Camp. Btichem. Physiol., 1975, Yof.
5OA,pp. 297 to 302. Pergamon Press. Printed in Great Britain
SOME BIOCHEMICAL PROPERTIES OF Na,K-ATPase IN FROG EPIDERMIS*t JUN KAWADA,~ R. E. TAYLOR, JR. AND S. B. BARKER Department of Physiology and Biophysics, The Medical Center, University of Alabama in Birmingham, Birmingham, Alabama 35233, U.S.A. (Received 30 October
1973)
Ah&act-l. Na,K-ATPase of frog epidermis was sensitive to proteases and easily destroyed. In contrast, Mg-ATPase was highly resistant towards proteolytic action. 2. The Arrhenius plot of Na,K-ATPase activity shows a sharp discontinuity near 23’C; however, Mg-ATPase has no such critical point. 3. Short-circuit current and Na,K-ATPase activity of the skin from a fold-a~limati~d animal was not sibilantly different from that observed in a moderate-temperatur5a~limatized animal. MgATPase activity was lost around 50 per cent in cold-acclimatized animals; however, oxygen consumption of the skin was not significantly changed. 4. The preparation obtained by Nakao’s NaI method showed a high substrate specificity for ATP. The activity with CTP was about 10 per cent of that with ATP as substrate, Almost no ADP was utilized. The I$, of the enzyme preparation for substrate ATP was calculated as 1.5 x 1O-8M.
INTRODUCTION OUABA~N-SENSITIVE Na,K-dependent ATPase was demonstrated in crude homogenates of lyoph~lized frog skin and related with cation flux in the tissue by Bonting et al. (1963). The difficulty to get consistent data with this tissue was pointed out at the same time. Considering the facts that the active sodium transporting mechanism is thought to reside in the epidermis and that the difficulty involved in getting consistent data is due to the hardness of dermal tissue and its high inorganic phosphate concentration (Taylor ef al., 19661, we separated the epidermal layer from the whole skin for the enzyme study using alkaline halide solutions (Kawada et al., 1969). Hitherto little has been known about the properties and the purifi~tion of ouabain-sensitive Na,Kdependent ATPase in frog skin. In this paper, the influence of tem~rature on the enzyme in oitro and in uivo system, the effect of proteases, partial purification of ouabain-sensitive Na,K-dependent enzyme and its substrate specificity are described. MATERIALS AND METHODS Runapigiens and Rana catesbeiana frogs obtained from Lemberger Co. Oshkosh, Wisconsin, were used. They
were kept in tap water at a temperature of 24+ 1°C at least 5 days before use. In the temperature-acclimatization experiment, A. pi&ens frogs were divided into two groups. They were all kept in artificial pond water. One group was at a temperature 10 _t2°C and the other was at 24+ 1°C for 3 weeks. In some experiments, rat kidney was used to compare the results with frog tissue. Separation of the epidermis from the ventral skin and the enzyme assay method have been described elsewhere (Kawada et al., 1969). The homogenization medium routinely used was 025 M sucrose s&ution, the pH being adjusteh to 7.4 with 1OOmM Tris-IX1 buffer unless otherwise mentioned. In temperature-acclimatized animals, oxygen consumption of the whole skin was determined by a differential manometer and the short-circuit current was also recorded using Ussing’s chamber (Ussing & Zerahn, 1951). Partial purifi~tion of the enzyme was tried by the method of treating with a high con~nt~tion of NaI as proposed by Nakao (1963, 1965). For the study of the effect of proteases, Trypsin 2X Crystalline and Chymotrypsin a were purchased from the Nut~tional Biochemicals Corp. As nucleotides, ATP (Tris salt, Sigma), ADP (Na salt, Sigma), CTP (T&K salt, Calbiochem), GTP (Tri-Na salt, Sigma), ITP (Tri-Na, salt, Calbiochem) and UTP (Tri-Na salt, Calbiochem) were used to examine the substrate specificity. The final concentration of each nucleotide in the incubation medium for enzyme assay was 3 pmoles. RESULTS
*This investigation was supported in part by grant AM-10436 from the National Institutes of Health, Bethesda, Maryland. t Address requests for reprints to Jun Kawada. $ Present address: Department of Biochemistry, Faculty of Pharmaceutical Sciences, University of Tokushima, Tokushima, Japan.
&%!ct 5f temperature The ATPase activity of the frog epidermis was determined at different incubation temperatures from 37 to 12°C. The results examined as Arrhenius plots are shown in Fig. 1. To compare this result 297
J~JNKAWADA,R. E. TAYLOR,JR.
298
v I/T,
I
,
I
I
3.3
34
3-5
3.6
I/T,
xIO-~
AND
S. B.
BARKER
chamber with the same skin between the two groups of animah. The values of the short-circuit current of the individual skin varied with a wide range. AIthough Na,K-ATPase activity measured at an incubation temperature of 32°C was not changed significantly by cold acclimatization, Mg-ATPase activity was about 50 per cent lower in the coldacclimatized group than in the 24°C group. Oxygen consumption of the skin was not changed significantly by cold acclimatization. These results are summarized in Table 1. As far as these experimental conditions are concerned, no m~ningful relationship can be demonstrated between the data obtained from the Arrhenius plot and the active sodiumtransport phenomenon observed in the animals acclimatized at the low temperature.
x10-3
Effect of proteases Fig. 1. Arrhenius plot of ATPase activity of R. catesbeiunu epidermis (a) and rat kidney (b): o-o, MgATPase and o-o, Na,K-ATPase. Incubation medium contained Na+, 1OOmM; K+, 10 mM; M2+, 6mM; EDTA, 0.1 mM; Tris-ATP, 3 mM and Tris (l~mM)-
HCI buRer, pH 7.4. with mammalian tissue enzymes, rat kidney ATPase was also determined in the same way. For these experiments, 900 g supernatants of the homogenates were used. The graph of log of Na,K-ATPase activity vs. the reciprocal of the temperature showeda discontinuity at around 23°C for both materials. However, in frog epidermis, the activity decreased more rapidly below 23°C and was extremely low at 16°C. The Mg-ATPase activity curve of frog epidermis has no distinct critical point. However, the Mg-ATPase activity curve of rat kidney may have a discontinuity of 25°C. Although it must be kept in mind that the properties of an enzyme or enzyme system in v&o may not be similar to that in vivo, it is of considerable interest that such a dramatic change in Na,K-ATPase activity occurs near 23”C, a temperature which is not an unusual condition for temperature poikilothermic vertebrates. Therefore the temperature acclimatization experiment was carried out in order to determine if the results shown in the Arrhenius plot had any biological significance. As shown in Fig. 2, there was no significant differencein short-circuit currents which were determined at different temperatures in the
Figure 3 shows the effect of incubating epidermal ATPase preparation with trypsin and chymotrypsin for 15 min at 23°C. Ouabain-sensitive ATPase activity was almost completely destroyed by both enzymes, but Mg-ATPase activity was not significantly changed even after combined treatment with a high concentration of trypsin and chymotrypsin. Fig. 4 follows the change in the enzyme during 80 min incubation with 0.01% trypsin. Mg-ATPase activity was not influenced si~ificantly. However, ouabain-sensitive ATPase activity was greatly decreased during the first 5 min. In order to compare this phenomenon with mammalian tissue, the 900 g supernatant of rat kidney homogenate was treated with O-Ol”Atrypsin. The results are shown in Fig. 5; in contrast to frog epidermal preparations, the MgATPase activity of rat kidney was usually decreased approximately by 30 per cent after 15 min incubation, Rat kidney ouabain-sensitive ATPase activity is also rapidly decreased by the proteolytic action of trypsin. An addition of 0.1 per cent of deoxycholate to the homogenization medium increased the destruction of Mg-ATPase activity after 15 min incubation with trypsin to about 50 per cent. Partial pur~~&atio~~ of ollaba~n-sensitive Na,Kdependent enzyme and its substrate specificity The results of three examples using different starting materials for partial purification of enzyme
Table 1. Enzyme activity and oxygen consumption of skin from acclimatized frogs Temperature acclimatized (“Cl
No. of frogs
Mg-ATPase Na,K-ATPase @moles Pi/me! protein per hr) @moles Pi/mg protein per hr)
5 6 * At WC, 760 mm Hg. i Mean& S.E.
3.09 F 0.24t 6.14+0.33
2.99kO.37 2.92kO.32
0, consumption (~1 Wcm2)* 8.91 It:0.53 9.612 0.76
299
Biochemical properties of N&K-ATPase in frog epidermis (b) (a) 200
200-
I50
y
1
:
1
I
1
I
8
0
20
40
60
I
’ 0
I
1
I
20
40
60
min
min
(a) Fig.
-,
2.
0
Short-circuit current of R, pip&s skin acclimatized at 24°C (a) and 10°C co> for 3 weeks: measurement was followed at 24°C and - - - -, followed at 10°C. Recording of short-circuit current was started 1 hr after mounting on the Using chamber.
by Nakao’s method are shown in Table 2. In the first example, O-25 M sucrose solution was used as the homogenization medium and 900 g supernatant was the starting material. Specific activity &moles Pijmg protein per hr) of ouabain-sensitive Na,Kdependent enzyme was increased only twice. However, the ratio of Na,K-dependent to Mg-dependent ATPase activity was changed several-fold. In the second instance, a 9000 g supernatant fraction of water homogenate containing O*l’? deoxycholate was used as the starting material. Although the Na,K-dependent to Mg-dependent ATPase activity ratio was again increased only several-fold, the specific activity was raised fifteenfold over the statting material. However, this preparation, being unstable, lost about 70 per cent of its activity during 5 days’ storage at -20°C. Loss of Mg-dependent activity was not so remarkable. In the last experiment, crude epidermal homogenate was used as the
starting material. Homogenization medium was 0.25 M sucrose solution containing 1 mM EDTA and 0.1% deoxycholate. In this case, the specific activity was not increased so much, since a large amount of precipitate including cell debris and nuclei was found in the final 20,000 g sedimented fraction. However, the percentage of ouabainsensitive Na,K-dependent activity in the. total activity was increased to over 85 per cent against 16 per cent in the starting material and the Na,Kdependent to Mg-dependent ATPase activity ratio was raised about thirty-sixfold over the original crude homogenate. The Lineweaver-Burk plot of the enzyme preparation using ATP as substrate is depicted in Fig. 6. In this experiment, partially purified enzyme from R.catesbeiana was used. The I&, value was calculated as 1-5x 10” M from this figure. The substrate specificity was examined using the last preparation. The results are summarized in
Table 2. Partial purification of Na,K-ATPase from frog epidermis Specific activity &moles PJmg protein per hr) Starting material
Frog R. cutesbeianu
R. catesbeiana R .pipiens
900 g supematant 9000 g supematant
Crude homogenate
Starting material 2.47 OS96 2.93
Final preparation 4.67 ( x 1*9)* 14.54 ( x 15.2) 3.85 (x 1.3)
* Values in parentheses indicate a multiple of starting material.
Ratio of Na,K-ATPase~Mg-ATPase Starting material 0.23 0.29 0.19
Final preparation 1.78 (x 7.7) 1*64(x5.7) 6*95(x 366)
JUN KIAWADA,
300
R. E.
TAYLOR, JR. AND S. B. BARKER
0.30 -
ii F x .a
0‘20 -
x c .2 t 0 (u ;;
0.10-
c Q
ok_
Control
Try~jsin
egotrypsin
Trypsin Chymotrypsi~
Fig. 3. Effect of trypsin andfor chymotrypsin on R. caresbeiana epidermal ATPase. Protease concentration was O-1per cent, incubation time was 15 min. Constituents of incubation medium were the same as described in Fig. 1: q, Mg-ATPase and a, Na,K-ATPase.
Gruener & Avi-Dor (1966) have shown that the inhibitory effect of ouabain on rat brain enzyme decreases sharply with reduction in incubation temperature. With frog epidermal enzyme, howindicate that ouabainever, our experiments inhibitory activity was not significantIy different even at 16°C as compared to the activity at 37°C. Therefore the inhibitory effect of ouabain is not temperature dependent where frog epidermal enzyme is concerned. In 1967, Smith reported the influence of temperature acclimatization on the temperature dependence of goldfish intestinal ouabain-sensitive ATPase and suggested that modification of the membrane ATPase system in goldfish intestinal mucosa may serve to regulate sodium ion transport at different enviro~ental temperatures. Also Bowler & Duncan (1968) compared the temperature dependence of Na,K-ATPase and Mg-ATPase activity of rat and frog brain microsomal preparations and discussed their physiological roles. Takenaka (1963) reported that in frog skin the short-circuit current is equal to the net sodium transport between 6 and 20°C and the temperature coefficient of the short-circuit current changes at a temperature around 14°C. Recently, Asano et al. (1970) found that Na,K-ATPase and the active sodium transport system of frog showed the same characteristic temperature dependence. These observations would suggest some physiological
Table 3, Substrate specificity of partially purified ouabain-sensitive Na,K-dependent enzyme from frog epidermis Substrate ATP ADP CTP GTP ITP UTP
Ouabain-se~itive activity (@molesPi/mg protein per hr) 3.85 (100%) 0*71(18-4%) 0+.x) (@O%) 035 (9.1%) O-16 (4.2%)
Table 3. Among five different nucelotides tested, ATP was the most available substrate for the ouabain-sensitive, Na,K-dependent enzyme. However, the activity with CTP or less extent with ITP was also observed. The enzyme activity with CTP was approximately 11 per cent of the value with ATP. DISCUSSION
According to our approximate calculation using the Arrhenius equation, the activation energy of Na,K-ATPase of frog epidermis was 7 kcal mole-l between 37 and 23°C and increased to 97 kcal mole-l between 23 and 16°C. This might suggest that a major conformational change of the Na,KATPase protein may occur at the critical temperature in V&O system. Ahmed & Judah (1965) and also
Na,K-de~ndent
activity
(,umoies Pi/mg protein per hr) 3.76 0.04 0.41 @oo 0.06 0.05
(100%) (1.1%) (11.0%) (0.0%) (1.6%) f l-3%)
significance of Na,K-ATPase for adaptation to the environmental temperature in poikilothermic vertebrates. However, our in vivo study using acclimatized frogs indicates no significant change in Na,KATPase activity and short-circuit current or active sodium transport across the skin. The remarkable loss of activity in Mg-ATPase from the coldacclimatized animals is hard to explain simply by the decreased rate of ATP synthesis and utilization resulting from the low body temperature of the animals, because oxygen consumption of the skin was not significantly changed from that observed with the animals kept in a moderate temperature. Trypsin has been used for separation of human epidermis (Szabo, 1955). We applied this method to frog skin and noticed that the epidermis separated by trypsin had almost no Na,K-ATPase activity.
Biochemical properties of Na,K-ATPase in frog epidermis
Incubation
time
with
trypsin,
301
min
Fig. 4. Time course of effect of trypsin on R. cutesbeiana epidermal ATPase. Trypsin concentration was 0.01 per cent: o-e, control and O-O, trypsin treated.
(bf
C%t
T;;p
.-..-_-
Control
Trypsin
-DOC
I Cz L--.. _.__,TGP t DOC
Fig. 5. Effect of trypsin on rat kidney ATPase: 900 g supematant of rat kidney homogenate was used as enzyme preparation and the trypsin concentration was0,1 per cent.(a). Effect of incubation timewith trypsinwas compared at 15 min and 135 min. (b). Effect of deoxycholate (DOC) in homogenizing medium on trypsin treatment: n, Mg-ATPase and U, Na,K-ATPase.
Marchesi & Palade (1967) reported that trypsin decreases the activity of transport ATPase in the mammalian erythrocyte membrane. The influence of some proteolytic enzymes upon rat brain Na,KATPase activity was compared by Somogyi (1968). Considering these facts and our data, it seems that a high susceptibility of Na,K-ATPase to proteases is a common property of this enzyme protein. At the same time, some protective effects against proteolytic
action by Mg and ATP (Marchesi & Palade, 1967) and Na-K (Somogqri, 1968) have been reported by those authors. We examined also these protective effects. However, the variation was so great that no decisive conclusions were given for the frog epidermal preparation. The partial purification method for Na,Kdependent enzyme using high concentrations of NaI is also very useful for the frog epidermal enzyme.
JUN KAWADA,
Reciprocal
of
ATP
concn.,
R. E. TAYLOR,JR. ANDS. B. BARKER
x106M
Fig. 6. Lineweaver-Burk plot of partially purified R. catesbeiana epidermal Na,K-ATPase for various ATP
concentrations.
ATP gave the highest activity as substrate for the ouabain-sensitive and Na,K-dependent enzyme from the frog epidermis. However, the activity with CTP was about 10 per cent of that with ATP. Almost no ADP was utilized. This fact may indicate that adenylic acid kinase activity was also eliminated in this preparation. These data coincide with the results reported by Nakao ef al. (1965). Acknowledgements-The authors would like to thank Messrs. Wayne Curles and Joseph Kidd for their technical assistance.
REFERENCES AHhEED K. & JUDAH J. D. (1965) On the action of strophanthin G. Can. J. Biochem. 43,877-880. ASANOY., MATS~ H., NAGANOK. & NAKAO M. (1970) (Na*+K+)-ATPase from the frog bladder and its relationship to sodium transport. Bioch~m. biophys. Acta 219, 169-178.
BUNTINGS. L. & CARAVA~IO L. L. (1963) Studies on sodium-potassi~-acti~t~ adenosine triphosphatase-V. Correlation of enzyme activity with cation flux in six tissues. Archs. Biochem. Biophys. 101, 3746. BOWLERK. & DUNCAN C. J. (1968) The temperature characteristics of the ATPase from a frog brain microsomal preparation. Comp. Biochem. Physiol. 24, 223227. GRUENERN. & AVI-DOR Y. (1966) Temperature-dependence of activation and inhibition of rat-brain adenosine triphosphatase activated by sodium and potassium ions. Biochem. J. 100,762-767. KAWADAJ., TAYLORR. E., JR. & BARKERS. B. (1969) Measurement of Na,K-ATPase in the separated epidermis of Rana cutesbeia~a frog and tadpoles. Camp. Biochem. PhysioI. 30,965-97X MARCHESIV. T. & PALADEG. E. (1967) Inactivation of adenosine triphosphatase and disruption of red cell membrane by trypsin: protective effect of adenosine triphosphate. Proc. nat Acad. Sci. U.S. 58, 991-995. NAKAOY., NAGANOK., ADACHIK. & NAKAO M. (1963) Separation of two adenosine triphosphatases from erythrocyte membrane. Biochem. biophys. Res. Commun. 13,&l-448. NAKAOT., TASHIMAY., NAGANOK. & NAKAOM. (1965) Highly specific sodium-potassium-activated adenosine triphosphatase from various tissues of rabbit. Biochem. hiophys. Res. Commun. 19,755-758. SMITHM. W. (1967) Influence of temperature acclimatization on the temperature-dependence and ouabainsensitivity of goldfish intestinal adenosine triphosphatase. Biochem. J. 105,65-71. SOMOGYIJ. (1968) The effect of proteases on the (Na++ K-+)-activated adenosine triphosphat~e system of rat brain. Biochim. biophys. Acfa 151,421-428. Sznti G. (1955) A modification of the technique of “skin splitting” with trypsin. J. Pathol. Bact. 70, 545. TAKENAKAT. (1963) Effects of temperature and metabolic inhibitors on the active Na transport in frog skin. Jap. J. Physiol. 13, 208-218. TAYLORR. E., JR., TAYLORH. C. & BARKERS. B. (1966) Chemical and morphological studies on inorganic phosphate deposits in Rana cutesbeiuna skin. J. enp. Zool. 161, 271-286. USSING H. H. & ZERAHNK. (1951) Active transport of sodium as the source of electric current in the shortcircuited isolated frog skin. Acta physiof. scand. 23, 110-127. Key Word Zttdex--Frog skin; Na,K-ATPase; short-circuit current.
protease;
5OA,pp. 297 to 302. Pergamon Press. Printed in Great Britain
SOME BIOCHEMICAL PROPERTIES OF Na,K-ATPase IN FROG EPIDERMIS*t JUN KAWADA,~ R. E. TAYLOR, JR. AND S. B. BARKER Department of Physiology and Biophysics, The Medical Center, University of Alabama in Birmingham, Birmingham, Alabama 35233, U.S.A. (Received 30 October
1973)
Ah&act-l. Na,K-ATPase of frog epidermis was sensitive to proteases and easily destroyed. In contrast, Mg-ATPase was highly resistant towards proteolytic action. 2. The Arrhenius plot of Na,K-ATPase activity shows a sharp discontinuity near 23’C; however, Mg-ATPase has no such critical point. 3. Short-circuit current and Na,K-ATPase activity of the skin from a fold-a~limati~d animal was not sibilantly different from that observed in a moderate-temperatur5a~limatized animal. MgATPase activity was lost around 50 per cent in cold-acclimatized animals; however, oxygen consumption of the skin was not significantly changed. 4. The preparation obtained by Nakao’s NaI method showed a high substrate specificity for ATP. The activity with CTP was about 10 per cent of that with ATP as substrate, Almost no ADP was utilized. The I$, of the enzyme preparation for substrate ATP was calculated as 1.5 x 1O-8M.
INTRODUCTION OUABA~N-SENSITIVE Na,K-dependent ATPase was demonstrated in crude homogenates of lyoph~lized frog skin and related with cation flux in the tissue by Bonting et al. (1963). The difficulty to get consistent data with this tissue was pointed out at the same time. Considering the facts that the active sodium transporting mechanism is thought to reside in the epidermis and that the difficulty involved in getting consistent data is due to the hardness of dermal tissue and its high inorganic phosphate concentration (Taylor ef al., 19661, we separated the epidermal layer from the whole skin for the enzyme study using alkaline halide solutions (Kawada et al., 1969). Hitherto little has been known about the properties and the purifi~tion of ouabain-sensitive Na,Kdependent ATPase in frog skin. In this paper, the influence of tem~rature on the enzyme in oitro and in uivo system, the effect of proteases, partial purification of ouabain-sensitive Na,K-dependent enzyme and its substrate specificity are described. MATERIALS AND METHODS Runapigiens and Rana catesbeiana frogs obtained from Lemberger Co. Oshkosh, Wisconsin, were used. They
were kept in tap water at a temperature of 24+ 1°C at least 5 days before use. In the temperature-acclimatization experiment, A. pi&ens frogs were divided into two groups. They were all kept in artificial pond water. One group was at a temperature 10 _t2°C and the other was at 24+ 1°C for 3 weeks. In some experiments, rat kidney was used to compare the results with frog tissue. Separation of the epidermis from the ventral skin and the enzyme assay method have been described elsewhere (Kawada et al., 1969). The homogenization medium routinely used was 025 M sucrose s&ution, the pH being adjusteh to 7.4 with 1OOmM Tris-IX1 buffer unless otherwise mentioned. In temperature-acclimatized animals, oxygen consumption of the whole skin was determined by a differential manometer and the short-circuit current was also recorded using Ussing’s chamber (Ussing & Zerahn, 1951). Partial purifi~tion of the enzyme was tried by the method of treating with a high con~nt~tion of NaI as proposed by Nakao (1963, 1965). For the study of the effect of proteases, Trypsin 2X Crystalline and Chymotrypsin a were purchased from the Nut~tional Biochemicals Corp. As nucleotides, ATP (Tris salt, Sigma), ADP (Na salt, Sigma), CTP (T&K salt, Calbiochem), GTP (Tri-Na salt, Sigma), ITP (Tri-Na, salt, Calbiochem) and UTP (Tri-Na salt, Calbiochem) were used to examine the substrate specificity. The final concentration of each nucleotide in the incubation medium for enzyme assay was 3 pmoles. RESULTS
*This investigation was supported in part by grant AM-10436 from the National Institutes of Health, Bethesda, Maryland. t Address requests for reprints to Jun Kawada. $ Present address: Department of Biochemistry, Faculty of Pharmaceutical Sciences, University of Tokushima, Tokushima, Japan.
&%!ct 5f temperature The ATPase activity of the frog epidermis was determined at different incubation temperatures from 37 to 12°C. The results examined as Arrhenius plots are shown in Fig. 1. To compare this result 297
J~JNKAWADA,R. E. TAYLOR,JR.
298
v I/T,
I
,
I
I
3.3
34
3-5
3.6
I/T,
xIO-~
AND
S. B.
BARKER
chamber with the same skin between the two groups of animah. The values of the short-circuit current of the individual skin varied with a wide range. AIthough Na,K-ATPase activity measured at an incubation temperature of 32°C was not changed significantly by cold acclimatization, Mg-ATPase activity was about 50 per cent lower in the coldacclimatized group than in the 24°C group. Oxygen consumption of the skin was not changed significantly by cold acclimatization. These results are summarized in Table 1. As far as these experimental conditions are concerned, no m~ningful relationship can be demonstrated between the data obtained from the Arrhenius plot and the active sodiumtransport phenomenon observed in the animals acclimatized at the low temperature.
x10-3
Effect of proteases Fig. 1. Arrhenius plot of ATPase activity of R. catesbeiunu epidermis (a) and rat kidney (b): o-o, MgATPase and o-o, Na,K-ATPase. Incubation medium contained Na+, 1OOmM; K+, 10 mM; M2+, 6mM; EDTA, 0.1 mM; Tris-ATP, 3 mM and Tris (l~mM)-
HCI buRer, pH 7.4. with mammalian tissue enzymes, rat kidney ATPase was also determined in the same way. For these experiments, 900 g supernatants of the homogenates were used. The graph of log of Na,K-ATPase activity vs. the reciprocal of the temperature showeda discontinuity at around 23°C for both materials. However, in frog epidermis, the activity decreased more rapidly below 23°C and was extremely low at 16°C. The Mg-ATPase activity curve of frog epidermis has no distinct critical point. However, the Mg-ATPase activity curve of rat kidney may have a discontinuity of 25°C. Although it must be kept in mind that the properties of an enzyme or enzyme system in v&o may not be similar to that in vivo, it is of considerable interest that such a dramatic change in Na,K-ATPase activity occurs near 23”C, a temperature which is not an unusual condition for temperature poikilothermic vertebrates. Therefore the temperature acclimatization experiment was carried out in order to determine if the results shown in the Arrhenius plot had any biological significance. As shown in Fig. 2, there was no significant differencein short-circuit currents which were determined at different temperatures in the
Figure 3 shows the effect of incubating epidermal ATPase preparation with trypsin and chymotrypsin for 15 min at 23°C. Ouabain-sensitive ATPase activity was almost completely destroyed by both enzymes, but Mg-ATPase activity was not significantly changed even after combined treatment with a high concentration of trypsin and chymotrypsin. Fig. 4 follows the change in the enzyme during 80 min incubation with 0.01% trypsin. Mg-ATPase activity was not influenced si~ificantly. However, ouabain-sensitive ATPase activity was greatly decreased during the first 5 min. In order to compare this phenomenon with mammalian tissue, the 900 g supernatant of rat kidney homogenate was treated with O-Ol”Atrypsin. The results are shown in Fig. 5; in contrast to frog epidermal preparations, the MgATPase activity of rat kidney was usually decreased approximately by 30 per cent after 15 min incubation, Rat kidney ouabain-sensitive ATPase activity is also rapidly decreased by the proteolytic action of trypsin. An addition of 0.1 per cent of deoxycholate to the homogenization medium increased the destruction of Mg-ATPase activity after 15 min incubation with trypsin to about 50 per cent. Partial pur~~&atio~~ of ollaba~n-sensitive Na,Kdependent enzyme and its substrate specificity The results of three examples using different starting materials for partial purification of enzyme
Table 1. Enzyme activity and oxygen consumption of skin from acclimatized frogs Temperature acclimatized (“Cl
No. of frogs
Mg-ATPase Na,K-ATPase @moles Pi/me! protein per hr) @moles Pi/mg protein per hr)
5 6 * At WC, 760 mm Hg. i Mean& S.E.
3.09 F 0.24t 6.14+0.33
2.99kO.37 2.92kO.32
0, consumption (~1 Wcm2)* 8.91 It:0.53 9.612 0.76
299
Biochemical properties of N&K-ATPase in frog epidermis (b) (a) 200
200-
I50
y
1
:
1
I
1
I
8
0
20
40
60
I
’ 0
I
1
I
20
40
60
min
min
(a) Fig.
-,
2.
0
Short-circuit current of R, pip&s skin acclimatized at 24°C (a) and 10°C co> for 3 weeks: measurement was followed at 24°C and - - - -, followed at 10°C. Recording of short-circuit current was started 1 hr after mounting on the Using chamber.
by Nakao’s method are shown in Table 2. In the first example, O-25 M sucrose solution was used as the homogenization medium and 900 g supernatant was the starting material. Specific activity &moles Pijmg protein per hr) of ouabain-sensitive Na,Kdependent enzyme was increased only twice. However, the ratio of Na,K-dependent to Mg-dependent ATPase activity was changed several-fold. In the second instance, a 9000 g supernatant fraction of water homogenate containing O*l’? deoxycholate was used as the starting material. Although the Na,K-dependent to Mg-dependent ATPase activity ratio was again increased only several-fold, the specific activity was raised fifteenfold over the statting material. However, this preparation, being unstable, lost about 70 per cent of its activity during 5 days’ storage at -20°C. Loss of Mg-dependent activity was not so remarkable. In the last experiment, crude epidermal homogenate was used as the
starting material. Homogenization medium was 0.25 M sucrose solution containing 1 mM EDTA and 0.1% deoxycholate. In this case, the specific activity was not increased so much, since a large amount of precipitate including cell debris and nuclei was found in the final 20,000 g sedimented fraction. However, the percentage of ouabainsensitive Na,K-dependent activity in the. total activity was increased to over 85 per cent against 16 per cent in the starting material and the Na,Kdependent to Mg-dependent ATPase activity ratio was raised about thirty-sixfold over the original crude homogenate. The Lineweaver-Burk plot of the enzyme preparation using ATP as substrate is depicted in Fig. 6. In this experiment, partially purified enzyme from R.catesbeiana was used. The I&, value was calculated as 1-5x 10” M from this figure. The substrate specificity was examined using the last preparation. The results are summarized in
Table 2. Partial purification of Na,K-ATPase from frog epidermis Specific activity &moles PJmg protein per hr) Starting material
Frog R. cutesbeianu
R. catesbeiana R .pipiens
900 g supematant 9000 g supematant
Crude homogenate
Starting material 2.47 OS96 2.93
Final preparation 4.67 ( x 1*9)* 14.54 ( x 15.2) 3.85 (x 1.3)
* Values in parentheses indicate a multiple of starting material.
Ratio of Na,K-ATPase~Mg-ATPase Starting material 0.23 0.29 0.19
Final preparation 1.78 (x 7.7) 1*64(x5.7) 6*95(x 366)
JUN KIAWADA,
300
R. E.
TAYLOR, JR. AND S. B. BARKER
0.30 -
ii F x .a
0‘20 -
x c .2 t 0 (u ;;
0.10-
c Q
ok_
Control
Try~jsin
egotrypsin
Trypsin Chymotrypsi~
Fig. 3. Effect of trypsin andfor chymotrypsin on R. caresbeiana epidermal ATPase. Protease concentration was O-1per cent, incubation time was 15 min. Constituents of incubation medium were the same as described in Fig. 1: q, Mg-ATPase and a, Na,K-ATPase.
Gruener & Avi-Dor (1966) have shown that the inhibitory effect of ouabain on rat brain enzyme decreases sharply with reduction in incubation temperature. With frog epidermal enzyme, howindicate that ouabainever, our experiments inhibitory activity was not significantIy different even at 16°C as compared to the activity at 37°C. Therefore the inhibitory effect of ouabain is not temperature dependent where frog epidermal enzyme is concerned. In 1967, Smith reported the influence of temperature acclimatization on the temperature dependence of goldfish intestinal ouabain-sensitive ATPase and suggested that modification of the membrane ATPase system in goldfish intestinal mucosa may serve to regulate sodium ion transport at different enviro~ental temperatures. Also Bowler & Duncan (1968) compared the temperature dependence of Na,K-ATPase and Mg-ATPase activity of rat and frog brain microsomal preparations and discussed their physiological roles. Takenaka (1963) reported that in frog skin the short-circuit current is equal to the net sodium transport between 6 and 20°C and the temperature coefficient of the short-circuit current changes at a temperature around 14°C. Recently, Asano et al. (1970) found that Na,K-ATPase and the active sodium transport system of frog showed the same characteristic temperature dependence. These observations would suggest some physiological
Table 3, Substrate specificity of partially purified ouabain-sensitive Na,K-dependent enzyme from frog epidermis Substrate ATP ADP CTP GTP ITP UTP
Ouabain-se~itive activity (@molesPi/mg protein per hr) 3.85 (100%) 0*71(18-4%) 0+.x) (@O%) 035 (9.1%) O-16 (4.2%)
Table 3. Among five different nucelotides tested, ATP was the most available substrate for the ouabain-sensitive, Na,K-dependent enzyme. However, the activity with CTP or less extent with ITP was also observed. The enzyme activity with CTP was approximately 11 per cent of the value with ATP. DISCUSSION
According to our approximate calculation using the Arrhenius equation, the activation energy of Na,K-ATPase of frog epidermis was 7 kcal mole-l between 37 and 23°C and increased to 97 kcal mole-l between 23 and 16°C. This might suggest that a major conformational change of the Na,KATPase protein may occur at the critical temperature in V&O system. Ahmed & Judah (1965) and also
Na,K-de~ndent
activity
(,umoies Pi/mg protein per hr) 3.76 0.04 0.41 @oo 0.06 0.05
(100%) (1.1%) (11.0%) (0.0%) (1.6%) f l-3%)
significance of Na,K-ATPase for adaptation to the environmental temperature in poikilothermic vertebrates. However, our in vivo study using acclimatized frogs indicates no significant change in Na,KATPase activity and short-circuit current or active sodium transport across the skin. The remarkable loss of activity in Mg-ATPase from the coldacclimatized animals is hard to explain simply by the decreased rate of ATP synthesis and utilization resulting from the low body temperature of the animals, because oxygen consumption of the skin was not significantly changed from that observed with the animals kept in a moderate temperature. Trypsin has been used for separation of human epidermis (Szabo, 1955). We applied this method to frog skin and noticed that the epidermis separated by trypsin had almost no Na,K-ATPase activity.
Biochemical properties of Na,K-ATPase in frog epidermis
Incubation
time
with
trypsin,
301
min
Fig. 4. Time course of effect of trypsin on R. cutesbeiana epidermal ATPase. Trypsin concentration was 0.01 per cent: o-e, control and O-O, trypsin treated.
(bf
C%t
T;;p
.-..-_-
Control
Trypsin
-DOC
I Cz L--.. _.__,TGP t DOC
Fig. 5. Effect of trypsin on rat kidney ATPase: 900 g supematant of rat kidney homogenate was used as enzyme preparation and the trypsin concentration was0,1 per cent.(a). Effect of incubation timewith trypsinwas compared at 15 min and 135 min. (b). Effect of deoxycholate (DOC) in homogenizing medium on trypsin treatment: n, Mg-ATPase and U, Na,K-ATPase.
Marchesi & Palade (1967) reported that trypsin decreases the activity of transport ATPase in the mammalian erythrocyte membrane. The influence of some proteolytic enzymes upon rat brain Na,KATPase activity was compared by Somogyi (1968). Considering these facts and our data, it seems that a high susceptibility of Na,K-ATPase to proteases is a common property of this enzyme protein. At the same time, some protective effects against proteolytic
action by Mg and ATP (Marchesi & Palade, 1967) and Na-K (Somogqri, 1968) have been reported by those authors. We examined also these protective effects. However, the variation was so great that no decisive conclusions were given for the frog epidermal preparation. The partial purification method for Na,Kdependent enzyme using high concentrations of NaI is also very useful for the frog epidermal enzyme.
JUN KAWADA,
Reciprocal
of
ATP
concn.,
R. E. TAYLOR,JR. ANDS. B. BARKER
x106M
Fig. 6. Lineweaver-Burk plot of partially purified R. catesbeiana epidermal Na,K-ATPase for various ATP
concentrations.
ATP gave the highest activity as substrate for the ouabain-sensitive and Na,K-dependent enzyme from the frog epidermis. However, the activity with CTP was about 10 per cent of that with ATP. Almost no ADP was utilized. This fact may indicate that adenylic acid kinase activity was also eliminated in this preparation. These data coincide with the results reported by Nakao ef al. (1965). Acknowledgements-The authors would like to thank Messrs. Wayne Curles and Joseph Kidd for their technical assistance.
REFERENCES AHhEED K. & JUDAH J. D. (1965) On the action of strophanthin G. Can. J. Biochem. 43,877-880. ASANOY., MATS~ H., NAGANOK. & NAKAO M. (1970) (Na*+K+)-ATPase from the frog bladder and its relationship to sodium transport. Bioch~m. biophys. Acta 219, 169-178.
BUNTINGS. L. & CARAVA~IO L. L. (1963) Studies on sodium-potassi~-acti~t~ adenosine triphosphatase-V. Correlation of enzyme activity with cation flux in six tissues. Archs. Biochem. Biophys. 101, 3746. BOWLERK. & DUNCAN C. J. (1968) The temperature characteristics of the ATPase from a frog brain microsomal preparation. Comp. Biochem. Physiol. 24, 223227. GRUENERN. & AVI-DOR Y. (1966) Temperature-dependence of activation and inhibition of rat-brain adenosine triphosphatase activated by sodium and potassium ions. Biochem. J. 100,762-767. KAWADAJ., TAYLORR. E., JR. & BARKERS. B. (1969) Measurement of Na,K-ATPase in the separated epidermis of Rana cutesbeia~a frog and tadpoles. Camp. Biochem. PhysioI. 30,965-97X MARCHESIV. T. & PALADEG. E. (1967) Inactivation of adenosine triphosphatase and disruption of red cell membrane by trypsin: protective effect of adenosine triphosphate. Proc. nat Acad. Sci. U.S. 58, 991-995. NAKAOY., NAGANOK., ADACHIK. & NAKAO M. (1963) Separation of two adenosine triphosphatases from erythrocyte membrane. Biochem. biophys. Res. Commun. 13,&l-448. NAKAOT., TASHIMAY., NAGANOK. & NAKAOM. (1965) Highly specific sodium-potassium-activated adenosine triphosphatase from various tissues of rabbit. Biochem. hiophys. Res. Commun. 19,755-758. SMITHM. W. (1967) Influence of temperature acclimatization on the temperature-dependence and ouabainsensitivity of goldfish intestinal adenosine triphosphatase. Biochem. J. 105,65-71. SOMOGYIJ. (1968) The effect of proteases on the (Na++ K-+)-activated adenosine triphosphat~e system of rat brain. Biochim. biophys. Acfa 151,421-428. Sznti G. (1955) A modification of the technique of “skin splitting” with trypsin. J. Pathol. Bact. 70, 545. TAKENAKAT. (1963) Effects of temperature and metabolic inhibitors on the active Na transport in frog skin. Jap. J. Physiol. 13, 208-218. TAYLORR. E., JR., TAYLORH. C. & BARKERS. B. (1966) Chemical and morphological studies on inorganic phosphate deposits in Rana cutesbeiuna skin. J. enp. Zool. 161, 271-286. USSING H. H. & ZERAHNK. (1951) Active transport of sodium as the source of electric current in the shortcircuited isolated frog skin. Acta physiof. scand. 23, 110-127. Key Word Zttdex--Frog skin; Na,K-ATPase; short-circuit current.
protease;
