105, 1855-1865. 10. Turi,A. and Torok,K. (1985) Biochim. Biophys. Acta 818,123-131 11. Turt,A. and Somogyi,J. (1988) Biochim. Biophys. Acta 940, 77-84. 12. Jorgensen,P.L. (1974) Biochim. Biophys. Acta 356,35-52 13. Lowry,O.H., Rosenbrough,N,J., Farr,A.L. and Randall,R.J.(1951) J. Biol. Chem. 193, 265-275. 14. Tsien,R.Y. and Rink,T.J. (1980) Biochim. Biophys. Acta 599,623-638. 15. Schmitt,C.A. and McDonough,A.A. (1986) J. Biol. Chem. 261,10439-10444. 16. Laemli,U.K. (1970) Nature 227,680-685. 17. Sackett,D.L., Bhattacharyya,B. and Wolff,J. (1985) J. Biol. Chem. 260, 43-45. 18. Karlish,S.J.D. (1980) J. Bioenerg. Biomemb. 12, 111-136. 19. Charlemagne,D., Mayoux,E., Poyard,M., Oliviero,P. and Geering,K. (1987) J. Biol. Chem. 262,8941-8943. 20. Matsuda,T., Iwata,H. and Cooper,J.R. (1984) J. Biol.Chem. 259,3858-3863 21. Matsuda,T. and Iwata,H. (1986) Biochim. Biophys. Acta 860,620-625. 22. Sweadner,K.J. and Gtlkeson,R.C. (1985) J. Biol. Chem. 260,9016-9022. 23. Lytton,J., Lin,J.C. and Guidotti,G. (1985) J. Biol.Chem. 260, 1177-1184.
974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
31, 1991
Pages
THE EFFECT OF MICROMOLAR Na+/K+-ATPase
Ca2 + ON THE ACTMTIES
969-974
OF THE DIFFERENT
ISOZYMES IN THE RAT MYOMETRIUM
Agnes Turi, Janos Somogyi and Nandor Mullner
Inst. Biochem. I., Semmelweis Univ. Sch. Med. POB 260 Budapest, H1444 Hungary Received
November
26,
1990
In the present work we show the existence of two Nat/K+-ATPase isozymes in rat myometrial microsomes and suggest that they have different Ca*’ sensitivities. The catalytic subunits (alphal, alpha2) of Na+/K+-ATPase were labelled by fluoresceinisothiocyanate and separated by SDS gel electrophoresis. The two isozyme Ca*+sensitivities were studied by comparing the kinetics of Ca*+, strophantidin, ouabain and N-ethylmaleimide inhibitions. Our results indicate that the activity of the high ouabainsensitive part (alpha2 type) of Na+/K+-ATPase enzyme could only be inhibited by micromolar Ca +. Furthermore, treatment of the rmcrosomal preparation wtth 1mM Nethylmaleimide selectively inactivated the high Ca*+ sensitive isoform of myometrial Na /K+-ATPase. B 1991~~~~~~~~press, I”~.
Recently in several tissues different isozymes of Na+ /K+ -ATPase are expressed (l-3). By application of a high-resolution SDS-gel electrophoretic system the separation of slower and faster migrating catalytic subunits of Na+ /K+ -ATPase was possible (1). The faster migrating subunit is homogenous alphal, but the slower migrating alpha proved to be heterogenods in different species containing alpha2 and alpha3 isoforms (4). The deduced amino acid sequence of the three isoforms showed 85% homology (5). The isozymes differ in several substantial properties. There is a significant difference between the Na+ /K+ -ATPase isozymes in their sensitivities to cardiac glicosides (1,6-7). Furthermore they have different susceptibility to several sulfhydryl reagents and proteases (WV
Two different alpha subunit mRNAs (a1 and aI1) have been detected in the myometrium (9). However the isozymes have been examined so far neither by comparing their gel electrophoretic mobility nor functionally. Na+ /K+ -ATPase activity of the myometrial microsomal or plasmamembrane fraction could be detected only after detergent treatment of the preparations (10). Previously we found the myometrial Na+ /K+ -ATPase activity inhibited about 50% in the presence of 35uM Ca2+, while the complete loss of enzyme activity occured only at loo-fold concentration (11).
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
In the present work we examined wether the different Nat /Kt -ATPase isoforms exist in the myometrium which could explain the observed biphasic inhibiton of Ca2+ on the enzyme activity. METHODS
AND MATERIALS
Preparation of microsomal fractions: Rat myometrium was homogenized in 0.25M sacharose, 30mM histidine-HCl (pH 7.2) containing medium, and the microsomal fraction was isolated by differential centrifugation of the homogenate at 12.00Oxg for 20 min. then lOO.OOOxgfor 60 min. Rat brain and kidney microsomal fractions were isolated on the same manner. The so called “Ca2+ insensitive” preparation was obtained by SDS-treatment of m ometrial microsomes at 0.35:l.O detergent/protein ratio as described by Jorgensen r 12). The solubilized portion was removed by centrifugation at lOO.OOOxgfor 60 min. We obtained the “Ca2+ sensitive” preparations by treating the myometrial microsomal fraction with DMSO at a final concentration of 40% (v/v) for 10 min at 25’C temperature. After DMSO-treatment 0.5mg SDS was added to l.Omg protein and the preparation was centrifuged as above. Purified Nat/KC-ATPase was isolated from pig kidne according to Jorgensen (12). Determination of Nat/K+-ATPase activity: The Nat /yK+-ATPase activity was measured as it was described earlier (10). Protein was determined by the Lowry method (13). For measuring the effect of Ca2+ at micromolar range a Ca2+-EGTA buffer system was used. The final concentrations of free Ca2’ in the Na+/K+- ATPase assay medium were adjusted using a Ca2+ ionselective electrode (Radelkis, Hungary). The measurements of free Ca2+ were calibrated by the method of Tsien et al (14). N-ethylmaleimide treatment: 3mg of microsomal protein of Ca2+ sensitive preparation was treated with 1mM N-ethylmaleimide in 3ml 30mM imidazole-HCl at pH 7.28, at 25’C temperature for 30 min, then centrifuged at lOO.OOOxgfor 60 min, washed and resuspended in the homogenization buffer. Labelling the catalytic subunits of Nat/K+-ATPase by fluorescein isyothiocyanate (FITC): lOtlug of miometrial microsomal preparation and 20ug of purified kidney Na+/K+-ATPase were labelled with FITC to a final concentration of 1OuM in the absence and presence of 10mM ATP as described by Schmitt and McDonough (15). The labelled samples were dissolved in Laemli sample buffer (16) and applied to 7.5% SDS polyacrylamide el. The resolution of alpha subunits was achieved using Laemli SDS-PAGE system as mo cfified by Sackett et al (17). The labelled gels were transilluminated by ultraviolet lamp and photographed through an ultraviolet filter. The negative images (which provided dark signals on a clear background) were scanned with LKB leser densitometer. Materials: DMSO was purchased from Fluka, EGTA was obtained from Serva. Bovin serum albumin and fluorescein isothiocyanate were Sigma products. N-ethylmaleimide was produced by Boehringer-Mannheim. The chemicals not listed above were from Reanal (Hungary) and were of analytical purity. RESULTS Ca*’ was ineffective on the Nat /K+-ATPase activity of the SDS-treated (“Ca2+ insensitive”) preparation at a concentration less than 1OOuM (Fig. 1). However, the enzyme activity of the DMSO t SDS-treated (“Ca2’ -sensitive”) preparation could be inhibited by 50% at a free Ca2+ concentration of 2-5uM. Moreover we observed that the complete inhibition could be accomplished only at milimolar concentration of Ca *+. Based on these results we supposed that the myometrial Na+/K+-ATPase comprises two enzyme fractions which can be influenced by low and high concentrations of Ca2+, respectively. FITC specificly binds to the catalytic subunits of the Nat /K+-ATPase in the presence of 1OOmM Nat at pH 9.2 (18). This observation could be exploited to the identification of 970
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
0.7
BIOPHYSICAL
I
RESEARCH
I
COMMUNICATIONS
1 iB
A 08
m 8 ; r
0.7
0.6 x
2 d
06
* 05
02
OL bz-3 28
29 Y-poshon
Firmre 1 The effect of Ca2+ on the Nat/K+-ATPase DMSO t SDS treated microsomal preparations (0).
30 immb------a
activity
31
1.
05 28 Y-start
-28
29 Y-stlx
* 30 =31
of the SDS- (x) and
lmg of microsomalprotein was treated with 0.35mgSDSfor 10mm at 25 OCin lml volume.The sameamountof protein waspreincubatedwith 40% (v/v) DMSO for 10 min at room temperatureand then was treated with 0.65mgSDS as above described.After the treatments the samplej weie centrifuged the pellets were resuspended m the hogemzatronbuffer and the Na /K ATPase activities were determined.The values expressedas percentageof Na+/K+ATPase activity measuredin the presenceof 1 mM EGTA are meansof 6 experiments (S.A. < 10%) The specificactivities of Nat /K+-ATPase in the SDS- and DMSO +SDS treated preparationswere 13.8and 17.1,respectively. Figure 2. Labelling of Nat /Kt -ATPase catalytic subunits with fluorescein-isothiocyanate. Myometrial microsomefraction (A) and kidney Nat/K+-ATPase (B) containing60 and 1Ougprotein, respectivelywere labelledwith FITC as describedin the Methods. The (*) indicatesthose sampleswhich were preincubatedwith 1OmMATT to demonstratethe competition betweenATP and FITC. The sampleswere applied to a 7.5% (w/v) SDS polyacrylamidegel (pH 9.2) transilluminatedand photographed.The negative imagesof FITC labelled proteins were scannedby LKB leser densitometer. Only 90-120kD of region of the gel is shownas it is indicated by the molecularweight standards(97.4 and 116.0kD).
alpha subunit isoforms even in a crude plasma membrane preparation as it was suggested by Schmitt et al. (15). Fig. 2 shows the FITC-labelled catalytic subunits of Nat /Kt ATPase of the myometrial DMSO t SDS-treated preparation compared with the purified kidney enzyme. The presented densitogram demonstrates the FITC-labelled gels in the area of alpha subunits. Kidney Nat /Kt -ATPase contained only
one type of catalytic
subunit (alphal), however, in the myometrium a slower and a faster migrating alpha isoform could be distinguished. The lighter component was comparable to the alpha subunit of kidney enzyme, and the other labelled protein with higher apparent molecular weight was suggested to be alpha2.
FITC labelling of the catalytic subunits was
significantly reduced in the presence of 10mM ATP in consequenceto their competition. From kinetic analysis of cardiac glycoside inhibition of the enzyme activity the existence of Nat /Kt
-ATPase isozymes can be concluded. Within a given speciesa direct correlation
was found between the strophantidin or ouabain affinity of Nat /Kt -ATPase and the combination of isoenzyme types (1,6,19). Fig 3. shows the inhibition of strophantidin on the Nat /Kt -ATPase activity of rat brain, kidney and myometrial microsomal preparation. Myometrial Nat /K+-ATPase
activity showeda biphasic inhibition curve in the function of
strophantidin concentration asit was found in brain preparation too, but not in kidney. Rat brain is known to contain different catalytic subunits (alpha2-3 and alphal) with high and low strophantidin sensitivity, while in the kidney Nat /Kt -ATPase alpha subunit is 971
31
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Old 168
IO-7 106 strophantidr
165
10-L 163 (M i
ouatmn
iM)
Firmre 3. The effect of strophantidin on the Na+/K+-ATPase activity of rat brain (o), kidney (x) and myometrium (0). The enzyme activity was determined in the DMSO+SDS
treated preparation obtained from brain, kidney medulla and miometrial microsome fraction as it was described in the Methods. (The values are means of 5 experiments +/S.A). Fi_eure4 The effect of Ca2+ on the ouabain inhibition of myometrial Na+/K+-ATPase Ouabain’inhibition of Na+/K+-ATF’ase wasdetermined in the SDS-treated ( anel a) and +SDS-treated (panel b) preparations in the presence of 1mM E8 TA (x) and (0). The difference between ouabain sensitivity in the absence and presence of by the dotted line in panel b. Data represent means of 3-4 determinations (S.A. < 10%). homogen (alphal) and the enzyme activity could be inhibited only at a high concentration of the cardiac glycosides. Biphasic inhibition of myometrial Na+/K+-ATPase activity by strophantidin also demonstrated the existence of different isozymes. Ouabain at low concentrations like strophantidin selectively inhibits the activity of Na+/K+-ATPase isoform containing alpha2.3 catalytic subunit. Furthermore isozymes can be distinguished by their different sensitivities to sulfhydryl reagents as Nethylmaleimide (1,8). We used these properties to obtain indirect evidences that Ca2+ sensitivities of the two isozzymes also differ from each other. Fig 4b. demonstrates inhibitory effect of ouabain on the myometrial Nat /K+-ATPase activity in the presence of 10mM EGTA or 5uM Ca2+. Ca2+ reduced the enzyme activity of DMSOt SDStreated microsomes and higher concentrations of ouabain (K05 = 10-4M) were needed to inhibit the residual Nat/K+-ATPase activity than in the presence of EGTA (K05 = 1.6~10-~). The plotted curve shows the difference between enzyme activity measured in the presence and absence of Ca2’ at increasing concentrations of the inhibitor representing ouabain inhibition of the Ca2+ sensitive enzyme fraction &5=5x10-6M). Ouabain at a concentration of 5~10-~M inhibited about 80% the Ca2+ activity. sensitive but only 20% the residual Ca2+ insensitive part of the Na+/K+-ATPase Fig 4a. illustrates that ouabain sensitivity of the Na+/K+-ATPase in the SDS-treated (“Ca2 + insensitive”) preparation was not influenced by micromolar Ca 2t. To confirm the above results the inverses of these experiment were also carried out. (Fig. Sa-b). Compared to the control samples (OmM ouabain) it can be seen, that ouabain at a concentration of 5~10-~M abolished the Ca2+ sensitivity of the myometrial Nat /Kt -ATPase in the DMSOt SDS treated microsomes [b 1. On the Nat/K+ -ATPase activity of SDS-treated preparation Ca2 + was inneffective in the presence of ouabain as well as in the control [a 1. 972
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
0'
06
05
COMMUNICATIONS
1
3
5
Ca*+
/ pM1
7
Fieu+re 3 Effect of ouabain on the Cazt sensitivity of myometrial Nat /Kt -ATPase. Na /K -ATPase activity of SDS-treated (panel a) and the DMSO+ YS-treated ( b) reparation was measuredin+ the absence.(x) and presence of 5x10- M ouabain dif Perent concentrations of Ca . The dotted lutes express the difference between the sensitivity of the total and the partially inhibited Nai /Kt -ATPase activity. Each are means of 3-4 determinations (S.A. < 10%). Fizure 6. Effect of Nje+tbylmaleimide on the Cazt sensitivity of Nat/K+-ATPase. sensitive” (DMSO + SDS-treated) preparation with 1mM NTreatment of the “Ca ethylmaleimide (NEM) was performed as described in the Methods. Na+/K+-ATPase acttvi of the control (x) and the NEM-treated sample (0) was measured. Data represent one o“f tree identical experiments.
Significant
differences
were observed in the sensitivity
of rat brain and kidney Nat /Kt
-
to inhibition by sulfhydryl reagents as N-ethylmaleimide (NEM). Fig 6. ATPase demonstrates that a milde NEM treatment which is described to selectively block alpha2 type isoform (l,S), influenced the Ca2+ sensitivity of Nat /K+ -ATPase in the myometrial DMSO t SDS-treated
preparation.
The NEM-treatment
reduced the Nat /Kt
-ATPase
activity and abolished Ca2+ sensitivity of the enzyme too. DISCUSSION Nat /Kt
-ATPase isozymes having different type of catalytic subunits (alphal, alpha2 and In many species and tissues alpha1 alpha3) differ from each other in several properties. appears less sensitive to cardiac glycosides than the other two. This isozyme is more protected against proteases (1). Alpha2 has more reactive sulfhydryl groups which were tested by measuring incorporation of the labelled N-ethylmaleimide and sensitivity of the enzyme activity against
the sulphydryl
reagent treatment
(1,8). An antimetabolic
agent
pyrithiamin selectively blocked the activity of alpha2 isozyme type (20). Changes in the lipid environment also have different effects on the activity of the isoforms (21). Tissue distribution of the Na+/K+-ATPase
isoforms shows wide variations. mRNA
hibridization studies concerning to myometrial Nat /K+-ATPase presumed the existence of alpha1 and alpha2 isoforms and our results supported this. Two distinct FITC labelled proteins were demonstrated by SDS gel electrophoresis in the area of catalytic subunit of Na+ /K+ -ATPase. These bands proved to be sensitive to preincubation with ATP showing the competition of these two substratesfor the binding site. Inhibiton of enzyme activity by strophantidin showed a biphasic sensitivity confirming the existence of different isozymes. 973
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Our further results showed that myometrial Na+/K’ -ATPase activity could be partially inhibited by micromolar Ca 2t and the high Ca2’ sensitivity is characteristic to the isozyme containing alpha2 catalytic subunit. Recently several results indicate that an alternate regulation of the different isoforms of Nat /Kt -ATPase can be possible in the tissues. Authors suggest that the slower migrating (alpa2-3) types hormonally-sensitive versions of the enzyme (22-23). We suppose that alfa2 isoform of myometrial Na+/K+-ATPase beeing sensitive to the alteration of intracellular Ca2 + level could participate in the hormonal regulation of uterus contractility.
REFERENCES Sweadner,K.J. (1979) J. Biol. Chem. 260,9016-9022. Siegel,G.J., Desmond,T. and Ernst,S.S. (1986) J. Biol. Chem. 261, 13768-13776. Lytton,J. (1985) J. Biol. Chem. 260, 10075-10080. Hara,Y., Nikamoto,A., Kojima,T., Matsumoto,A. and Nakao,N. (1988) FEBS Letters 238,27-305. p7;ge;en,P.L.and Collins,J.H. (1986) Biochim. Biophys. Acta 860, Feige,G:, Leuter T. and dePover,A. (1988) The NaC,Kt-ATPase (Skou,J.C., Norby,J.G., Maunsbach,A.B. and Esmann,M. eds.) pp. 377-384. Lelievre,L.G., Charlemagne,D., Mouas,C. and Swynghedauw,B. (1986) Biochem. Pharmacol. 35,3443-3453. Matsuda,T., Iwatw,H.and Cooper,J.R. (1985) Biochim.Biophys. Acta. 817, 17-24. Herrera,V.L.M., Emanuel,J.R.,Ruiz-Opozo,N., Levenson,R.and Nadal- Ginard,B. (1987) J. Cell. Biol. 105, 1855-1865. 10. Turi,A. and Torok,K. (1985) Biochim. Biophys. Acta 818,123-131 11. Turt,A. and Somogyi,J. (1988) Biochim. Biophys. Acta 940, 77-84. 12. Jorgensen,P.L. (1974) Biochim. Biophys. Acta 356,35-52 13. Lowry,O.H., Rosenbrough,N,J., Farr,A.L. and Randall,R.J.(1951) J. Biol. Chem. 193, 265-275. 14. Tsien,R.Y. and Rink,T.J. (1980) Biochim. Biophys. Acta 599,623-638. 15. Schmitt,C.A. and McDonough,A.A. (1986) J. Biol. Chem. 261,10439-10444. 16. Laemli,U.K. (1970) Nature 227,680-685. 17. Sackett,D.L., Bhattacharyya,B. and Wolff,J. (1985) J. Biol. Chem. 260, 43-45. 18. Karlish,S.J.D. (1980) J. Bioenerg. Biomemb. 12, 111-136. 19. Charlemagne,D., Mayoux,E., Poyard,M., Oliviero,P. and Geering,K. (1987) J. Biol. Chem. 262,8941-8943. 20. Matsuda,T., Iwata,H. and Cooper,J.R. (1984) J. Biol.Chem. 259,3858-3863 21. Matsuda,T. and Iwata,H. (1986) Biochim. Biophys. Acta 860,620-625. 22. Sweadner,K.J. and Gtlkeson,R.C. (1985) J. Biol. Chem. 260,9016-9022. 23. Lytton,J., Lin,J.C. and Guidotti,G. (1985) J. Biol.Chem. 260, 1177-1184.
974
