Biochem. J. (1990) 266, 611-614 (Printed in Great Britain)

A continuous fluorimetric

assay

611

for ATPase activity

Utpal BANIK and Siddhartha ROY* Department of Biophysics, Bose Institute, P1/12, C.I.T. Scheme VII M, Calcutta 700 054, India

A new continuous coupled fluorimetric assay is described for ATPases in general. Thus phosphate released from ATP hydrolysis is coupled to the nucleoside phosphorylase reaction using 7-methylguanosine as a fluorescent substrate for the nucleoside phosphorylase reaction. The hydrolysis of 7-methylguanosine leads to 7-methylguanine, which has lower quantum yield and hence can be used to monitor ATP hydrolysis continuously. The method has the potential to be extended to GTPase and nucleotidyltransferase assays.

INTRODUCTION Hydrolysis of ATP and GTP to nucleoside diphosphate and Pi is an energy-producing step and is widely used in biological systems to drive many energy-requiring reactions. ATPase and GTPase activities are widely distributed in Nature, and many such enzymes have been studied intensively. Many assay systems have been described to monitor the hydrolysis of ATP, e.g. using 32Plabelled ATP to measure phosphate release (Brattin & Waller, 1983), coupling of ADP produced to the pyruvate kinase (PK) and lactate dehydrogenase (LDH) reaction to NAD+ production (Anderson & Murphy, 1983), by pyruvate quenching (Ando & Miyata, 1983) or measuring Pi produced by colorimetric titrations (Chan et al., 1986). Recently, Shugar and his colleagues have shown that calf spleen nucleoside phosphorylase can utilize 7-methylguanosine (m7Guo) and 7-methylinosine as substrates (Kulikowska et al., 1986). Both of these substrates are fluorescent, and phosphorolysis produces 7-methylguanine (m7Gua) and 7-methylhypoxanthine respectively, which have a much lower quantum yield. So the nucleoside phosphorylase reaction can be monitored fluorimetrically by using these alternative substrates. We have utilized this fluorimetric method to couple phosphate production by ATPase (using ATPase activity myosin as a model) to the nucleoside phosphorylase reaction in the following manner: ATPase

ATP-

ADP + P

Nucleoside phosphorylase

o

m7Gua + ribose 1-phosphate We have shown that, in the presence of excess nucleoside phosphorylase, phosphate production can be monitored fluorimetrically.

Pi + m7Guo

MATERIALS AND METHODS ATP, m7Guo, nucleoside phosphorylase, LDH rabbit muscle myosin, PK and phosphoenolpyruvate were from Sigma Chemical Co. Tris (four times recrystallized) was purchased from Spectrochem, and CaCl2,2H20 was from Glaxo Laboratories.

Continuous monitoring of ATPase activity The assay was performed with a Hitachi F 3000 fluorimeter. A typical assay mixture contained 45,/Mm7Guo, 0.1 unit of nucleoside phosphorylase, 1 mMATP in 0.1 M-Tris HCl buffer, pH 7.5, containing 4 mmCaCl2 (buffer A). Myosin was added to initiate the reaction. The decrease in fluorescence was observed at 410 nm (the excitation wavelength was 300 nm). Purine nucleoside phosphorylase was dialysed three times, for 12 h each time, against 1 litre of 0.1 M-Tris/HCl buffer, pH 7.5, before use. The PK/LDH assay was performed as described by Anderson & Murphy (1983). Calibration of fluorescence change To 45 /tM-m7Guo and nucleoside phosphorylase (0.1 unit/ml) in buffer A, a predetermined amount of Pi in buffer A was added. After 10 min, when no further change of fluorescence occurred, the fluorescence value was noted (F). The fluorescence value before any addition of Pi is Fo. Finally, excess phosphate was added and fluorescence value was noted (F,). This fluorescence value (Fc,,), which corresponds to the condition where all the m7Guo has been hydrolysed, was subtracted from all fluorescence values.

RESULTS Fig. 1 shows the trace of the fluorimeter as a function of increasing myosin concentration. It is clear that increasing myosin concentration increases the rate of decrease of fluorescence. The decrease is linear up to at least 5 min. Slight non-linearity in the beginning is not unexpected for a coupled assay during which the steadystate is established. Fig. 2 shows the protein concentration-dependence of the initial rate of decrease of fluorescence. As the Figure shows, the rate increase is proportional to the protein concentration over the range tested, i.e. 1-130 ,tg/ml. Fig. 3 shows the time course of decrease of fluorescence over a much longer time scale. It can be seen that fluorescence decreases almost linearly, until the substrate, m7Guo, is exhausted. The residual fluorescence may be explained by the diminished, but finite, fluorescence of the product, m7Gua.

Abbreviations used: m7Guo, 7-methylguanosine; m7Gua, 7-methylguanine; PK/LDH, pyruvate * To whom correspondence and reprint requests should be addressed.

Vol. 266

kinase/lactate dehydrogenase.

612

U. Banik & S. Roy

a1) U

a) U a) 0

0

1

2

3

4

5

Time (min)

Fig. 1. Rate of hydrolysis of ATP as determined by loss of fluorescence of m7Guo The excitation wavelength was 300 nm and the emission wavelength 410 nm. Each solution contained I mM-ATP, 45 /tM-m7Guo and 0.1 unit of nucleoside phosphorylase/ml in buffer A. Trace 1, No myosin; 2, 2.6 ,ug of myosin/ml; 3, 13 ,ug of myosin/ml; 4, 26 ,ug of myosin/ml; 5, 39 ,ug of myosin/ml.

Clen cu

-

2.0

50

C-

cn

-0

~0 -0

c

1.0

U a) CA

Cu

a) 0

a)

Cu

0

0

26

52 78 [Myosin] (pg/ml)

15

30 Time (min)

45

60

Fig. 2. Myosin-concentration-dependence of the initial rate Solution conditions were the same as in Fig. 1.

Fig. 3. Time course of loss of fluorescence of m7Guo in the coupled assay The solution conditions are 1 mM-ATP, 0.1 unit of nucleoside phosphorylase/ml, 45 1a6m-m7Guo and 130 ,ug of myosin/ml.

If the assay is working according to the stated principle and not as a result of any contamination, then withholding any component should result in a complete loss of activity. Fig. 4 shows the effect of withholding different components of the assay mixture. It is clear that, without nucleoside phosphorylase or ATP, no reaction takes place. This rules out any possibility of phosphate contamination from either myosin or nucleoside phosphoryl-

the source of phosphate. Similarly, in Fig. 1, the almost zero rate without myosin indicates that phosphate contamination from ATP is also not a serious problem. To convert fluorescence change into units of concentration, we have determined fluorescence change as a function of added Pi in a solution of 45 ,uM-m7Guo and nucleoside phosphorylase (0.1 unit/ml in buffer A). Fig. 5 shows the fractional fluorescence change plotted against ase as

0

Time (min)

Fig. 4. Effect of withholding various assay components on the rate The excitation wavelength was at 300 nm, with emission at 410 nm. Solutions contained; 1, 1 mM-ATP, 45 M_M-m7Guo and 65 ,ug of myosin/ml in buffer A; 2, 45 ,uM-m7Guo, 0.1 unit of nucleoside phosphorylase/ml and 65 ,ug of myosin/ml; 3, ATP added to solution 2 to make it 1 mM.

1990

A continuous fluorimetric

assay

for ATPase activity

613

PK/LDH assays. Four such determinations were made and average values were calculated. Specific activities determined by both methods correspond well, with the fluorimetric method giving an approx. 12 % lower value. Fig. 6 shows the effect of pH and ionic strength on myosin activity as determined by the fluorimetric method. Increase of both pH and ionic strength leads to a decrease in myosin ATPase activity. Such a decrease of ATPase activity of myosin with increasing pH (over the pH range 7-8.5) and ionic strength has been observed previously (Driska & Hartshorn, 1975).

0.8

0.6

0.2

0

10

20

[Pi] (PM)

Fig. 5. Fluorescence change as a function of added Pi To 45 /,M-m7Guo and 0.1 unit of nucleoside phosphorylase/ml in buffer A, a predetermined amount of Pi was added. The excitation wavelength was at 300 nm and emission was at 410 nm.

0

[NaCI] (M) (A) 0.1 0.2 0.3 0.4

c

2.0

en +.

_3

1.0

+-,

0

7.0

8.0 pH (0)

9.0

Fig. 6. Effect of pH (e) and ionic strength (-) on ATPase activity of myosin Solution conditions are same as in Fig. 1, except the pH of the buffer or NaCl concentration, as noted in the Figure.

added-P1 concentration. The plot is linear at least up to about 60 % of total fluorescence change, indicating that rates measured during the first half of the reaction can be reliably converted into concentration units. We have compared the fluorimetric assay with the PK/LDH coupled assay. Table 1 shows the specific activity of myosin as determined by fluorimetric and Table 1. Comparison of the fluorimetric ATPase assay with the PK/LDH-coupled assay

Specific activity (nmol/min per mg of protein) Observation no.

2

3 4

Average Vol. 266

...

Fluorimetric

PK/LDH

214 211 176 155

226 244 212 173 214

189

DISCUSSION In general, fluorimetric assays are inherently more sensitive than spectrophotometric assays and hence are desirable in many cases. However, the problem of finding a suitable coupled system and fluorescent substrate is not straightforward for the reaction: ) ADP + Pi ATP The problem is further complicated by the fact that ATP has a high absorbance below 300 nm, and any fluorescent substrate having an excitation wavelength below 300 nm would suffer from a high inner-filter effect. m7Guo, which has an excitation wavelength at 300 nm and above, does not suffer from such a drawback. In addition, phosphate production is coupled by a single enzyme to the measured reaction. This is always the desirable situation in a coupled assay system. As shown in the Results section, the initial rate is linear up to 5 min, and the initial rate is proportional to protein concentration. To estimate the precision of the assay, we have determined the equivalence of the P1 liberated and the observed fluorescence change. Half of the total fluorescence change occurs at approx. 18 /tM-phosphate, as opposed to the expected value of 22.5 ,UM. The difference could be due to the small amount of hydrolysed m7Guo which may be present in the initial sample. Comparison indicates that, except for a small difference (approx. 12 %), the fluorimetric and PK/LDH assays yield similar results. The small difference may be attributed to different substrates present in the two assays. The effect of ionic strength and pH on myosin ATPase activity as determined by the fluorimetric method corresponds to that determined by other assay methods. Only at pH 9.0 is there a substantial difference. Driska & Hartshorn (1975) reported that there is an activity increase beyond pH 8.5 which peaks at pH 10. The present results indicate a sharp decrease in activity on going from pH 8.5 to pH 9.0. This could be due to loss of activity of the coupling enzyme, nucleoside phosphorylase. Kulikowska et al. (1986) has reported a severalfold decrease in Vmax /Km of nucleoside phosphorylase with m7Guo as substrate on going from pH 7.5 to 8.5. Thus it is possible that ATP hydrolysis no longer remains the rate-determining step at pH 9.0, and hence the validity of the assay beyond pH 8.5, under the present conditions, should not be taken for granted. The assay is a general one and is capable of measuring all ATP hydrolysis that produces P. It has also been found to work as an assay of GTPase activity (M. Z. Papastavrs, D. Lowry, A. G. Redfield & S. Roy, unpublished work). In addition, it should be possible in principle to use the method for assaying nucleotidyltransferase activity by inserting a hydrolysis step, i.e.

614

hydrolysing the liberated pyrophosphate to Pi by inorganic pyrophosphatase. The sensitivity of the assay under these conditions can be estimated from Fig. 5, and the activities are reported in Fig. 1. We estimate that activities of 0.05-0.1 /uM * min-' can be measured very easily, which would make it considerably more sensitive than the PK/LDH coupled assays. In a dehydrogenase-coupled spectrophotometric assay, it is difficult to measure a change of less than 0.5 tM *min-1 (which translates to 0.003 A/min at 340 nm). We thank Dr. Susweta Biswas for the sample of myosin, Dr. B. Bhattacharyya for stimulating discussions and all-around help and Professor B. B. Biswas for allowing us to use the

U. Banik & S. Roy fluorimeter. We also thank all members of the Department of Biochemistry for their co-operation.

REFERENCES Anderson, K. W. & Murphy, A. J. (1983) J. Biol. Chem. 258, 14276-14278 Ando, T. & Miyata, H. (1983) Anal. Biochem. 129, 170175 Brattin, W. J. & Waller, R. L. (1983) J. Biol. Chem. 258, 6724-6729 Chan, K., Delfert, D. & Junger, K. D. (1986) Anal. Biochem. 157, 375-380 Driska, S. & Hartshorn, H. J. (1975) Arch. Biochem. Biophys. 167, 203-212 Kulikowska, E., Bzowska, A., Wierzchowski, J. & Shugar, D. (1986) Biochim. Biophys. Acta 874, 355-363

Received 11 July 1989/27 November 1989; accepted 22 December 1989

19.90