Measurement of adenosine triphosphate content of crayfish stretch receptor cell preparations

ANALYTICAL

BIOCHEMISTRY

24,

Measurement Crayfish

of Adenosine Stretch

SPING Division

531-540 (1968)

of Neurology,

LIN

Triphosphate

Receptor AND

University

Cell

HAROLD of Minnesota,

of

Preparations1 P. COHEN Minneapolis,

Received November

Content

Minnesota

55456

24, 1967

For the past few years we have been carrying out ATP determinations in single crustacean stretch receptor cells (1, 2) as part of a comparative study of their biochemical and electrical properties. The method employed was based on the luciferase-luciferin procedure of Strehler and Totter (3)) utilizing a Nuclear-Chicago liquid scintillation spectrometer as quantum counter. Since liquid scintillation counters are increasingly available in many laboratories, the high sensitivity, specificity, and rapidity of the method would make it the method of choice for assay of very low levels of ATP (down to 2 x 10-l* mole). This communication reports the conditions under which high sensitivity of the method can be attained together with some of the results obtained by this method. A less sensitive version of the method has been reported in brief (4). MATERIALS

AND

METHODS

instrument we used was a Nuclear-Chicago liquid scintillation spectrometer model 725. Since the spectrometer was also used for isotope counting, it was modified only slightly for measurement of luminescence. It was operated in single channel (DATA) with the original photomultiplier (PM) tube replaced by an EMl? type 60975 PM tube having low thermionic emission so that the spectrometer could be operated at room temperature, which is close to optimal for the enzyme luciferase (5). Our instrument was later equipped with fast printout3 so that counting of ATP samples could be made in even shorter time. Luciferuse-Zuciferin. The source of the luciferase-luciferin was the fireLiquid

scintillation

spectrometer. The

‘This investigation was supported by Public Health Service Grants NB 5223, NB 5229, and NB 7192 from the National Institute of Neurological Diseases and Blindness. *EM1 Electronic Ltd., England; Whittaker Corp., Gencom Div., Plainview, N. P. 3By replacing 2 No. 826732 circuit cards with No. 827662 and No. 827572 cards the printout required only about 1 set instead of 8 sec. 531

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fly lantern extract obtained from Sigma Chemical Co. The extract was reconstituted with glass-distilled water according to the supplier, and then left in a refrigerator for two or three days to decrease the endogenous luminescence associated with ATP stores of the lantern. Before use, the extract was centrifuged in an International clinical centrifuge and the insoluble residue was discarded. The reaction mixture consisted of: 50 mM glycylglycine buffer, pH 7.5, 29 parts; 0.1 1M MgSO,, 2 parts; reconstituted firefly lantern extract, about 1 part. The amount of lantern extract was varied to give appropriate light output (between 1 x 105 to 2 X lo5 counts per 0.20 min4 at 1700 V for 6 X 10-12 mole ATP), depending on the enzymic activity and luciferin content of the lantern extract, which varied from batch to batch. For counting, 2 ml of the reaction mixture was pipeted ino the regular 20 ml sample counting vials. We also found it helpful to let the reaction mixture stand at room temperature for an hour or two to stabilize count rates. This is especially important when there was not enough time to allow for decay of luminescence under refrigeration. Other reagents. All the chemicals used were reagent grade, when available. Besides firefly lantern extract (FLE-50)) sodium ATP and Tris were also obtained from Sigma Chemical Co. Sodium ADP was from Pabst Laboratories, glycine and glycylglycine were from Calbiochem, MgSO, was from Merck, and sodium barbital was from Mallinckrodt. Water was glass-distilled. Preparation of samples. The slow adapting stretch receptor cells were dissected from live crayfish (mostly Procumbarus and 0rconectu.s spp.). Those that were capable of displaying impulse activity upon stretching were trimmed to 800 X 560 ,p (6): They were then extracted in either of two ways. (1) At first, the receptor cell preparation was frozen with dry ice onto a 2 X 3 mm piece of microscopic cover slip and then dropped into 0.3 ml of 2 mM glycine buffer, pH 9, maintained at 100” for 5 min (some earlier samples were extracted with 0.5 ml of buffer and heated for 10 min), immediately cooled in ice water, and kept in a deep freeze at -60” until use. (2) In later assays, the extraction with boiling glycine buffer was done after the frozen receptor cell preparation had been freeze-dried and treated with 0.25 ~1 of 0.2 N hydrochloric acid. The later method gave much higher ATP values (see Table 1). Measurement of ATP. The sample ATP content was determined along “The over-all resolving time of the spectrometer is 1 ,usec; the coincidence of counts at these rates would be 1 to 2%, respectively. ‘The authors wish to thank Miss E. Handelman for skillful dissection preparation of some of the stretch receptor cell preparation samples during early part of this study.

loss and the

ATP

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CRAYFISH

STRETCH

533

RECEPTOR

TABLE 1 and Net Sample Count Rates at Different Voltages Applied to the Photomultiplier Tube The counts were from 3.55 X 10-l* mole ATP in 0.3 ml glycine buffer, pH 9, added to 2 ml of reaction mixture (for details see “Methods”). Counting time, 0.20 min. Background

Voltage 1075

1400

1700

406 13.1

248 3046 12.3

3905 57,480 14.7

6321 97,340 15.4

5.3

37.4

1000

Background (B) Net count (S) S/B

Figure of merit: se/B, 103

31

846.1

1499.0

with ATP standards interspersed among the samples. The standard ATP solution as well as the receptor cell preparation extracts were all made in 2 m&I glycine buffer, pH 9, the pH around which ATP was found to be most stable (7), despite the slight depressing effect of this buffer on count rate (see “Results” below). These solutions were kept ice cold to minimize degradation. Working standards were made up just before assay by diluting a standard stock solution. The latter was prepared with crystalline ATP disodium salt at 6 X D5 M in glycine buffer and kept frozen at -60” in small portions. The actual ATP concentration was checked by measuring the UV absorption and the acid-labile phosphates (8). Before sample was added, two 0.20 min background counts were made of the vial containing the reaction mixture. The sample was then added with a pipet, fitted with a rubber pipetter for quick additions, mixed thoroughly by rapidly twirling the vial for about 2 set, and lowered into the counting chamber; this required about 10 to 11 sec. Counting for 0.20 min was then begun manually, 11 set after the sample or standard was added. ATP standards (up to 6 X lo-l2 mole) were added with a Hamilton microsyringe with appropriate additional amounts of 2 mM glyeine buffer. Continuous counting may be made if one wishes to check luminescence decay characteristics. With experience, 30 to 35 samples and standards can be counted within one hour. Net sample count was obtained by subtracting the average background count from the gross sample count before converting to ATP value. RESULTS

Since high sensitivity of voltage applied to the PM shown in Figure 1, the count voltage applied. There was

the photomultiplier tube is desirable, tube was varied to increase count rate. rate increased in a sigmoid fashion with a relatively small concomitant increase

the As the in

534

LIN

AND

Volt, DATA

COHEN

PM Tube

FIO. 1. Count rate (counts per 0.20 min) versus voltage applied to PM tube: (0) net sample counts; (Xf background counts; (6) PM tube noise. Note the large portion of background count due to noise of PM tube.

background counts. With our instrument, a series of linear curves of count rate versus ATP concentration were obtainable at different voltages. Some examples are shown in Figure 2 and Tables 1 and 2. For routine counting of low-level ATP, the voltage adopted was 1700 or 1750 V. Lower voltages can be used for higher concentrations of ATP. Although the background counts were high with high voltage, their variation between samples was rather small. Thus, the standard deviation of the group of 16 average 0.20 min background counts in Table 2 was equivalent to only 3 X lo-l5 mole ATP or less, while the coefficient of variation (i.e., the ratio of standard deviation to the mean) was 1.4%. The deviation from the mean of duplicate sample counts was also rather small, amounting to 3% or less. (Precaution should be taken to minimize exposure to light; otherwise spurious counts, presumably from phosphorescence, will be added to the background or the sample counts. We routinely do the counting in semidarkness.) The use of glycylglycine buffer seemed to give the highest count rate. While glycine buffer of similar concentration also gave good count rates,

1.45 5674 39,630 5

ATP, 10-n mole Background counts” Net counts Order of counting

counts.

2.91 5740 77,770 4

0.58 5881 15,910 9 4.36 5912 115,050 3

0.87 5730 22,430 8 4.36 5876 110,290 15

0.87 5789 23,720 12 5.82 5656 150,280 2

01.16 5781 28,800 7 7.27 5813 192,560 1

1.45 5675 37,960 6

7.27 5864 190,540 16

1.45 5837 38,970 13

PM tube high volt,

counts per sample. The mean and standard deviation for the 16 samples are 5796 f 80

1.45 5828 39,710 14

0.29 5828 8028 11

a Values are averages of two 0.20 min background

0.29 5855 7567 10

ATP, lWe mole Background counts0 Net couIlts Order of counting

TABLE 2 ATP Quantity and Count Rates (Counts per 0.20 min) ATP in 0.3 ml of 2 m&f glycine buffer, pH 9, was added to 2 ml of reaction mixture (for details see “Methods”). 1700 v.

m

i 2

E

8g w

B $ 3 : VI

tiD g

536

LIN

AND

COHEN

6 4

“0

I

2

3 4 ATP, 10m” Mole

5

6

7

FIG. 2. Count rate versus ATP quantity at different voltages applied to the PM tube. Note different count rate scales on the Y axis. In each scale, count rates at higher or lower voltages are also shown in thin lines for comparison. The main line at 1700 V was plotted from data in Table 2. Count rates were not corrected for coincidence loss (see footnote 4).

it is not effective a buffer as glycylglycine at pH 7.5. This may be the reason that McElroy has recommended using the latter (9). While increasing the amount of reaction mixture in the counting vial from 2 to 3 ml increased the background count 65%, the net sample count increased only 10%. It is thus preferable to increase count rate by increasing the firefly lantern extract in the reaction mixture instead of increasing the volume of reaction mixture. The concentration of arsenate buffer recommended in the original method of Strehler and Totter (3, 10) resulted in considerable quenching of the luminescence, giving less than 10% of the count recorded with glycylglycine buffer . Phosphate buffer of equivalent concentration

ATP

OF

CRAYFISH

STRETCH

537

RECEPTOR

inhibited light output, as was found previously by McElroy and associates (5), reducing the output by 75%. Tris buffer likewise reduced the light output by 700/o, and so did choline arsenate or choline chloride buffers; 50 mM sodium barbital buffer, pH 7.5, effected almost complete quenching. In view of the above it was no surprise to find that the quantum output of the reaction mixture is quite sensitive to the composition of the test sample. For instance, in 2 ml of reaction mixture, the addition of 0.2 ml of 2 mlM glycine buffer, pH 9, or water, reduced the count rate by 10 and 15% while the addition of 0.3 ml reduced the count rate by 20 and 40%, respectively. Likewise, the presence of neutralized 5% perchloric acid solution also affected the count rate: the addition of 0.1 ml reduced count rate by 15-200/o, while 0.15 ml reduced it by 50%. Similarly, addition of 0.2 ml of 70% ethanol reduced count rate by 40%. Even higher concentration of MgSO, (44 m&Q reduced the count rate almost by 50%. Therefore, the volume and composition of the sample and the standard should be strictly maintained as constant as possible. The amount of acid used (0.25 JJ of 0.2 N HCl or perchloric acid) to inactivate the lyophilized receptor cell preparations did not affect the light output significantly. TABLE 3 ATP Content of Crayfish Stretch Receptor Preparations Medium

In crayfish saline

No. of samples

Method of extraction

274

Frozen, then boiling Freeze-dried before boiling Freeze-dried before boiling

SO In crayfish saline plus 1 m&f glucose

32

ATP

content,~ 10-u per preparation

mole

1.64 rf: 0.05 4.24 f 0.20 5.02 + 0.25

0 ATP values are mean f S.E.

Another factor that affected the output was mixing of the sample with the reaction mixture. Adding sample by injection alone may not give sufficient mixing and would result in nonlinearity of count rate in relation to ATP concentration. Results of assay of ATP in the resting crayfish stretch receptors are recorded in Table 3. The amount of ATP is presented as picomoles per preparation rather than per unit weight. It is technicahy difficuh to prepare the microscopic sample for weighing, either fresh or dried, free from adhering saline or salt crystals. Washing the preparation with water invariably depolarized the cells and was therefore not satisfactory. Thus,

538

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in a separate series of determinations, a brief water rinse of the stretch receptor cell preparations before freezing significantly (P < 0.001) decreased the mean dry weight (N = 32) from 1.58 to 0.56 ,ug; at the same time, the ATP content was also significantly reduced (P < 0.02) from 4.52 X lo-l2 to 2.74 X 10-l’ mole. A dry weight of 0.5 ,pg for preparations of such dimension as described above has also been reported (11). Assuming that the water-rinsed sample weight is more representative of the true value, and that the dry weight is 20% of the wet weight, the ATP concentration of the stretch receptor preparation would be about 2 &moles per gram fresh weight, which is within the same order of magnitude as that of many other animal nervous tissues. DISCUSSION

Although an increase in the voltage applied to the photomultiplier (PM) tube increases both background and sample count rates, the absolute increase of sample count rate is much more pronounced (Table 1). If we take some values from Figure 2 and compute the “figure of merit” as commonly used in liquid scintillation counting to indicate the maximization of count rate, we will see (Table 1) that increasing voltage from 1000 to 1700 V raised the “figure of merit” about 300 times. This increase in counting efficiency, together with the small variation of background counts, permits the attainment of sensitivity that is seldom achieved by a relatively simple enzymatic method. Even with the high background, we can calculate from the data in Table 2 that 2 x 10-14 mole of ATP would give, at 5% confidence level, a net count of 530 + 216 which should be detectable from a background count of 5800 + 149 at the same confidence level6 Addanki, Sotos, and Rearick (13) reported a similar procedure for ATP determination. However, theirs is a much less sensitive method. Furthermore, their results showed linearity of count rate held only with ATP quantity above 20 picomoles, with considerable deviations below that. The method described here, on the other hand, gives linear proportionality and is particularly suitable to cover the range below 10 picomoles. By applying lower voltage to the PM tube, larger quantities of ATP can also be measured. According to the literature of the manufacturer, the quantum efficiency of the PM tube used (EM1 60975) is about 16% at peak response of ‘With a single background count of 5800, the standard error at 5% confidence level is (5800)v” X 1.96= 149. A net count of 530 above background would give a sample count of 5800 $- 530 = 6330 with a standard error of 80. The standard error for the 530 net count would then be (5800 + 6330)“’ X 1.96 = 216 at 5% confidence level (12).

ATP

OF

CRAYFISH

STRETCH

RECEPTOR

539

380 rnp and about 3.7% at 562 n+~, the latter being the peak of the emission spectrum of the firefly luciferase-luciferin bioluminescence (5). Other PM tubes with better matching of quantum efficiency and the luminescence emission peak, and with lower thermonoise at room temperature, should be able to improve the sensitivity. Scintillation counters having faster loading characteristics would also improve counting performance by including more of the light output during the early portion of the slow decay, the half-time of which has been reported as 13 set (5). The Nuclear-Chicago instrument has a base level discriminator fixed at 0.5 V and use of the coincidence circuit cut down the count rate tremendously. It is possible that other liquid scintillation spectrometers without such fixed base discriminator could be so adjusted as to give lower background counts without lowering the sample count rates. Extraction of ATP by boiling alone was not satisfactory even for microscopic tissue samples such as the crayfish stretch receptor cell preparation. Whether this is because of occlusion of ATP in the heat-coagulated tissue sample or because of heat destruction of ATP was not ascertained. More likely, however, it is probably due to enzymic degradation of ATP during the instant when the sample was warmed up and before heat inactivation of the ATPase took place, as was found in extraction of ATP from frozen brain powder (8). It is possible that improved procedure of extraction would give still higher values of ATP. The use of 2 mM glycine buffer, pH 9, for preparing working standards apparently help maintain ATP concentration: such solutions were found to give stable count rates when used within l-2 hr of preparation. The small amount of firefly lantern extract used not only makes this assay method economical as well as sensitive and rapid, but also minimizes other interfering enzymes such as adenylate kinase that are known to be present in such extracts (14). Under our counting conditions, ADP gave only 1.5-2s of count rate of an equivalent amount of ATP, which was probably due to ATP contaminant in the ADP used. We have not tried using purified or partially purified luciferase and luciferin for this method; it is conceivable that by eliminating some of the endogenous luminescence, the background could he reduced to values approaching the thermonoise level (Fig. 1). SUMMARY

A sensitive and rapid method for the assay of ATP down to 2 X10-14 mole was presented. This was achieved by the use of firefly luciferaseluciferin reaction and a scintillation spectrometer operated on single channel at high voltage. .The ATP content of single resting crayfish stretch receptor cell prepa-

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rations was determined. It was found to be 4.24 picomoles in the absence, and 5.02 picomoles in the presence, of 1 mM glucose. REFERENCES 1. ~ZUOLO, C. A., BONEWELL, G., GUCOBINI, E., HANDELMAN, E., AND LIN, S., Federation Proc. 23, 113 (1964). 2. ROSSINI, L., COHEN, H. P., HANDELMAN, E., LIN, S., AND TERZUOLO, C. A., Ann. N. Y. Acad. Sci. 137, 864 (1966). 3. S~~HLFR, B. L., AND TOTTER, J. R., Arch. Biochem. Biophys. 40, 28 (1952). 4. COHEN, H. P., AND HYDE, J. R., INeurology 13, 355 (1963). 5. MCELROY, W. D., AND SELIGER, H. H., in “Light and Life” (McElroy, W. D., and Glass, B., eds.) p. 219. Johns Hopkins Univ. Press, Baltimore, 1961. 6. GIACOBINI, E., HANDELMAN, E., AND TERZUOLO, C. A., Science 140, 74 (1963). 7. “Methods of Enzymatic Analysis” (Bergmeyer, H. -II., ed.), p. 15. Academic Press, (Verlag Chemie) New York, 1963. 8. LIN, S., AND COHEN, H. P., Arch. Biochem. Biophys. 88, 256 (1962). 9. MCELROY, W. D., in “Methods in Enzymology” (Colowick, S. P., and Kaplan, N. D., eds.), Vol. 6, p. 445. Academic Press, New York, 1963. 10. STREHLER, B. L., AND TOTTER, J. R., in “Methods of Biochemical Analysis, (Glick, D., ed.), Vol. 1, p. 341. Interscience, New York, 1954. 11. GIACOBINI, E., AND GRASSO, A., Acta physiol. &and. 66, 49 (1966). 12. TITTLE, C. W., Nuclear-Chicago Tech. Bull. No. 14, 4 pp. (1964). 13. ADDANKI, S., SOTOS, J. F., AND REARICK, P. D., Anal. Biochem. 14, 261 (1966). 14. BALFOUR, W. M., AND SAMSON, F. E., JR., Arch. Biochem. Biophys. 84, 140 (1959).