Erik Sall, C. Delp Givens, Ronald P.Taylor' a n d Charles M. Grishamz University of Virginia, Charlonesville. 22901
I
Determination of Rate Constants for Ouabain Inhibition of Adenosine Triphosphatase An undergraduate biological chemistry laboratory
In the process of developing an undergraduate laboratory course in biological chemistry a t Virginia, we discovered that there exist few experiments which can introduce the student to an interesting biological system and a t the same time illustrate an imp&ant principle from physical chemistry. In the experiment described below, we have adapted the kinetic studies of Barnett3 to orovide the student with an examole of pseudo-first-order kihetics, the cardiac glycmide inhibition o f mammaliam sodium and ootassium transoort. This entire experiment (including the enzyme preparation) can be comoleted in two laboratorv oeriods and requires onlv a low speed .. preparative centrifuge and a spectrophotometer with recorder. Background K+)-ATPase (known to physiolugists as the The (Naf "sodium pump") is a protein-lipid complex which is found in the plasma memhrane of all animal cells.' This remarkable enzyme system is one of nature's most interesting examples of energy conversion. The enzyme, in the presence of Mg2+, Na+, and K+, catalyzes the hydrolysis of adenosine triphosphate (ATP)
+
ATP4- + 2H2O
ATPase. ME'+. Na*. Kt
+
Figufe 1. Reaction and transport scheme far the (Na+ + K+)-ATPare.
ADP3- + HPOa2- + HaOt
The reaction is exothermic as written and has a free energy change, AG, of about -7 kcallmole under physiological conditions. The ATPase uses this free energy to transport Na+ and K+ ions across the plasma memhrane, both against concentration gradients, as shown in Figure 1. This conversion of the chemical energy of ATP hydrolysis into the mechanical enerw nrovides reeula... of ion transnort . . tion and control d a vllri~ty1 8 f wllular functioni in such organ.. 33 tht: hrain. heart. and kidnev. The heart ofl'ers an ooonrtunity for an interesting medical lesson since the class ofdrugs known as cardiac elvcosides, which are commonlv used to treat heart disorders,aie potent poisons of the ( ~ a + K+)ATPase. A common heart disfunction known as fibrillation (weak, rapid heart heats) results when the normal Na+ and K+ gradients in the heart are altered. Small doses of cardiac glycosides can restore normal heart function by partially inhibiting the cardiac (Na+ + K+)-ATPase. The structures of two typical cardiac glycosides, digitoxin and ouabain, are shown in Figure 2. The inhibition of the ATPase by ouabain is a second-order reaction overall: first-order in enzyme (E) and first-order in ouabain (I)
+
OH
OH
OUABAIN
Figure 2. The structures of two common cardiac glycosides,digitoxin and ouabain. The R arWD consists of a chain of three dioitoxose suoars. - . on dioitoxin " Common characteristics of cardiac olvcasides include the steroid nucleus lme
Research Career Development Awardee of the National Institutes of Health.
2This work was supported by National Institutes of Health Grmt AM-19419 to this author, to whom correspondence should be addressed (at Department of Chemistry). "arnett, Ronald E., Biochemistry, 9,4644 (1970). Hakin and Dahl, .I.. in "Metabolic Pathways, VI, Metabolic Transport," (Editor:Hokin, L. E.) Academic Press, New Yurk, 1972, p. 269.
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122 ! Journal of Chemical Education
rate of = u = ki[E][I] inhibition If, as is often the case in such a system, the enzyme concentration is very small compared to the inhibitor concentration, then [I] is nearly constant over the course of the in-
hihition. W e can use a n apparent r a t e constant, kspp, where
kgpp= kiU1 T h e n we have a case of pseudo-first-order kinetics U = k.,,[El Then k., will be linearly dependent on ouabain concentration a n d .a olot of k.,, versus ouahain concentration will be a strai& line w i t g a slope of k , . In thisexwriment the ( N a . KT)-ATPaseactivity isassayed usingbyruvate kinase a n d lactate dehydrogenase i n a c o u ~ l e denzvme assav. T h e sequence of reactions is shown
+
ADP3Pyruvate-
-
+ PEP2- Pyrvvat. Kinam, M P ,K+ ATP4- + Pyruvate+ NADH + H30C
Lactat. Dohydrogenaas --f
Ladate-
+
NAD+
+ Hz0
-
I -time
I
Figure 3.A typical recorder chart trace for the ouabain inhibition studies. The absorbance will cominue to change linearly with time beyond point b, due to the presence of ouabain-insensitive ATPases in the preparation.
Clearly for every A T P hydrolyzed in t h e first reaction, one
PEP (phosphoenol pyruvate) is converted t o pyruvate, a n d one molecule of N A D H is oxidized. Whereas NADH absorbs strongly a t 340 nm, NAD+ is almost transparent, and thus the ATPase can he followed spectrophotometrically a t 340 nm. This couoled assav is used t o characterize t h e inhibition of t h e ATPase by ouabain. T h e enzyme preparation t o b e used is a microsomal preparation from kidney. T h e enzyme is isolated during one lab period and the kinetics can h e carried out on t h e next lab dav. T h e enzvme can b e safelv frozen between preparation a n d use. Enzyme Preparation Althoueh sheen kidnev was used in the emeriment described here. ~, almust any mnmmalian kidnry wold he used, including mhbit, dog, or ply. F n u m kidneys ran lw thawed uvwnight in aadution ofO.:Il JI sucnae. 1 m4f KIYI'A,pll :.tl~z5°CI.'l'l~rsuluti~~n will I s referrrd to herein as "suerase." The kidney can be dissected on a wooden or plastic block covered with filter paper and soaked in sucrose. Crwssectional slices of the kidney will reveal the dark brown cortex surrounding the red medulla and white or pink inner tissue. The cortex and inner tissue can be discarded, along with the white, tough fibers which penetrate the medulla. The dissected medulla is then homogenized in nine volumes of sucrose at high speed in a Waring blender for a total timeof 2 min (8 periods of 15 s with intermediate periods of cooling in ice works well). The homogenate is then centrifuged at 600 X g., (2700 rpm in a Beckman JA-20 rotor) for 10 min. The supernate is saved and the pellet is resuspended in sucrose and centrifuged a t 600 X g.,for 10 min. The suoernates from the two snins are oooled and centrifuged ~~~~
-...,...
~
~.~
--.-,--,~~
~~~
~~
then centrifuged at 20,000 X g, (16,000 rpm) for 6gmin and the pellet is resuspended in sucrose. Fifteen grams of starting medulla can be resuspended in 15 mlaf sucrose. Theenzymecan bestored at -20°C or lower. Procedure for Kinetics Prepare five assay solutions in small test tubes as follows: Volume 0.1 mi 10.2 mi 0.6 ml 0.3 ml 1 pi 1.79ml
Solution 5 mM Nadh 22.5 mMPEP ATP mixS 0.1 M lmidezale (pH 7.2 at 2S°C) F'yruvate Kinase, 3000 ulmi Lactic Dehydrogenase. 8000 ulml 0.1 MEDTA (pH 7.0) Distilled HzO
These solutions should be mixed well and kept in a test tube rack at room temperature until use. If the speetrophotometers are not thennostated, it will be important in this experiment to carefully measure the ambient temperature in the spectrophotometer cell compartment and to preadjust the assay solutions to this temperature as carefully as possible. Once this has been done, set the spectrophotometer wavelength to 340 nm
1
0
~
" ' 30
~
'
'
60
time
' 90
'
'
' 120
'
~
(set)
Figue 4. Two typical first-order inhibition plots obtained as desdbed in the text. M(A) and 3.6 X lo-' M (Bl. ouabain concentrations were 1.4 X
and place one of the assay solutions in the compartment and measure the absorbance for 10 min to get an accurate measure of baseline drift. Then add 4 rcl of the kidnev micrasomes. Mia the solution by covering theeuvette kith Parafilm and invertingseveral times and record A& for 3-5 minor until the absorbance reaches 0.5 A, whichever occurs first. Add 30 pl of 1mM ouabain solution (CAUTION-ouahain is anextremely toxic heart and nervoussystem poison!), mix and record A310 until the plot has been linear for 10 min. Repeat the procedure with the other assay tubes, adding (in this order) 20,15,10, and 4 pl of the ouabain solution to the various assay tubes. In these latter assays the auabain should be added quite early, so that the plots can reach linearity before all the NADH is oxidized. Particular care should be taken that the olots are ahsolutelv linear before ending a particular run. Sighting along the plot with the eye close to the chart paper will help to distinguish linearity and nearlinearity. Data Analysis An example of t h e t y p e of d a t a one should obtain is shown in Figure 3. Values which will b e referred t o as AA are ohtained from the plots between points a and b by extrapolating 5 ATP mix, prepared in advance, cvnsists oE 15 mM ATP, 15 mM MgCL, 500 mM NaCI, and 50 mM KCI. 6 Fieser, L., and Fieser, M., "Steroids," Reinhold, New Yark, 1959, p 727-809. Jargensen, P. L., "Methods in Enzymology," 32B. 277 (1974). SJencks, W. P., "Catalysis in Chemistry and Enzymology," MeGraw-Hill, 1969.
Volume 55, Number 2,February 1978 / 123
versus time on semilog graph paper and determines a halftime, t ~ / z , and a rate constant, k.,, in the usual way (Fig. 4 and footnote 8). The k,,values obtained in this way are then plotted versus ouabain concentration (Fig. 5). The slope of this plot is ki, the second-order rate constant for ouabain inhihition. Discussion
[OUABA IN]
(n lo6 M )
Figure 5. The dependence of k,, on ouabain concentration. The slope of this line is the true second-der rate constant for ouabain inhibition of the ATPase.
the straight line obtained beyond point b back in time as shown. The heights AA are then measured as shown between a and b. Using any starting point after a, one then plots AA
124 1 Journal of ChemicalEducation
This experiment not only introduces the student to an interesting and important biochemical system, but also demonstrates the use of a coupled enzyme assay and some important, practical kinetics. The student should be encouraged to think about the mechanism of action of ouahain (which binds only to the outside surface of the plasma membrane). I t is interesting to note that this mechanism is not at all understood a t the present time.4 Many variations of this experiment can be explored by the student, possibly as special projects. For example, the second-order inhibition constant, ki, is quite sensitive to sodium and potassium concentrations. On the other hand, the student may wish to use any of several published procedures for purifying the ATPase7 and determine whether the sensitivity of the ~urifiedenzvme differs from that of the microsomal prepnration used sburr. The coupling rnzynlcs, pyruvate kinnsr and lactic dehvdnzensse arr not inhibited by ouabain. but the student may wish to verify this in separate experi: ments.
