Buffer systems variably affect the interaction of

Buffer systems variably affect the interaction of norepinephrine with brain Na + -K * ATPase S. J. MIHIC,P.W. WU,'

AND

H. KALANT

Department of Pharmaeslogy, Faculty ofkledicine, Medical Sciersces Building, University of Toronto, Toronto, Ont., Canada M5S l A 8 and Addiction Research Foundation, Toronto, Bnt., Canada

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Received October 20, 1987 MIHIC,S.J., Wu, B. H., and KALANT, H. 1988. Buffer systems variably affect the interaction of norepinephrine with brain Na+-K+ ATPase. Cm. J. Physiol. Pharmacol. 66: 1035-1040. The reported effects of norepinephrine (NE) on brain Na+-K+ ATPase are quite variable, Different investigators have reported activation, inhibition, or no effect. An investigation of the importance of reaction conditions on brain Na'-K' ATPase activity was undertaken to resolve some of these discrepancies. Using porcine cerebral cortical Na+-K' ATBase and rat brain synapbsomal membrane preparations, it was observed that NE strongly inhibited brain Na+-K+ ATPase in Tris-HC1 buffer. This inhibition of the enzyme was reversed by the addition of EBTA. In contrast, NE did not significantly inhibit Na'-K+ ATPase in imidazole-glycylglycine and Krebs-Ringer-phosphate buffers. This buffer dependence of NE inhibition of the enzyme was consistently demonstrated with three different established methods for phosphate measurement. Kinetic analysis indicated that NE, in Tris-HC1 buffer, inhibited the enzyme noncompetitively at high affinity, and competitively at low affinity, ATP substrate sites.

Mr~ae,S.J., Wu, P. H., et KALAWT, H. 1888. Buffer systems variably affect the interaction of norepinephrine with brain Na'-K+ ATBase. Can. J . Physiol. Phmacol. 66 : 1035- 1040. k s effets Be la nor6pinCphine (NE) sur la Na+-K+ ATPase cCrCbrale varient beaucoup. Divers chercheurs ont par16 d'activation, d'inhibition ou d'absence d'effet. Une Ctude de l'importance des conditions de la rCaction sur 15activitt5de Na'-K+ AWase cCrCbrale a CtC effectuCe pour r6soudre certaines de ces contradictions. En utilisant des prCparakions de Na'-K' ATPase corticales cCrCbrales porches et de membranes synaptosomales de cerveau de rat, on a observC que la NE inhibait fortement la*Na+-K+ ATPase cCrCbrale dans un tampon Tris-HCB. Cette inhibition de l'enzyme a kt6 renvers6e p a l'addition d'EDTA. A 170pposC,la NE n'a pas significativementinhibC la Na+-K+ ATPase dans des tampons d9imidazoleglycylglycine ek de phosphate-Keebs-Ringer. Cette dkpendance, vis-a-vis le tampon, de l'inhibition de l'enzyme par aha NE a CtC montr6e de manibe continue avec trois mCthodes de mesure du phosphate reconnues. Une analyse cinCtique a indiquC qrae Ba NE, dans ran tampon Tris-HCl, inhibaik l'enzyme aux sites des substrats de l'ATP, de mani5re non comNtitive ceux de haute affinitk et de mani5re comgtitive a ceux de faible affinitk. [Traduit p a la revue]

Introduction Catecholamines have been reported to stimulate (Yoshimura 1973; Godfraind et a&. 19'74; Logan and B'bnovan 1976; Desaiah and Ho 1977; Lee and Phillis 1977) and to inhibit (Vysoehina and Popova 1978; Rodriguez de Lores Arnaiz and Antonelli de Gomez de Lima 1986; Rodriguez de Lores h a i z and Mistrorigo de Pancheco 1978) brain N ~ + - K +ATPases in different species and tissue preparations. The catecholamine stimulation of Na+-Kf ATPase activity has been attributed to the presence of vanadium in comercial ATP preparations; catecholamines reduce v5+ to v 3 + , rendering it inactive in inhibiting the enzyme (Cantley et a&.1977; Hudgins and Bond 1977;Josephson and Cantley 1977; Rangaraj and Kalant 1979). The recent-finding of cytosolic soluble factors that promote the stirnulatory effects of catecholamines on brain N ~ + - K + ATPase may suggest the existence of a different mechanism for stimulation (Rodriguez de Lores h a i z and Antonelli de Gomez de Lima 1986). The inhibitory effect of norepinephrine (NE) on brain synaptosomal N ~ + - K + ATPase has been reported by Vysoshina and Popva (1978), and this has dso been demonstrated in brain homogenates (Rodriguez de Lores Arnaiz and htoneHHi de Gomez de Lima 1986). In contrast,Rangaraj and ' ~ u t h s rto whom correspndencc m y be sent at the University of Toromto adchess.

Kalant (1979), Nhamburo et ak. (I 986), awd Syapin et ak (I 985) were unable to demonstrate any significant effeft of catecholamines on Na+-K+ ATPase in homogenate preparations. The discrepancies in the reported results may be due to differences in incubation conditions, animal species used, and (or) tissue preparation. Two buffers have primarily been used in the assay of Na+-K+ ATPase activity: Tris-PIC1 and imidazole -glycylglycine (IGG). The purpose of this investigation was to study the Na+-K+ ATPase activity in both of these buffers and in a buffer system resembling that found in vivo, i.e. KiebsRinger-phosphate (KRP), and to examine more closely the mechanisms by which buffers modify enzyme activity and NE-enzyme interactions.

Materials and methods Materials Male Wistar rats weighing 250-350 g were obtained from Taconic F m s (Gemantown, New York). Vanadium-free Na2ATP (Sigma A-5384), norepinephrine hydrochloride, porcine brain Na'-K+ ATPase, imidazole, and glycylglycine were purehasd from Sigma Chemical Co. Trizma base and ultra pure Tris (base) were obtained from Sigma Chemical Co. and Schwarz/Mmn Co., respectively. [ ' 2 ~ (specific ] ~ ~ activity ~ 789 Ci/mmol) (1 Ci = 37 GBq) was obtained from ICN (Iwine, CA). All other chemicals were of ACS analytical grade.

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CAN. J. PWYSIOL. PHARMACOL. VOL. 64, 1988

Preparation ea$rat bmin enzyme Rats were maintained on laboratory chow and water ad libitum. They were killed by cervical dislocation and decapitated. The brain was rinsed in cold saline solution and the meninges were removed. The whole brain (minus cerebellum) was homogenized in 10 vol. (w/v) of chilled 0.32 M sucrose solution in a glass Potter-Elvehjem homogenizer equipped with a Teflon pestle. The homogenate was centrifuged to yield a crude mitochondrial fraction which was resuspended in 16)voli. of 0.32 M sucrose solution and further fractionated in a discontinous sucrose gradient system (Gray and Whittaker 1962). The synaptosomal fraction was isolated and Iysed in 5 rrah% M g S Q and 18 mM Tris-HC1 buffer (pH 7.4) for 5 min in an ice-bath. and the membranes were then washed with distilled water and centrifuged at 24) 000 X g for 20 min twice. Protein content of the membranes was detemined by the method of Lswrgr et ah. (1951), using bovine serum albumin as the reference standard. Norepinephrine hydmhloride, Na2ATP. and EDTA were dissolved in cold distilled H 2 0 and pH values were adjusted to 6-$, so that the addition of these solutions into the incubation mixture would not alter the pH of the final incubation medium (pH 7 2-7.4). The norepinephrine and Na2ATP solutisns were prepared fresh on each day the experiments were conducted. ATPase measurement Brain Na'-K' ATPase activity was detemined by measuring inorganic phsspate (Pi) released during the enzymatic cleavage of ATP. Inorganic phosphate levels were measured by the coliorimetpic methods of Fiske and S u b b a o w (1925) and of Post and Sen (19671, md (or) by the radioisotope method of Siegel and Albers (1967). Three buffers were used in the experiments: BOO mkl Tris-HCl, 30 nib4 IGG, and KWP buffer. In all cases, with the exception of KWP solution, electrolytes were added to the buffer for the following concentrations (in mM): Na', 72.5; K+,6.25; Pdg2+,5.6);andNa2ATP,2.5. TheKRP solution contained the following (in d4):Na+, 154; K' ,6.0; Pdg2', 1.21; PO^^-, 16.5; and Na2ATP, 2.5 (Dawson et al. 1969). The M g ' + - ~ ~ P a sactivity e was measured in buffer systems containing (in M)NaCl, 14; MgCI2, 5; and ouabain, I . The final incubation volume was 68.5 d. Na+-K' ATPase activity was calculated by subtracting ~ g " - ~ ~ P a sactivity e from total ATPase activity. The rate of nonenzyrnatic hydrolysis of ATP was also detemined. This was subtracted from the rate measured in the presence of enzyme. For studying Na+-K' ATPase kinetics, 38 p g (protein) of porcine ATPase was incubated with varying concentrations (0.872, 1.744, 3.488,8.72, 17.44 W1WB) of Na2ATP. Pi released was measured by the Fiske and SubbaRow (1925) method. The reaction was initiated by the addition of the enzyme preparation or ATP and the mixture was incubated for 20-30 min at 37'C in a shaking water bath, During the 38-wnin incubation, approximately 6% of the ATP was hydrolyzed. The use of several incubation periods indicated a linearity in the reaction rate over time, under our experimental conditions. The reaction was stopped by adding 0.5 mL sf 1- 2 M HC104 sollution. Measurement of inorganic phosphate F i s h and SubbaRow ( I 925) method After the addition of HC%C14,1.5 mL of 1.25% (w/v) ammonium molybdate in 1 M H2S04-14 d v f FeS04 solution was added to each sample. After waiting 15 f i n for completion of colour development, the phosphomolybdate absorbance was read at 700 nm in a Gilford 256) spectrophotometer. A standard curve was prepared using NaP12P04. Mod@ed Post and Sen (1967) method After termination s f the reaction with perchloric acid, 1.5 mL of I .45% (w/v) solution of sodium molybdate (pH 7.8) was added to f o m phosphomolyMate9 which was fhen extracted into 4 mL of a-butyl acetate. The n-butyl acetate phase was separated from the aqueous phase by cen~fugation.To a 2-wnE sample of the organic phase was added 4 d of isopmpmol and 5 glb.L of rnercaptsethanol; the sample absorbance was read at 825 m.

Radioactive phosphate method (Siegel and Albers 1967) Since KRP solution contains a higher concentration of phosphate than other buffers, a higher concentration of molybdate was needed to ensure complete formation of phssphomolybdate; 4.2% sodium molybdate in WCI04-NaOH solution was used. The [3"]phosphomolybdate formed was extracted into I mL of a-butanol. Following centrifugation to separate the aqueous phase, 400 FL of the n-butanol phase was added to 5.0 d.,of Aquasol (New England Nuclear) for scintillation counting (efficiency >92%) in a SexBe ANALYTIC 81 scintillation counter.

Results Measurement of porcine bmin ~ a-K+ + ATPcase activity Porcine brain Naf -Kt ATPase activity was measured by spectrometric and radioisotope methods in three different buffer solutions. The basal enzyme activity was similar irrespective of the measurement technique employed (Table 1). Na+-K+ ATPase activity was markedly elevated in IGG buffer as ccsmpaed with activity measured in Tris-MCl or KRP buffers. The ratio of the specific activity of the enzyme in IGG buffer and Tris buffer was 1.9 using the spectrometric method and 1.8 using the radioisotope method. The enzyme showed similar specific activity in KWP and Tris-HCI buffers. In all experimental conditions tested, there was a strong correlation among the three methods used to measure Pi (Table I). Eflects of NE on porcine Na+-K' ATPase activiq in three diflerent bufier solutions The effects of NE (100 p M ) on porcine N ~ + K +ATPase activity were investigated. Figure 1 shows that NE (I00 pM) inhibited N~'-K+ ATPase by 58.3% in Tris-HCl buffer; the same concentration of NE only marginally inhibited enzyme activity in IGG buffer, when enzyme activity was detemined by the spec&ome~cmethods. Using the radioisotope method, 100 p M NE inhibited the enzyme by 53% in Tris-HCl buffer but not in the IGG and KRP buffers. Using the spectrometric method, the inhibitory effect of NE on porcine ~ a + - K +ATPase in Tris buffer was seen (Fig. 2) to be dependent on the NE concentration: 58.3 8.7% ( n = 12) inhibition at 100 p%MNE, 44.0 6.2% ( n = 4) inhibition at 18 p M NE, and 19.5 + 7.9% ( n = 3) inhibition at 1p M NE (df = 2,16, F = 28.89, p = 8.0000). In IGG buffer, 180 FM NE inhibited the enzyme 8.6 k 6.5% (n = 8), 10 p M NE inhibited 1.2 & 5.8% ( n = 3 , and 1pM NE inhibitedO.94 + 2.8% (n = 5) (df = 2,15, F = 5.42, p==-0.0169.) Eflecgs of EE)TA on NE inhibition of porcine brain Na -K+ ATPase in Trls-HC&bufler NE (100 pM) potently inhibited Na+-K+ ATPase activity in Tris-HC1 buffer. Addition of 200 pM EDTA to the incubation mixture almost completely abolished the inhibition of the enzyme by 108 p M NE (see Fig. 3). One-way ANOVA showed a concenkation-depndent reversal of NE inhibition by EDTA in the Tris-H@l buffer (df = 3,8, F = 247.3, p = 0.0800). The concentration-response curve indicated that the ECSofor EDTA reversal was 12.5 p M . EDTA (200 pM) also increased the N ~ + - H ( + ATgase activity in Tlds-He1 buffer in the absence of NE, raising enzyme activity very close to that seen in the IGG buffer (58.9 ~ m o Pi/(mg l protein h). Addition of 200 pM EDTA had little effect on Na'-Kf ATPase activity in IGG buffer, in the presence or absence of BOO FM NE. Since EDTA increases basal Na'-K' ATBase activity, the reversd of NE inhibition might be thought to be due to a suprimposd elevation of the enzyme activity by EDTA in the Tris buffer. However, SehefE's post hoc eompkson of the

*

MIHIC ET AE.

TABLE1. Comparison of Na+-K+ ATPase activity measured by thee established methods Porcine Na+-K+ ATPase activity ( pmol Pi/(mg protein h))

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-

Buffer system

Post and Sen

Fiske and SubbaRow

Siegel and Albers

IrnidmsleglycyIglycine THis-WCI Krebs-Ringer

58.92~7.1 30.6+$.3

57.320.4 31.1.28.3

51.7k7.2 28.5k0.5 25.55~1.5

-

-

NOTE:Porcine I V ~ + - M + ATBase was incubated with ['2P]ATP and A n at a final concentrationof 2.5 d A for 2Q-30 min at 37°C. The reaction was terminated by adding 1.2 M HC1O4. Pi released during the reaction was converted into phosphomolybdate md was determined according to the methods d Post and Sen (1967), Rske and SubbaRow (19251, and Siegel and Albers (1967). The results are mean 9SD sf three triplicate experiments.

-:60

60

+

g

-

50

g

43

% .- 40

30

5

\

M

9

Q) 0

e

50

r

30

LC-

-,

20

20

10

10

E

%

n

n

IGG

buffer

Tr i s b u f f e r

KRP b u f f e r

FIG.1. Effects sf buffer composition on Na+-K+ ATPase activity. The basal activity of porcine brain Na+-K+ ATPase was measured in iddazole-glycylglycine (IGG), Tris-WCl, and kbs-Ringer-phosphate buffers (solid bars). The effect of NE (100 pM) on enzyme (hatched bars) in the thee buffers was also determined. Enzyme activity in d Tris buffers was measured spectrometricbuffer was measured using the radioisotope of thee triplicate determinations.

100 blsl HE

10 UM HE

1 uY NE

Re. 2. Effect of norepinephrine on Na+-K+ ATPase activity in Tris and imidazole-glycylglycine (IGG) buffers. The inhibitory effect of NNE (1-188 pM) on porcine brain Na+-K+ ATPase was measured in Tris (solid bars) and I6;e; (hatched bars) buffers. The number of triplicate measurements is indicated over each bar. Results are the mean k SD.One-way ANOVA was used to analyse the results in both buffers (see text). sites. Analysis of the data showed that the high affinity site had a Kmvalue of 1.74 0.23 nmnM and a Vm, value of 29.8 + 2.4 pmol Pi/(mg protein h). The addition of NE (100 FM) resulted in a noncompetitive inhibition of the high affinity substrate site resulting in a reduction of V,, to 2 1.6 ? 2.9 pmol Pi/(mg protein h) . In the absence of NE, the low affinity ATP substrate site showed a K, of 10.43 2 2.47 mM and a V,, of 75.6 ? 25.0 pmol Pi/(mg protein. h). This low affinity ATP substrate site was competitively inhibited by 100 pM NE (see Table 2).

*

means of the enzyme activity, in the presence and absence of NE, indicated significant differences in Na+-K+ ATPase activity only at EDTA concentrations of 8, 1, and 18 pM. Eflects ofNE on kinetic parameters oborcine NCE+-K+ ATBase in Tris-HCl bufler The effects of NE on the ATP substrate sites of porcine Na+K+ ATPase in Tpis-HC1 buffer were investigated. ATP concentrations ranging from 8.872 to 17.44 nmnM were incubated with porcine Na+-Kf ATPase in the presence or absence of 1 0 0 pM NE. Figure 4 shows that in the absence of NE, the Na+-K+ ATPase had two substrate sites for ATP. The Lineweaver-Burk plot was analyses% using linear regression analysis. After determining the coefficients of correlation of all five points of the line, and sections consisting of three or four points, it was fmnd that the best coefficient of correlation was obtained by breaking each line into two components. Each of these components consisted of t h e points, i. e. , the middle p i n t of the line was c o m n to both components. These two components were referred to as the low and high affinity ATP substrate

Efects of NE on rat brain ~napbosomalNa+-K+ ATPase The ability of NE to inhibit Na+-K+ ATPase was also tested in rat brain synaptossmal preparations. Norepinephrine ( 100 pM) inhibited the synaptosornalNa+-K+ ATPase by 46 2 14% (n=7) in the Tris-HCl buffer but only 5.4 ZZ! 4.7% (n=7) during incubation in the HGG buffer. Synthetic organic buffers free sf inorganic phosphates have been used for the determination of brain Na+-K+ ATPase activity in preparations of varying purity. Pi-free buffers are used because Pi released by the enzyme is detected through a

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CAN. J . PHYSIOL. PHARMACOL. VOL. 66, 1988

EDTA Concentrat i on (pM1 FIG. 3. EBTA reversal of norepinephrine inhibition of Na+-K' ATPase in Tris buffer. The activity of the porcine Na'-K9 ATPase was measured in the absence (0) and presence ( 0 )of 1W pM norepinephrine at EBTA concentrationsranging from 0 to 200 p M . Results are mean & SD. The inset graph shows the s m e data expressed as the percent inhibitionof porcine brain Na+-K' ATPase activity by norepinephrine. NE ( 1 0 0 pM) inhibition of the enzyme (solid bar) could be reversed by the addition of EDTA (1-200 pM) to the incubation medium (hatched bars). The number of triplicate measurements is indicated over each bar. Results are mean SD. One-way ANOVA was used to analyse the data (see text).

*

-0.6

-0.4

o

-0.2 I/S

0.2

0.4

0.6

0.8

I

1.2

(ATP concentrat i on ,MI-'

FIG. 4. ine ewe aver-Ihrk plot of the effect of NE On porcine brain Nal-K' ATPase activity in Tris-HC1 buffer. The ATP concentration ranged from 0.872 to 17.44mM. Activity was measured in the absence (0) and presence (A)of 100 p M NE. Results are mean + SB of three trip%icateexperiments.

colour reaction that involves the conversion of Pi to phosphomolybdate; the presence of even trace amounts of Pi in the buffer solution will thus complicate the measurement of enzyme activity. As a result, N ~ + - K + ATPase activity has been determined in artificial buffer solutions not resembling the biological fluid. Although P4a+-Kf ATPase behaves optima^ly'yn these biochemical assays the "optimal9' enzyme activity may not represent the actual enzymic activity in its biological environment. In addition, modification of a biological system or an enzymatic reaction by a phmacologic agent can be altered by the reaction conditions employed. Brain Na+-K+ ATPase

has been shown to be activated (stimulated) by catecholamines; subsequent analysis of enzyme activation has, however, indicated that most of the enzyme activation was due to chelation of vanadium (present in equine ATP preparations) which inhibits Na+-K+ ATPase in nanomolar concentrations (Cantley et al. 1977). Norepinephrine reduces vanadate from VS+, to V3+,a form that is incapable of inhibiting the Na+-K+ ATPase (see review, Wu 1986). There is evidence to suggest that in addition to reversing vanadate inhibition of the Na+-K+ ATPase, catecholamines may also stimulate the enzyme though a receptor-mediated mechanism (Wu and Phillis 1980). Rodriguez de Cores Amaiz and Antowelli de Gomez de Lima (1986) showed that catecholamines could stimulate Na+-K+ ATPase in the presence of soluble factors in brain fractions. These soluble factors were also able to inhibit the enzyme if allowed to age 24 h a t 4'C. In isolated brain synaptic membrane preparations, catecholamines were found to inhibit Naf -K+ATPase activity (Antowelli de Gomez de Lima and Rodriguez de Lores Amai; 1987).On the other hand, ~~~~~d et (1983) were unable to see the enzyme inhibition by catecholamines. No apparent differences in tissue preparation a d reaction conditions could account for the differences in the results seen, except that Tris-HCl buffer was used in the first case and IGG inthe latter. The present results indicate that the composition of the buffer can modify catecholamine effects on the Na+-K+ ATPase. Two buffers have primarily been used in the assay of Naf -K+ ATPase activity: Tris-HC1 and HCG. Syapin et a!. (1985) and Syapin and Alkana (1986) reported higher basal activity in the Tris-HCI buffer than in IGG. It was d s o noted that NE stimulated Naf -K+ ATPase activity in the HGG but not the Tris-HCl buffer. The different enzyme source and tissue preparation may account for the discrepancies between their results md ours. Gilbert et ak. (1988), for example, found that different tissue preparations modified the effect of NE on the

MIHIC ET AL.

TABLE 2. Effects of norepinephrine (NE) on Na+-K+ ATPase kinetic parameters Parameters

Control

NE (188 pA4)

High affinity substrate site KI( M I I/*, (pmol ~i*rng-'-h-')

1.7458.23 29.8L2.4

1.74k0.23 21.452.9

Noncompetitive inhibition

10.4L2.47 75.4?25

1 4 . 4 t 1.47 75.4525

Competitive inhibition

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Low affinity substrate site mu, ( M I

I/-('rnsl~i-mg-~-h-')

Type of interaction

NOTE:Porcine ~ a + - K +ATPase was incubated with varying concentrations of ATP (0.872- 17.44 mM) for 20 min at 37°C. The reaction was terminated by adding B .2 M HC104. Pi concentration was detennined using the hiske and SubbaRow (1925) m e t h d . The K, and Vma, values were obtained from each experiment, using Lineweaver-Burk plots. Results are mean 2 SD of three triplicate experiments.

system for the study of the effects of various pharmacological N ~ + - K +ATPase; NE inhibited isolated membrane Na+-K+ agents on brain ~ a + - K +ATPase activity. ATPase but stimulated synaptosomd ~ a + - K +ATPase. A radioisotope measuring technique was used to investigate the enzyme activity in Krebs-Ringer buffer. Naf -K+ ATPpase Acknowledgement activity was nearly twice as high in the IGG buffer as in the This project was supported by the Medical Research Council Tris-HC1 buffer, measured by either the spectrometric methods of Canada (grant MA-9613). or the radioisotope method. The enzyme showed similar activities in the KRP and Tfis HCl buffers. This suggests that the ANTONELLIBE GOMEZBE LIMA,M . , and RODR~GUEZ BE LORES specific activity of Na+-K+ ATPase measured in Tris-HCI A~NAIZ, G. 1987. Inhibition of synaptosornal Naf , Kf -ATPase by buffer gives a more accurate estimate of enzyme activity under an aged soluble fraction from brain. Possible involvement of a biological conditions. IGG seemed to stimulate the enzyme plypeptide factor. J. Neurwkem. 48: 5 152. activity. Further analysis of several buffer systems revealed that BEAUGB, F., STIBLER, H.,and MALANT, H. 1883. Brain synaptosomd many m i n o acids and small peptides, such as glycylglycine, (Na+ and K+) ATPase activity as an index of tolerance to ethanol. were able to increase Na+-K' ATPase activity in the Tri%-HC1 Phmacol. Biochem. Behav. lS(Supp1. 1): 5 19-524. CANTLEY, L. D., Ja., JOSEPHSON, L., WARNER, R., YANAGISAWA, buffer (Mihic et al. 1988). Histidine has also been reported to Ma, LECHENE, C., and GUIBOTTI, G. 1977. Vanadate is a potent activate N~'-K+ ATPase (Specht and Robinson 1973;Luly and inhibitor found in ATP derived from muscle. J. Biol. Chem. 252: Verna 1974). 7421-7423. The pH of the incubation mixture could not account for the DAWSON, a . M. C., ELLIOTT, D., ELLIOTT,W. PI., ~ ~ ~ J Q PK.NM. ES, differences in enzyme activity between the buffer systems 1969. Data for Biochemical Research. CIarendon Press, Oxford. because, in both the Tris-HC1 and IGG buffers, the final pH of BESAIAH, D., and Mo, I. M. 1977. Kinetics of catecholamine sensitive the reaction mixture was 7.4-7.5. Also, the addition of NE (188 Na+-M+-ATPase activity in mouse brain synaptosomes. Biochem. pM) to the incubation mixture did not change the pH value. Phmacol. 26: 2029-2035. We observed two ATP substrate sites on the porcine ~ a + - K + FISKE,C. M., and SUBBAROW, Y. 1925. The colorimetric determinaATPase, the high affinity site having a K m of 1.74 HLM and a tion of phosphorus. J. Biol. Chem. 66: 375-400. GILBERT, 9. C., SAWAS, A. H., and WYLLIE, M. G. 1980. Stimulation Qmax of 29.8 pmol Pi/(mg proteinm h), and the low affinity site and inhibition of synaptosome ATPases by noradrendine: the having a Km of 18.43 Hah.I and a Vm,, of 75.6 pmol Pi/(mg involvement of cytoplasmic factor. Arch. Hnt. Phmacodyn. Ther. proteineh), in Tris-HC1 buffer. In our experiments, the high 3-45: 42-47. affinity site was saturated when 2.5 Hah.I ATP was used. NE G~DF~%AIND, T.,MOCH,M., and VERBEKE, N. 1974. The action of (100 pM) inhibited ATP interaction at the high affinity site EGTA on the catecholamines stimulation of rat brain Na-Knancompetitively and reduced the V,, value. The exact ATPase. Biochem. P h m a c o l . 23: 3505-35 11. mechanism for the NE inhibition of ATP Burnover at the high GRAY,E. G., and WHITTAKER, V.P. 1362. The isolation of nerve affinity site is not clear at this time. The low affinity site does not endings from brain: an electron microscopic study of cell fragments likely contribute significantly to the Tris-NE interaction, since derived by h~mogenizationmd centrifugation. J . Anat. (London), 96: 79-88. that site was f a from k i n g saturated under our experimental HEXUM, T. D. 1977. The effect of catecholamines on transport (Na,K) conditions. This NE-induced inhibition of Na+-K+ ATPase adenosine triphosphatase. Bi~ckem.P h m a c o l . 26: 1221- 1227. activity in Tris-HC1 buffer was seen not only in porcine Na+ MUDGINS, P.M., and BOND, G. H. 1977. (Mg2+-K+)-~e~endent -K+ ATPase preparations but also in rat brain synaptosomal inhibition of Na-K-ATPase due to a contaminant in equine muscle Na'-K+ ATPase as well. ATP. Biochem. Biophys. Res. C o m u n . 77: 1824-1029. In conclusion, we have demonstrated that NE inhibition of JOSEPHSON, k.,and CANTLEY, E. C., Ja. 1977. Isolation of a potent Na'-K' ATPase activity occurs in Tris-HC1 buffer but not in (Na-K)ATPase inhibitor from striated muscle. Biochemistry, 16: IGG or KRP buffers. This inhibition can be reversed by EDTA, 4572-4578. suggesting the involvement of metal ions. The inhibition of LEE,S. E., and PHILLIS, 9. W. 1977. Stimulation sf cerebral cortical Na+-K' ATPase by several metal ions has been reported by synaptosomd Na-K-ATPase by biogenic mines. Can. J. Physiol. Hexnm (1977). Care must be taken in the selection of a buffer P h m ~ 0 1 55: . 941-964.

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CAN. J. PHYSIBL.BHARMACOL.VBL.46, 1988

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