530

SPECIALIZED TECHNIQUES

[48]

without significantly affecting the observed values of 3zPt bound to F1). A concentrated protein sample emerged from the column followed by a volume of buffer which may have been held within the gel beads in the lower portion of the column. It would appear from these considerations that the centrifuge column differs in important ways from the usual type of Sephadex gel chromatography. The success of the centrifuge column procedure in measuring azPi binding by F1 may be in part related to the relatively slow rate at which azpi dissociates from the Fx-Pi complex. 5 In general, it may be anticipated that this binding procedure will underestimate ligand binding if the "off" rate of the ligand from the protein is rapid in relation to the initial penetration of protein into the column. The centrifuge column procedure readily lends itself to two additional uses. First, it may be used to desalt small samples of protein under exactly the same conditions described in steps 1 and 2. The Sephadex to be used is preequilibrated with any buffer of interest. Second, the procedure may be used to concentrate small samples of protein. Concentration results when the centrifugation time of step 2 is reduced to about 30 sec. Alternatively, it is useful to carry out centrifugation in step 1 at high speed (1800 rpm) for 2 min and, after applying the sample, to carry out centrifugation in step 2 at 450 to 900 rpm for 30 to 60 sec. Optimum conditions for any given protein must be determined empirically. Concentrations of fiveto tenfold may be achieved but losses of protein of 30% or more should be expected.

[48] C o n t i n u o u s M e a s u r e m e n t o f A d e n o s i n e T r i p h o s p h a t e with Firefly Luciferase Luminescence

By JOHN J. LEMASTERSand CHARLES R. HACKENBROCK Firefly luciferase has long been used for accurate and sensitive determinations of adenosine triphosphate (ATP) in biological samples. 1 Recently, we have adapted the luminescence technique to continuous measurement of ATP concentration in metabolically active suspensions of mitochondria and submitochondrial particles. 2,3 This method, when corni B. L. Strehler, Methods Biochem. Anal. 16, 99 (1968). 2 j. j. Lemasters and C. R. Hackenbrock, Biochem. Biophys. Res. Commun. 55, 1262 (1973). a j. j. Lemasters and C. R. Hackenbrock, Ear. J. Biochem. 67, 1 (1976).

METHODS IN ENZYMOLOGY, VOL. LVI

Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181956-6

[48]

ATP

MEASUREMENT W I T H FIREFLY LUCIFERASE

531

bined with oxygen electrode technology,4 permits direct and simultaneous observation of mitochondrial oxidative and phosphorylative activities. Firefly luciferase catalyzes the following reactions in the production of luminescence.5 Mg~+

E + LH2 + A T P ~ E'LH2AMP + PPi E-LH2AMP + 02 -~ oxyluciferin+ AMP + COz + hv

(1) (2)

The initial activation step (reaction 1) is the formation of enzyme-bound luciferyl adenylate (E-LH2AMP) and pyrophosphate from ATP and firefly luciferin (LH2). Divalent cation is an absolute requirement. The enzyme complex subsequently reacts with molecular oxygen (reaction 2) to produce a quantum of light, AMP, CO2, and the decarboxyketo derivative of luciferin, oxyluciferin. The latter compound is a potent inhibitor of luminescence and is responsible for product inhibition of the reaction. Materials and Instrumentation Purified luciferase and synthetic luciferin are obtained premixed from DuPont Corp., (Instrument Division, Wilmington, Delaware) as vials of lyophilized powder. The contents of a vial are dissolved in 3 ml of buffer (0.1 M Tris-HEPES or KH~PO4-K2HPO4, pH 7.4) to yield a solution containing approximately 710/zM luciferin and 1000 U/ml luciferase. A more precise determination of luciferin may be made by measuring absorbance at 327 nm where the extinction coefficient is 18,000. ~ We find that 1 unit of enzyme as supplied by DuPont equals 1.6-1.9/zg of protein, r The vials of powdered luciferase-luciferin must be stored frozen, but once reconstituted in buffer the enzyme-substrate solution should be stored at 00-4 ° and shielded from light. At this temperature the enzyme is stable for several weeks. Luciferase and luciferin may also be prepared as an aqueous extract of firefly lanterns. Approximately 100 mg of dessicated firefly tails (Sigma Chemical Co., St. Louis, Missouri) are homogenized in 10 ml of 0.1 M NaH2PO4-NazHPO4 buffer, pH 7.4, 0°. The homogenate is filtered through Whatman No. 1 paper and centrifuged at 12,000g for 15 min. The yellow layer that forms on top of the supernatant is suctioned off, and the remaining supernatant is stored at 0° until use. Aqueous stock solutions of adenine nucleotides (AMP, ADP, and ATP) are made up as 0.1 M and adjusted to pH 7.4. Their concentrations are 4 R. W. Estabrook, Vol. 10, p. 41. 5 M. DeLuca, Adv. Enzymol. 44, 37 (1976).

6 R. A. Morton, T. A. Hopkins, and H. H. Seliger,Biochemistly 8, 1598(1%9). r O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall,J. Biol. Chem. 193, 265 (1951).

532

SPECIALIZED TECHNIQUES

[48]

off

Light l

Off

Inten$ityJ

°° j off

1

FIG. 1. Luminescence response to constant, continuous ATP addition. ATP is added at the rate indicated on each tracing to a final concentration of 120 ~M. Reaction medium is 100 mM sucrose, 5 mM sodium succinate, 5 mM MgClz, 1 mg/ml bovine serum albumin, aqueous extract of 0.5 mg/ml firefly lanterns, 5 mM KH2PO4-KzHPO4 buffer, 5 mM NaH~PO4Na~HPO4 buffer, pH 7.4, 23 °.

determined by absorbance at 259 nm using an extinction coefficient of 15,400. Rat liver mitochondria are prepared by differential centrifugation in 0.25 M sucrose to a stock concentration of 50-100 mg protein per milliliter, s Inner membrane vesicles are prepared by sonication of the mitoplast (inner membrane-matrix) fraction" to a stock concentration of 20-50 mg protein per milliliter. ~° We use the photometer of a Brice-Phoenix Light Scattering Photometer (Virtis Co., Gardiner, New York) to measure luminescence. However, the photometers of various spectrophotometers or fluorometers may be adapted to this purpose. A magnetic stirrer is necessary to assure rapid, W. C. Schneider, J. Biol. Chem. 176, 259 (1948). 9 C. Schnaitman and J. W. Greenawalt, J. Cell Biol. 38, 158 (1968). lo C. R. Hackenbrock and K. Hammon, J. Bh~l. Chem. 250, 9185 (1975).

[48]

ATP

MEASUREMENT WITH FIREFLY LUCIFERASE

533

even mixing of added reactants. Light-tight additions to the reaction medium are made with microliter syringes through a rubber stopper fitted in the lid of the photometer compartment. The reaction medium is contained in a glass, water-jacketed cell (Gilson Medical Electronics, Model OX705, Middleton, Wisconsin) fitted with an oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, Ohio). 4 A syringe pump (Harvard Apparatus Co., Millis, Massachusetts) is used to add reagents continuously to the reaction medium. Rapid mixing experiments are carried out in an Aminco-Morrow stopped flow apparatus. Luminescence during these experiments is detected by the photometer of an Aminco-Chance spectrophotometer (American Instrument Co., Silver Springs, Maryland). Computational chores are considerably eased by the use of a programtriable desk top calculator. Qualitative Aspects The response of luciferase to constant addition of ATP via syringe pump is illustrated in Fig. 1. Luminescence increases with increasing ATP at a rate which is roughly proportional to the rate of ATP addition. Although ATP increases linearly in these experiments, luminescence does not, and a progressive decline in luminescence begins as soon as ATP addition is terminated. Product inhibition of the luminescence reaction is responsible for these effects. Because of product inhibition there is no constant proportionality between ATP concentration and luminescence. Despite the lack of an exact proportionality between ATP and luminescence during the continuous recording, qualitative information concerning mitochondrial oxidative phosphorylation can readily be obtained. As an example, Fig. 2 illustrates the effects of calcium ion accumulation on oxidative phosphorylation. Mitochondria in these experiments are incubated in the presence of phosphate, respiratory substrate, and luciferase. This alone produces a small amount of luminescence due to the production of endogenous ATP. Active oxidative phosphorylation is then initiated by the addition of external adenine nucleotide as ADP and AMP. A rapidly rising luminescence signal demonstrates ATP synthesis. When the added adenine nucleotide is completely consumed by conversion to ATP, the luminescence signal stops rising and begins to decline. It is apparent that calcium addition to these respiring mitochondria interrupts (Fig. 2c and d) and even reverses (Fig. 2a and e) ATP synthesis by oxidative phosphorylation. This interruption is short-lived, and once the calcium is actively taken up by the mitochondria, ATP synthesis begins again. A control experiment (Fig. 20 demonstrates that calcium does not affect the luminescence reaction itself.

534

SPECIALIZED TECHNIQUES

[48]

Co2*I

m,

d~d

(0) ~

~ /

(d)

Ca2*0~F mito

,.,°i,o

)/

If

A ")

mito

Light Intensity

(f)

/

/ z~tM/sec

-

',?" or/

2 rnin FIG. 2. Luminescence tracings of calcium ion interruption of oxidative phosphorylation. Reaction medium is 106 mM sucrose, 5 mM sodium succinate, 5 mM MgC12, 1 mg/ml bovine serum albumin, 1 mg protein per milliliter mitochondria [except (f)], aqueous extract of 0.5 mg/ml firefly lanterns, 5 mM NaH2PO,-Na~HPO4 buffer, 5 mM KH2PO,-K2HPO, buffer, pH 7.4, 23°. Ca ~+ (when added) is 200 t~M as CaCIz. Adenine nucleotide (AdN) (when added) is composed of 106/~M AMP and 30/~M ADP. The rate of ATP infusion in (f) is 2/.tM/sec.

Quantitation of ATP Concentration Quantitation of ATP concentration during ongoing luminescence is based upon an understanding of the kinetic behavior of the luciferase enzyme system during product inhibition. Addition of an aliquot of ATP, [S], to a mixture of luciferase, luciferin, and magnesium causes a rapid rise in light production known as the flash height (Fig. 3). The flash height displays classical Michaelis-Menten kinetics with respect to either ATP or luciferin as substrate. The rapid exponential decay subsequent to the flash height is not due to substrate consumption. Rather, it is due to specific inhibition by oxyluciferin, an end product of reaction (2). Light production remains sensitive to substrate concentration and increases sharply after the addition of a second aliquot of ATE [.t"]. Product inhibition during luciferase luminescence obeys noncompetitive inhibitory kinetics. 2"11Therefore, ATP concentration, [S], in the reac11 j. j. Lemasters and C. R. Hackenbrock,Biochemistry 16, 445 (1977).

[48]

A T P MEASUREMENT WITH FIREFLY LUC1FERASE

535

Is]

L,o., t

[]

Intensity

"1"

Vs+x

I rain t

FIG. 3. Luminescence response of luciferase-luciferin to ATP. 239/xM ATP is added at [S] and again 4 min later at [X]. Reaction medium is 10 U/ml luciferase, 7.1/.tM luciferin, 5 mM MgCI2, 70 mM sucrose, 220 mM mannitol, 2 mM HEPES, 7.5 mM K2HPO4, 5 mM sodium succinate, 0.5 mM EDTA, 6.25/zM rotenone, 1 mg/ml bovine serum albumin, pH 7.4, 23°. From Lemasters and Hackenbrock. 2

tion medium may be determined after addition of ATP standard, IX], using the equation

[X])2 - (KSl)+ IX])2 )~12 (3) IS] _ -(Ks 2+ [X]) + ~{VJVs+x(Ks4-(V--~/V----~+~where V~ and Vs+x are the respective reaction velocities just before and just after the addition of [X]. The only constant in Eq. (3) is K~, the Michaelis constant, which must be determined independently. K~ is readily determined following two successive additions of ATP to an unreacted luciferase-luciferin solution as in Fig. 3. K8 is then given by the expression [S](1 - Vs/V,,+~)

K~ = •

v~/v~+x-

[s]/([s] +

(4)

ix])

Values forK~ based on Eq. (4) agree closely with those derived from flash height determinations. For greatest precision, [S] and [X] should be close to the anticipated value of K~ (200-300 /zM). Many variables affect K~. Therefore, K~ should be estimated in a complete reaction medium including mitochondrial protein. Oligomycin (1 /xg/ml) is employed to inhibit mitochondrial ATPase during the K~ determination. When Ks is determined, Vmax, the maximum velocity of the reaction, may also be calculated: Vmax -----Vo(Kfl[S] + 1) where Vo is the flash height.

(5)

536

SPECIALIZED TECHNIQUES

[48] 48 pM

ATP

ATP

LIGHT INTENSITY

, Li;ht Intensity

~

/

/ / AdN/ ~ ' M , t ~ '

I

i Oxygen: ...... .

'

I

,Y

,Y

2 min

169 pM

'

JlO[pM

I

0

100

2OO

34 p a t / ~

-

"n

2mi

300

AdN added |pM)

FIG. 4. Oxidative phosphorylation of AMP and ADP by intact mitochondria. Reaction medium is 155 mM sucrose, 5rmM MgCI2, 5 mM sodium succinate, 5 p.M rotenone, 10 U/ml luciferase, 7.1 /zM luciferin, I mg protein per milliliter mitochondria, I1 mM KH2PO4K2HPO4 buffer, pH 7.4, 23°. In (A) mitochondria, adenine nucleotide (AdN) composed of 113/~M AMP and 14/~M ADP, and 38 # M ATP are added where indicated. The solid line is luminescence and the dotted line is oxygen concentration. In (B) AdN and ATP are added in the amounts indicated. AdN is composed of 89% AMP and 11% ADP. In (C) AdN added to mitochondrial suspensions in (B) is plotted versus net ATP synthesis. ATP is estimated b'y Eq. (3) after addition of ATP standard. Ks for luminescence is 268/zM.

Under some experimental circumstances, ATP concentration IS] is known and the ATP equivalence [X] of some incremental change in luminescence (V~+x - V J is desired. This is calculated by the following equation: IX] = [S]

V,/V~+x(K~ + [S]) - [S] - 1

(6)

The above expressions may be employed to measure ATP concentration in suspensions of mitochondria catalyzing oxidative phosphorylation (Fig. 4). Isolated mitochondria contain large amounts of adenylate kinase

[48]

ATP

MEASUREMENT

WITH

FIREFLY

LUC1FERASE

537

which reversibly convert ADP to ATP and AMP. In order that luminescence reflect only ATP synthesis by oxidative phosphorylation, adenine nucleotide is added as AMP and ADP in concentrations that are in adenylate kinase equilibrium with ATP already present. Since the equilibrium constant for adenylate kinase is approximately 1,12 concentrations of ADP and AMP are selected such that [ADP]2/[ATP] = [ATP]~endogenous~ Endogenous ATP concentration is determined in a parallel experiment. AMP and ADP added this way to mitochondria in the presence of respiratory substrate and phosphate cause a smooth progressive increase in luminescence which coincides with an increase in oxygen consumption (Fig. 4A). Mitochondria under these conditions completely convert adenine nucleotide to ATE Figure 4C shows the equality of added adenine nucleotide concentration with ATP concentration calculated by Eq. (3) at the completion of oxidative phosphorylation. Above 200 g M added adenine nucleotide, the calculated ATP values underestimate the expected ones. These poor estimates occur when the luminescence response to ATP standard addition is altered, as evidenced by a blunt instead of a sharp peak after ATP addition (top two traces of Fig. 4B). Empirically, this phenomenon is related to the age of the luciferase preparation and its exposure to light. When light shielded luciferase solutions less than 1 month old are used, ATP can be accurately measured at concentrations in excess of 200 gM. In any case, the shape of the curve after ATP standard addition is an indication as to the accuracy with which Eq. (3) will measure ATP concentration. Rate of Change of ATP Concentration If a luciferase-luciferin medium is unreacted, the initial rate of ATP generation, d[S]/dt, can be calculated from the initial rate of luminescence increase, dV/dt. The Michaelis-Menten equation is differentiated to obtain d[S] dV dt = dt

Km'Vmax (Vma x -

V ) '~

(7)

where V is the velocity of the reaction. When V = O, the equation reduces to diS]

d~-

dV

K ,,,

dt Vmax

(8)

12 L. Noda, in "'The E n z y m e s " (P. D. Boyer, ed.), 3rd ed., Vol. 8. p. 279. Academic Press. New York, 1973.

538

SPECIALIZED TECHNIQUES

(A)

[48]

19.4IJMATP

i50 ~M

02

"'"'"

Intensity

"'"'"'"'""'-...........

Vesicles ;

i / /

I

. I min

I

I

I

(el

c

1 ,0 F

J

--

E::L

0.5 I

00

-

0.05 II[AEP] (IJ.M "I )

-

0.10

FIG. 5. Oxidative phosphorylation by sonicated inner membrane vesicles. (A) The solid line is luminescence, and the dotted line is oxygen concentration. (B) The initial rates of ATP formation as estimated by Eq. (8) are plotted in double reciprocal fashion versus added ADP. Ks for ADP is 13.1 - 2.2 (S.E.)/~M and Vm~xis 1.57 -+ 0.06 (S.E.)/zmole ATP per second per gram of protein. Reaction medium is as Fig. 4 with the exception that mitochondria are replaced by vesicles, 0.5 mg protein per milliliter.

In the event that the luciferase and luciferin mixture has already commenced reacting, d[S]/dt may still be determined with Eq. (7), provided ATP concentration [S] is known and dV/dt is large in comparison to product inhibition-mediated decline in luminescence. Vmax must be redetermined and substituted into Eq. (7). Vmax = V ( K s "k IS]) IS]

(9)

[48]

ATP

MEASUREMENT W I T H FIREFLY LUCIFERASE

--t

539

48.5jzM

50 j~atm

[Oxygen

~, \ \ \

"

%

1

I Light

Intensity

195 ~ M ADP

mito 4

2 rain

FlG. 6. ATP formation during oxidative phosphorylation of ADP by intact mitochondria. Reaction medium is as Fig. 4. From Lemasters and Hackenbrock. a

Figure 5 shows oxidative phosphorylation by sonicated inner membrane vesicles. This membrane fraction lacks adenylate kinase, and ADP alone is used to initiate oxidative phosphorylation. The addition of ADP produces a rapid increase in light production which lasts over 1 min and ends with a return of luminescence decay. ATP addition and the appropriate calculation show that 64% of the ADP originally added has been phosphorylated to ATP. In this instance ATP formation cannot be inferred from the oxygen electrode recording, since ADP fails to stimulate respiration. Since there is no endogenous ATP, the initial rate of ATP synthesis can be calculated with Eq. (8) from the slope of the luminescence recording, dV/dt, immediately following ADP addition. A double reciprocal plot of ADP concentration and the rate of ATP synthesis is linear with a K , for

540

SPECIALIZED TECHNIQUES

[48]

ADP of 13.1 ~M and a V m a x of 1.57 ~moles ATP per second per gram of protein. Equations (7) and (9) must be applied in more complicated circumstances. Figure 6 illustrates their use. ADP is added to mitochondria incubated as before. In order to determine the rate of ATP production, endogenous ATP concentration [S] must be known just prior to ADP addition and is determined in a parallel experiment. With this value and V, which is the rate of the reaction just prior to ADP addition, Vmax is calculated with Eq. (9). Vma×as given by Eq. (9) represents a product inhibited maximum velocity of the reaction and decreases progressively as luminescence continues. The initial rate of ATP production d[S]Mt is then calculated with Eq. (7) from the slope of the luminescence recording dV/dt immediately following ADP addition. V in Eq. (7) is the luminescence just prior to ADP addition, and Vmax is given by Eq. (9). In Fig. 4, these calculations give an initial rate of ATP formation of 8.3 /.tmoles/sec/g protein. The overall rate of oxidative phosphorylation is 2.8 p~moles ATP/ sec/g protein, and the difference between these two values represents the initial rate of adenylate kinase-mediated ATP formation.

Rapid Kinetics The continuous nature of the luminescence signal lends itself to examination of relatively rapid changes in ATP concentration when conventional sampling techniques would be inadequate. The time resolution of the luminescence method is limited by the kinetic characteristics of the light reaction itself (Fig. 7). After rapid mixing of ATP with luciferase and luciferin, light production begins after a lag interval of about 40 msec during which virtually no light is produced. Half-maximal luminescence requires about 190 msec. Both the lag and half-times are invariant with ATP concentration. 3,5 Figure 8 illustrates the use of luminescence to observe the rapid kinetics of ATP synthesis. Anaerobic, sonicated inner membrane vesicles are rapidly mixed with aerobic luciferase and luciferin in the presence of ADP, respiratory substrate, and phosphate. The resulting luminescence signal is linear with a lag time of 40-60 msec (Fig. 8A). This lag time approximates that of the luminescence reaction time itself and indicates that ATP synthesis must begin within 20 msec or less of mixing. The ensuing linearity of the luminescence signal indicates that a maximal rate of ATP synthesis is rapidly achieved and then maintained. Since ATP concentration is low in these experiments (< 5 /~M), luminescence is linearly proportional to ATP concentration, and product inhibition is negligible over the first 5 sec of luminescence.

[48]

ATP

MEASUREMENT W I T H FIREFLY LUCIFERASE

541

! 0

5OO

1000

Time (msec)

FIG. 7. Rapid reaction kinetics of luciferase luminescence. ATP is rapidly mixed with

luciferase and luciferinin a stopped-flowapparatus. Mixedreaction mediumis 5 I.LMATP, 125 U/ml luciferase, 89 /~M luciferin, 170 mM sucrose, 5 mM MgSO4, 5 mM sodium succinate, 5 #M rotenone, 10mM KH~PO4-K2HPO4buffer,25 mM Tris-HEPES buffer,pH 7.4, 23°. From Lemasters and Hackenbrock2 In Fig. 8B oxygen is pulsed to antimycin tetramethylphenylenediamine-treated vesicles. Within a few milliseconds such mixing produces oxidation of respiratory components on the oxygen side of the antimycin-inhibited site. Following this fast flow of reducing equivalents is a considerably slower steady-state flow whose rate is determined by the tetramethylphenylenediamine-mediatedbypass of the antimycin inhibited site. This fast then slow passage of reducing equivalents is reflected in the luminescence recording. An initial burst of luminescence is followed by a steady state increase similar to Fig. 8A. By subtracting the linear portion from the overall recording, we can generate a derived signal which represents an initial burst of ATP synthesis (Fig. 8C). The magnitude of the derived signal is equivalent to 0.08 /~moles ATP/g protein, and its half-maximal rise time is 300 msec. Since the half-maximal rise time of the luminescence reaction itself is 190 msec, the difference between these two values, approximately 100 msec, may be considered an order of magnitude value for the half-time of the initial burst of ATP synthesis. Precautions The selection of lucifefin and luciferase concentrations depends largely on the sensitivity required. In general, larger concentrations provide

542

SPECIALIZED TECHNIQUES

[48]

2"""

0 320

E

24.O 160 80

8

0

0

1

2

3

4

5

I00 '

8 g

I

'

8O 60 40 2O

O0

I I

2

Time (sec}

FIG. 8. Rapid kinetics of oxygen-pulsed ATP synthesis by sonicated inner membrane vesicles. Luminescence is measured vertically in units equivalent to 1 nmole ATP/g protein. In (A) oxygen is pulsed to reduced vesicles with succinate as respiratory substrate. The recording is the result of mixing an anaerobic suspension containing 170 mM sucrose, 5 mM MgSO,, 5 mM sodium succinate, 5/zM rotenone, 97.4/xM ADP, 4 nag protein/ml vesicles, 10 mM KH2PO4-K~HPO4 buffer, 25 mM Tris-HEPES buffer, pH 7.4, with equal parts of an aerobic solution containing 170 mM sucrose, 5 mM MgSO4, 5 mM sodium succinate, 5/zM rotenone, 250 U/ml luciferase, 177.5/~M luciferin, 10 mM KH2PO4-K2HPO4 buffer, 25 mM Tris-HEPES buffer, pH 7.4, 23°. In (B) oxygen is pulsed to antimycin-inhibited and tetramethylphenylenediamine-bypassed sonicated inner membrane vesicles. Antimycin blocks site II of the respiratory chain. Tetramethylphenylenediamine is added in sufficient quantity to restore respiration to 20% of its original rate by bypassing the antimycininhibited site. The recording is the result of mixing an anaerobic suspension containing 162.5 mM sucrose, 5 mM MgSO4, 5 mM sodium succinate, 5/xM tetramethylphenylenediamine, 0.25/zg/ml antimycin, 97.4 tzM ADP, 2.5 mg protein/ml vesicles, 10 mM KH~PO4-KzHPO4 buffer, 25 mM Tris-HEPES buffer, pH 7.4, with equal parts of an aerobic solution containing 162.5 mM sucrose, 5 mM sodium succinate, 5 mM MgSO4, 0.25/~g/ml antimycin, 5 / z M tetramethyipheylenediamine, 250 U/ml luciferase, 177.5 /zM luciferin, 10 mM KH2PO4K2HPO4 buffer, 25 mM Tris-HEPES buffer, pH 7.4, 23°. (C) is a derived plot showing the luminescence produced by the initial burst of ATP synthesis in (B). From Lemasters and Hackenbrock. 3

[48]

A T P MEASUREMENT WITH FIREFLY LUCIFERASE

543

greater sensitivity but also a greater amount of product inhibition which may in turn obscure variations in ATP concentration. At lower concentrations sensitivity is decreased but product inhibition proceeds more slowly. It is best to employ luciferase and luciferin concentrations that are as low as adequate sensitivity allows. Although a crude aqueous extract of firefly tails may be employed, it is preferable to use purified luciferase, since the crude extract contains contaminating adenylate kinase, pyrophosphatase, and apyrase. 1 Moreover, we find that the aqueous extract decreases mitochondrial A T P : O and respiratory control ratios by 20 to 50%. This latter effect can, however, be reversed by 1 mg/ml bovine serum albumin. Luminescence decreases in solutions of increasing ionic strength. The reaction is also inhibited by several monovalent anions.13 Since this inhibition cannot be prevented or reversed, it is simply tolerated, and moderate amounts of salt and chloride anion are included in the reaction medium in order to prevent any small change in ion concentration from being significant. Similarly, oxygen concentration must be maintained at levels which saturate the enzyme (> 50 /zM Oz), so that changes in oxygen content will not occur which will produce significant changes in luminescence. Several compounds produce specific and potent inhibition of luminescence, including anesthetics 14,15 and the fluorescent probes, 1,5anilinonaphthalenesulfonate (ANS) and 2,6-toluidinonaphthalenesulfonate (TNS).16 Additionally, we have noted that uncouplers of oxidative phosphorylation are inhibitory at concentrations which uncouple mitochondria. For the uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), this inhibition appears to be competitive with respect to luciferin (J. J. Lemasters, unpublished observations). In a continuous luminescence recording this type of specific inhibition of luminescence is usually obvious, since inhibitor addition results in a rapid decrease of luminescence that cannot be accounted for by enzymatic hydrolysis of ATP. Luciferase is extremely specific for ATP as substrate in the luminescence reaction. No other naturally occurring nucleoside triphosphate has significant activity. AMP and ADP at high concentrations can act as competitive inhibitors of ATP in the luminescence reaction, 17 but we have not encountered this as a problem at AMP and ADP concentrations usually employed to study oxidative phosphorylation. The ease and simple in13 j. L. Denburg and W. D. McElroy, Arch. Biochem. Biophys. 141,668 (1970). t4 I. Ueda and H. Kamaya, Anesthesiology 38, 425 (1973). is I. Ueda, H. Kamaya, and H. Eyring, Proc. Natl. Acad. Sci. U.S.A. 73, 481 (1976). 1~ M. Deluca, Biochemistry 8, 160 (1969). ~r R. T. Lee, J. L. Denburg, and W. D. McEIroy, Arch. Biochem. Biophys. 141, 38 (1970).

544

SPECIALIZED TECHNIQUES

[49]

strumental requirements of the luminescence assay lend themselves to routine use and to application in other biological systems where ATP is of importance.

[49] M e a s u r e m e n t o f M a t r i x E n z y m e A c t i v i t y in S i t u in Isolated Mitochondria Made Permeable with Toluene By M. A. MATLm, W. A. SHANNOr~, JR., and P. A. SRERE

It has been long appreciated that studies on isolated enzymes cannot accurately reflect their precise in vivo behavior. As our knowledge concerning the concentrations of metabolic intermediates has increased, it has become possible to show that regulatory data obtained from studies on enzymes in vitro do not agree with the known metabolic behavior of enzymes in tissues. In an attempt to explain the apparent discrepancies, the existence of microenvironments within cells has been postulated.~-3 The major difficulty with testing such hypotheses in animal cells stems from the impermeability of cells to most cofactors and substrates so that behavior of enzymes in vivo cannot be tested. Toluene has previously been employed to make isolated microbial cells permeable to normally nonpenetrating substrates and cofactors. Since these cells have rigid cell walls, no problem was encountered in relation to cell breakage. In a similar manner, yeast cells, which also possess a rigid wall, have been made permeable to metabolic intermediates by toluene treatment, and the control properties of several enzymes were studied, 4,s In addition, hepatocytes have been made permeable to tRNA by the use of toluene. 6 In this Chapter we present a method of making rat mitochondria permeable to substrates and stabilizing them so that the individual properties of Krebs cycle enzymes can be studied. Reagents and Solutions Isolation Medium A

Sucrose, 70 mM o-Mannitol, 220 mM i p. A. Srere, Proc. Natl. Acad. Sci. U.S.A. 70, 2534 (1973). 2 p. A. Srere and K. Mosbach, Annu. Rev. Microbiol. 28, 61 (1974). P. A. Srere, Life Sci. 15, 1695 (1975). 4 R. E. Reeves and A. Sols, Biochem. Biophys. Res. Commun. 50, 459 (1973). 5 p. D. J. Weitzman and J. K. H e w s o n , FEBS Lett. 36, 227 (1973). GR. H. Hilderman and M. P. Deutscher, J. Biol. Chem. 249, 5346 (1974).

METHODS IN ENZYMOLOGY.VOL. LVI

Copyright © 1979by Academic Press Inc. All rightsof reproduction in any form reserved. ISBN 0-12-181956-6

Continuous measurement of adenosine triphosphate with firefly luciferase luminescence.

530 SPECIALIZED TECHNIQUES [48] without significantly affecting the observed values of 3zPt bound to F1). A concentrated protein sample emerged fro...
873KB Sizes 0 Downloads 0 Views