A microassay for ATPase

ANALYTICAL

BIOCHEMISTRY

169,3

12-3 18 (1988)

A Microassay RICHARD

D. HENKEL.*

for ATPase

JOHN L. VANDEBERG,*

Received

AND RICHARD

A.

WALSH*+

July 27. 1987

A newly developed microtechnique for quantitating activity ofmyosin ATPase (EC 3.6.1.32) is more sensitive and less time-consuming than existing spectrophotometric methods. Measurement of ATPase activity using the new method can be accomplished in a final volume of 0.25 ml, allowing the assay to be conducted in individual wells of 96-well microplates commonly used for the enzyme-linked immunosorbent assaq (ELBA). The microassay is performed by adding purified myosin to microplate wells followed by addition of ATP to initiate the enzymatic reaction. The reaction is subsequently terminated by addition of an acidic solution containing malachite green and ammonium molybdate. The level of inorganic phosphate produced by enzymatic hydrolysis of ATP is measured by scanning the microplates using a microELBA plate reader. An entire 96-well microplate can be scanned in less than 2 min. and data from the microassay can be transferred directly to a microprocessor for statistical analysis. The microassay is capable of detecting between 0.2 and 3 nmol of inorganic phosphate in a reaction volume of 50 ~1, and the ATPase activity of as little as IO ng of rat cardiac myosin can be measured. The increased sensitivity compared with that of other spectrophotometric assays and ease of performing the microassay enable a detailed analysis of the enzymatic properties of cardiac myosin to be conducted on large numbers of small tissue specimens. Several kinetic properties of rat cardiac myosin were determined using this technique. ‘K. 1988 Academic Press. Inc. KEY WORDS: ATPase: phosphatase: microassay: myosin: phosphate detection.

Force generation in skeletal and cardiac muscle during contraction is the result of the movement of interdigitating thick myosin and thin actin filaments. According to Huxley’s sliding filament model (1). myosin filaments slide along actin fibers via the action of flexible bridges that protrude from the myosin molecules and attach to regularly spaced sites on the actin filaments. After binding, these structures, referred to as myosin heads. swivel into a more conformationally stable configuration that results in the movement of myosin relative to the actin filament. The attachment, translocation, and detachment cycle is repeated many times during muscle contraction. The translocation of myosin along actin filaments is an energy dependent process that is driven by the hydrolysis of ATP at a catalytic site located in the myosin head (3). The velocity of

muscle contraction is directIy related to the ATPase activity of myosin (3). Variable contractility among muscles may be associated with differences in myosin ATPase activity and is characteristic of different muscle types. Fast-twitch skeletal muscle contains myosin that exhibits a high level of ATPase activity, whereas slow-twitch muscle hydrolyzes ATP at a much lower rate but more efficiently (4). Thus, myosin ATPase activity is a major determinant of muscle performance. The standard spectrophotometric techniques used to measure myosin ATPase activity involve cumbersome procedures in which inorganic phosphate released by ATP hydrolysis is measured after termination of’ the reaction with perchloric or trichloroacetic acid. This approach requires the removal of protein by acid precipitation and centrit’u-

A MICROASSAY

gation, which have been reported to produce erroneous results (5). In this manuscript, we describe a microassay that is more sensitive and less time-consuming than existing spectrophotometric methods. Optimal ranges for the reaction time, myosin concentrations, and substrate concentrations are presented for rat cardiac myosin using this assay.

FOR

ATPASE

313

was allowed to stabilize for 5 min before it was scanned with the microELISA’ plate reader. By staggering the initiation and termination of the ATPase reaction column by column, a uniform enzyme reaction time for all 96 wells on the microplate was obtained. ncterr,2irlLtfio)l

c!f’ inorganic~ phosphutc~.

Levels of inorganic phosphate were determined using the method of Fiske and Subbarow (9). Briefly, the Fiske-Subbarow MATERIALS AND METHODS method was performed by mixing 280 ~1ofa Pur$cation c?f’myosin. Cardiac myosin sample containing inorganic phosphate and was purified as described by Pope et al. (6) 7.5% perchloric acid with 357 ~1 of distilled from 0.5-g samples of ventricles from 12 water. 267 ~1 of 1%)ammonium molybdate age-matched adult male (13 weeks old) F, (Aldrich Chemical Co.), and 48.5 ~1 of a rehybrid rats produced by crossing ACP fe- ducing solution containing 0.35~, (w/v) Imales with KGH males. Purified myosin was amino-3-naphthol-4-sulfonic acid (ANSA), stored at -20°C in 5 ml of 300 mM KC1 and 14.61? (w/v) sodium bisulfite, and 0.5? 45%) (v/v) glycerol. (w/v) sodium sulfite (Fisher Scientific Co.). Protri~l c.or?~cJntrrrtion.The protein conThe formation of phosphomolybdic acid centrations of purified myosin samples were complexes and subsequent reduction with determined by the method of Lowry (7) ANSA was quantitated after a IO-min incuusing bovine serum albumin as a standard. A bation using a Beckman DU-40 spectrophoreagent blank containing KC1 and glycerol at tometer equipped with a Quant II linear fit the concentrations described above was in- program at 660 nm. Inorganic phosphate cluded as an indicator of background absor- standards were prepared as described by bance. Tietz ( 10) for comparison with samples. :I 7’Pa.w assays.Calcium activated myosin Inorganic phosphate levels were also deATPase assays were conducted at 25°C as termined using the method described b> described by Litten (8) with minor modificaChan ct (11.(5). In this assay, 0.5 ml ofsamtions. Briefly, the microassay was performed ples or standards was mixed with 2 ml of by diluting 10 to 325 ng of purified myosin malachite green reagent. This reagent is preto a final volume of 25 ~1 in a final concen- pared from stock solutions of ammonium tration of 20 tIIM CaC&, 100 mM Tris-HCl, molybdate (5.72%). w/v. in 6 N HCI). 1.32”; pH 7.6, and 660 mM KC1 to microplate (w/v) polyvinyl alcohol (Sigma Chemical wells. followed by addition of 25 ~1 of ATP. Co.). 0.08 13!“1~(w/v) malachite green (Sigma The enzyme reaction was initiated by adding Chemical Co. 1.and distilled water mixed at a the ATP to columns of eight wells at 5-s in- ratio of I : I :3:2, respectively. After the malatervals using an Octapette multichannel chite green reagent was added to samples and pipet (Costar) moving in a left to right orienstandards. the re1atiL.e absorbance of the tation across the plate. It was necessary to samples compared to a reagent blank was gently tap the microplate between successive measured at 630 nm using the spectrophoadditions of ATP or to mix the reagents with tometer described above. Inorganic phosa Minimix apparatus (Fisher Scientific). The phate levels were also measured on a microenzyme reaction was terminated by adding scale using 50 ~1 of sample and 300 PI of 200 ~1 of malachite green reagent to successive columns of wells at 5-s intervals using a ’ Abbreviations used: ELISA, enzyme-linked immumultichannel pipet in the same orientation nosorbent assay: ANSA. I-amino-2-naphthol-4-sulfonic used for initiation of the reaction. The color acid.

314

HENKEL.

VANDEBERG.

malachite green reagent in lmmulon 2 flat bottom microplates (Dynatech). The absorbance of samples in the microplates was measured using a Dynatech MR 580 microELISA plate reader using dual wavelength settings of 630 and 490 nm for test and reference filters. respectively. Inorganic phosphate standards for the microassay were prepared using 0- 13 nmol of phosphate per 50 ~1 diluted in ATPase buffer. Standards were mixed with 200 ~1 of malachite green reagent and incubated for 5 min along with microplates containing ATPase samples. The microELlSA plate reader was blanked against the standard lacking inorganic phosphate before the remaining standards and samples were scanned. This procedure was found to minimize variation between batches of ATPase samples. The extent of nonenzymatic hydrolysis of ATP by the acidic malachite green reagent was determined for each microplate. One well in each column of eight wells contained ATP diluted in ATPase buffer without myosin, providing an estimate of column to column variation in addition to measuring the lcvcl of background ATP hydrolysis. (‘~1~~f~~~~1~o11.s. The relative absorbance values for the inorganic phosphate standards and ATPase samples were transferred either manually or automatically via an RS-232-C interface to a microprocessor (Micro Decision. Morrow. Inc.). The data were processed and analyzed using the I-2-3 software program (Lotus Development Corp.). Seven absorbance values were collected for each myosin sample from which an average and a standard error of the mean were calculated. The average absorbance of noncnzymatic ATP hydrolysis for each microplate was subtracted from the mean of each myosin sample on the same plate. The resulting net absorbances were transformed into values indicative of the level of inorganic phosphate produced by myosin ATPase activity by linear regression using the values obtained from the phosphate standards. The number of nanomoles of phosphate calculated for each sample was divided by the enzymatic reac-

AND

WALSH

tion time in minutes and further divided by the number of micrograms of myosin used in each assay. The resulting values were cxpressed as micromoles phosphate released per minute per milligram of myosin. Kinetic parameters of K,,, and I ‘,,,,, were determined using the theory of Lineweaver and Burk ( 18) by linear regression of reciprocal values of enzyme velocity versus substrate concentration at eight concentrations of ATP. Each data point in the regression represents the average of seven replicates for 12 rats (11 = 84). Both .v-and J‘ intercepts from the regression line were estimated from the best tit of the regression line. RESULTS

AND DISCUSSION

Several approaches are commonly used for quantitating the activity of ATPases. These methods generally involve the spectrophotometric measurement of inorganic phosphate (P,) released from ATP ( 1 1) or the measurement of radioactive phosphate released from [“PIATP ( 12). Spectrophotometric measurements off’, tend to be more widely used than radioactive methods although both approaches have shortcomings (5,13-15). In spectrophotometric assays, P, is quantitated indirectly by measurement of phosphomolybdic or molybdovanadophosphoric acid complexes (5.9,13-l 5. reviewed in 16). Two spectrophotometric assays described by Fiske and Subbarow (9) and by Chan (5) were compared with respect to sensitivity and stability of color formation through time as shown in Fig. 1. In these experiments. gravimetrically measured V, standards were assayed by both methods at several time points after addition of appropriate reagents for each assay. The sensitivity of the malachite green assay (Fig. IB) was approximately 10 times greater than that of the Fiske-Subbarow method, and the formation of phosphomolybdate-malachite green complexes was considerably more stable than the molybdenum blue product measured in the Fiske-Subbarow method. The measurement of P, in 96 well microplates with the mala-

A MICROASS.41’

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1000

1500

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readings of P, standards distributed in seven rows across I:! columns of wells on a microplate. Variation between assays was estimated by comparing values obtained for P, standards from four assays conducted over a I?-day period. The microplate assay produced linear results for P, concentrations from 0.40 up to 3.15 nmol P,/50 ~1. A coeRicicnt of determination (r’) of 0.999 I was obtained when the standards in the range of O-3.15 nmol PI/50 ~1 were compared with the means of the absorbance units for the column to column values. and an 1.’ of 0.9990 was observed for day to day variation with the same range of I’! concentrations. Lower 1.’ values of 0.8854 and 0.8403, respectively, \vert: obtained when O-l 3 nmol

7‘ABI.E VARIATIOU -

IN D~TECTIONOF~NORGANIC B\ THEMICROPIATFASSA'~

nmol Pi/ml FIN;. I. Inorganic phosphate detection using the Fiske-Subbarow and malachite green methods as describrd in the text. (A) The relationship between a series of inorganic phosphate standards and the absorbance at 660 nm produced by the Fiske-Subbarow method after incubations of IO. 20. 30. and 40 min (0. A, 0. and 0. respectively). (B) and (C) The absorbance values at 630 nm for phosphate standards using the malachite green assay after the sdme incubation periods described above. Note the IO-fold lower phosphate concentrations used in the malachkte green assay. Data for (B) were obtained with an assay volume of 2.5 ml and measured using a Beckman DU-40 spectrophotometer. whereas the data for(C) were measured in a final volume of0.15 ml using a microELISA spectrophotomcter in the total absorbance mode.

chite green assay (Fig. IC) closely paralleled the results obtained by the standard method of Chan in Fig. 1B. The microplate method, however, permits the screening of 96 samples in approximately 90 s. greatly reducing the time required to perform the assay. The extent of variability in the measurement of Pi with the microassay is indicated in Table 1. Variation within an assay was determined by comparing the absorbance

(‘olumn nmol

p,/SO ~1 0 0.30 0.80 I .hO 3.75 6.50 I 3 .OO

I

__-

to column (H = II)

14 93i 1x62 360 h7lc 1067 1277

i

X” 5 5 k 5 6 I I-1 i 36

PHOSPHATE -Day to day (If = 4) xiSh1 180 t 370 k 700 + IO97 t 1710 It

8 6 IO 6 II 43 44

:Vo!o. The average absorbance values and the standard errors of the means for inorganic phosphate standards were determined using the assay described in the text. The standards expressed as nmol PI/50 ~1 were prepared from a stock solution immediateI! prior to use in the assay and distributed into 96-well microplates. Malachite green reagent was subsequently added to the microplates and incubated for 5 min. The plates were spectrophotometrically scanned using a microELISA plate reader with a dual wavelength setting of630 and 490 nm for test and reference filters. respectively. Column to column variation was determined by comparing the absorbance values from 12 replicates for each phosphate standard distributed in rows across the microplate. Day to day variation was determined by comparing the ahsorbance values of phosphate standards from four experiments conducted over a 12-day period using a single batch of assay reagents. “Average absorbance units at 630 nm t I standard error i 1000.

316

HENKEL.

VANDEBERG.

pi/.50 ~1 was used in the analysis. We conclude that the measurement of Pi by the microplate method is highly reliable in the linear range of O-3.25 nmol Pi/50 ~1. Based on these observations, results from assays where the concentration of Pi in samples was unknown were accepted only if the absorbance values for the unknowns were within this range. After it was demonstrated that Pi could be measured rapidly and accurately in the 96well microplates, the feasibility of conducting ATPase assays and measuring the levels of Pi produced by the enzymatic reaction in the microplates was tested. A major advantage of using the phosphate detection method described by Chan (5) is that the direct measurement Of Pi in solutions containing as much as 50 pg of protein can be made by using polyvinyl alcohol as a stabilizing agent. Other methods for measurement of P, require deproteinization of the enzyme reaction mixture before addition of molybdate due to turbidity caused by the presence of protein (9,17). Removal of protein by acidic precipitation and centrifugation has been reported to reduce color intensity causing erroneous measurement of Pi in the methods (5,13). The use of polyvinyl alcohol to stabilize protein in the ATPase assay also reduces the number of steps in the procedure. The ATPase assay can be terminated by addition of the malachite green solution and scanned 5 min later without further manipulation. In order to evaluate the precision of the microassay, the ATPase activity of cardiac myosin from 12 F, hybrid rats that were age and sex matched was purified and used in the microassay. Variation observed in the results from using these samples could be attributed primarily to differences in the assay or purihcation techniques. The linearity of the microassay through time was determined using 40 ng of purified rat cardiac myosin (Fig. 2). In this assay. the calcium-activated myosin ATPase activity was determined at l-min intervals for 12 min by mixing myosin and ATP in microplate wells for the indicated length of time. The malachite green reagent

AND

WALSH 061

Reactton

Time

(m!n

)

FIG. 2. Linearity ofthe microassay through time. The ATPase activity of 40 ng of rat cardiac myosin was determined at I-min intervals for I2 min using the microassay. The graph depicts the average absorbance values at 630 nm of seven replicates obtained at the time intervals indicated and the standard errors of the means for each time point.

was then added to each well to stop the enzymatic reaction and measure the level of P, released by the reaction. When the net absorbance values obtained from these reactions were compared by regression analysis with the reaction times. an r’ value of 0.9966 was observed, indicating a highly linear relationship between length of reaction time in the specified range and the absorbance values obtained from the microassay. Next, the linearity and sensitivity of the microassay with respect to the myosin concentration used were determined at two time points in the linear range defined above by varying the amount of myosin used in the assay. As illustrated in Fig. 3, a linear relationship was observed between myosin concentrations from IO-325 rig/assay and the &,3o values derived from the microassay (r’ = 0.9893) with a reaction time of 2 min. With an S-min reaction time period, a linear relationship between the myosin concentration up to 160 rig/assay and the ,&,30 values was observed ( rz = 0.9999). Therefore, the microassay is sensitive to both the reaction time period and the amount of enzyme used in the assay within the range of l-l 2 min and between 10 and 325 ng of myosin (Figs. 2 and 3). Subsequent experiments have shown that no detectable level of inactivation of myosin ATPase occurs due to binding of the enzyme to the polystyrene wells of the microplates (data not shown).

A MICROASSAY

FIG. 3. Sensitivity of the microassay. The ATPase activity of rat cardiac myosin at concentrations between 10 and 325 ng per assay was determined using the microassay described in the text. The ATPase reaction was initiated by adding 25 /*I of 2 mM ATP to an equal volume of myosin in 96-well microplates. The enzymatic reaction was terminated after 2 (a) or 8 min (0) by adding malachite green reagent. The average absorbance at 630 nm for seven replicates for each myosin concentration and the standard errors of the means reflect the level of inorganic phosphate produced by myosin-catalyzed ATP hydrolysis,

The sensitivity of the microassay to changes in substrate concentration between 15 and 2000 ELM is shown in Fig. 4. The enzyme activity, expressed as micromoles Pi released per minute per milligram of myosin, is shown both with and without the subtraction of background levels of P,. The increase in the velocity of the ATPase reaction was linear, with substrate concentrations up to 0.5 rnM (I .2 = 0 . 8620). Further increases in substrate concentration did not significantly increase the net level of enzymatic hydrolysis of ATP, indicative of substrate saturation. The background represents the levels of Pi released by nonenzymatic hydrolysis of ATP due to the acid-labile nature of triphosphates (9). This is a minor component of the total P, measured in the assay at substrate concentrations less than 1 mM. After the optimal ranges of time, enzyme, and substrate concentrations for the microassay were established, the K, and VmaXparameters for calcium-activated rat cardiac myosin ATPase were determined ( 18) after background sub-

FOR

317

ATPASE

traction to be 82 PM and I .25 pmol Pi/mitt/ mg myosin. respectively. Values of 46 PM and 0.90 pmol/min/mg myosin, respectively. have been reported for Cal+-activated rat cardiac myosin ATPase (22). In other experiments the microassay produced a specific activity measurement of myosin ATPase that was approximately 25% greater than that obtained using the Fiske-Subbarow method on the same enzyme preparation. The basis for the difference between the results from the two assays is not known although acid precipitation of protein in the Fiske-Subbarow method has been reported to reduce the intensity of the molybdenum blue product measured in the assay ( 13). The present microassay is a sensitive and rapid spectrophotometric method for mea-

Substrate

Concenlrat~on

I mM 1

FIG. 4. Effects of substrate concentration on rat cardiac myosin ATPase activity. The cardiac myosin ATPase activity of I? male F, hybrid rats was determined using the microassay described in the text at eight substrate concentrations ranging from 2 to 0.015625 mM ATP. The values shown for each substrate concentration plotted on a log,,, scale represent the means and standard errors of the means of the 12 rat myosin preparations from the ATPase assay. The graph illustrates the total P, produced (A) and the amount of enzymatically hydrolyzed P, (0) for each substrate concentration. The latter value was obtained by subtracting the P, hydrolyzed in ATP blanks from the total P, detected in the assay.

318

HENKEL.

VANDEBERG.

suring the catalytic activity of cardiac rnyosin. The enhanced sensitivity decreases the amount of myosin needed to measure ATPase activity from previously reported concentrations ranging from 100 to 400 pg per assay (8,19.20) to submicrogram levels (Fig. 3). The increased sensitivity permits a thorough analysis of ATPase activity to be conducted with smaller tissue samples. Given the limited availability of cardiac tissue from experimental animals. the increased sensitivity of the microassay provides a valuable tool for assessment of cardiac muscle function. The sensitivity of the microassay is comparable with values reported for radioactive ATPase assays utilizing the release of radioactive phosphate from [3’P]ATP (2 I ). The sensitivity of the radioactive assays, being dependent in part upon the specific activity of the radioactive ATP used in the assays. however, can exceed that of the microassay under certain conditions (21). The microassay. unlike radioactive ATPase assays. can be rapidly performed on a large number of samples using technology developed for ELlSA methods. The microplate format allows the use of multichannel pipets and microELISA spectrophotometers in the assay to handle and measure the activity of the samples rapidly. Data from the microassay can be transferred directly from the spectrophotometcr to a microprocessor for statistical analysis. further decreasing the time required to obtain results from the assay. The microassay may be applicable for measuring other ATPases in addition to the Ca’+-activated ATPase activity of rat cardiac myosin. The increased sensitivity and ease of performing the assay make this technique an alternative to the more cumbersome approaches widely used for measurement of inorganic phosphate.

AND

WALSH

ACKNOWLEDGMENTS We mcrer.

thank Drs. Bennett and R. Mark Sharp

processing ported Heart.

and

Dykc. for their

statistical

Candace assistance

analyses.

in part by Grant lung. and Blood

This

HL3357Y Institute.

M. Kamwith data

study

from

was sup-

the

National

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