Journal of Biochemical and Biophysical Methods, 1 (1979) 1.63--1 69 © Elsevier/North-Itolland Biomedical Press

163

PHOTOKINETIC MICROASSAY OF ADENYLATE KINASE USING TIlE FIREFLY LUCIFERASE REACTION

SVEN E. BROLIN, ERIK BORGLUND and AMBJ()RN ~t[GREN Department of ltistolol.~y, Biomedicum, University of Uppsala, Uppsala, Sweden (Received 21 November 1978; accepted 2 April 1979)

A new rapid photokinetic method is described for determining the activity of adenylate kinase (ATP:AMP phosphotranspherase, EC 2.7.4.3) in 0.1--5.0 pg of freeze-dried tissue. This represents a weight range far below that obtainable by fine-needle biopsy. The reaction 2 ADP ~ AMP + ATP was employed and the ATP formed assayed with firefly luciferase as light yielder. The light emission was recorded on a multi-channel scaler. The adenylate kinase activities found in tissues of mice were in the same range as previously described in a study using fluorometric microassay. Key words: bioluminescence analysis; adenylate kinase; firefly luciferase; photokinetics; bioenergetics

INTRODUCTION When ATP p r o d u c t i o n is insufficient m a n y cells m a y enter a critical period and need a short c u t to a transient energy supply. This c o n c e r n s particularly muscles and cells engaged in the delivery of signals or release o f h o r m o n e s , i\ s h o r t - t e r m energy d e m a n d can be m e t b y a d e n y l a t e kinase (EC 2.7.4.3) w h i c h catalyzes the i n t e r c o n v e r s i o n o f a d e n i n e nucleotides, t h e r e b y p e r m i t t i n g e n e r g y transfer f r o m A D P to ATP. In o r d e r to assay this e n z y m e in small samples, the possibilities o f using either A D P or A T P f o r m a t i o n c o u p l e d to d e h y d r o g e n a s e r e a c t i o n s were evaluated and c o m p a r e d in f l u o r o m e t r i c m e a s u r e m e n t s [ 1 , 2 ] . T h e A D P p r o d u c t i o n o b t a i n e d f r o m a substrate o f A T P was d e t e r m i n e d using c o u p l e d p y r u v a t e kinase (EC 2 . 7 . 1 . 1 ) lactate d e h y d r o g e n a s e (EC 1.1.1.27) reactions. The NAD* f o r m e d in the latter r e a c t i o n was used as the measurable p r o d u c t after c o n v e r s i o n to a highly f l u o r e s c e n t c o m p o u n d b y t r e a t m e n t with s t r o n g alkali. T h e alternative possibility of m e a s u r i n g t h e a d e n y l a t e kinase activity was based u p o n A T P f o r m a t i o n f r o m a substrate o f ADP. By m e a n s o f the h e x o k i n a s e (EC 2.7.1.1) and g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e (EC 1.1.49) reactions, the N A D P H t h a t was f o r m e d was m e a s u r e d b y m a k i n g use o f its native fluorescence. Results o b t a i n e d with the t w o m e t h o d s were in g o o d a c c o r d a n c e w h e n i n t e r f e r e n c e b y p h o s p h a t a s e s was t a k e n into a c c o u n t . This source o f error was p a r t i c u l a r l y e v i d e n t w h e n A T P was used as substrate and m a d e an

164

assay for the opposite direction preferable. The sensitivity and reproducibility of the fluorometric assay based upon the ATP formation was sufficient for determination of the adenylate kinase activity in small samples of cells from the granular layer of the cerebellum, which emits fast impulses governing the coordination of movement, and from the Islets of Langerhans [2], which secrete insulin, often at a high rate [3]. The cerebel!ar and islet samples showed much higher activity values than parenchymatous cells from the exocrine pancreas and the liver, but were far from reaching the extremely high values of muscle cells. It is thus possible to use a fluorometric technique for analyzing adenylate kinase in small samples composed of isolated cells of the same kind. However, the bioenergetic importance of this enzyme and a growing interest in its occurrence in specialized cells, which only can be isolated in small numbers, have drawn attention to the convenience and sensitivity of bioluminescence analysis. We describe, therefore, determination of the adenylate kinase activity by measuring ATP formed in a coupted assay employing firefly lueiferase. MATERIALS AND METHODS

Animals Female mice with the obese-hyperglycemic syndrome (gene symbol

ob/ob) and their female lean litter mates were used. The obese mice, originally obtained from the Jackson Laboratory, Bar Harbor, Maine, U.S.A., have been bred at the Department of ttistology since 1959 and used for studies of their comparatively big insulin-secreting pancreatic islets. The animals, all aged about 6 months, were allowed free access to food and water [4].

Chemicals Collagenase was obtained from the Worthington Biochemical Corp., Freehold, N.J., U.S.A. and balanced ttank's salt solution was supplied by Statens Bakteriologiska Laboratorium, Stockholm, Sweden. Tris(hydroxymethyl)aminomethane, firefly luciferase and apyrase (EC 3.6.1.5) were purchased from Sigma Chemical Company, St. Louis, Mo., U.S.A. ATP and ADP were obtained from Boehringer and Soehne, Mannheim, F.R.G. Ethylenediaminetetraacetic acid (EDTA) and other salts, which were of analytical grade, were from A.G. Merck, Darmstadt, F.R.G. Doubly distilled water was used t h r o u g h o u t all experiments.

Preparation of tissue samples The animals were killed by decapitation under ether anaesthesia. The pancreas, left liver lobe and the upper pole of the kidney were quickly

165 removed and placed on c r yos t a t chucks, using brain paste as adhesive, and rapidly frozen in cold isopentane chilled to --160°C with liquid nitrogen. T h e frozen samples were sectioned (20 pro) in a cryostat (--25°C) and then freeze-dried and stored at --25°C in argon gas containing less than 1 ppm oxygen. The cell samples (pancreatic islets, liver parenchym a and kidney cortex) were dissected free hand f r om the freeze-dried sections under a stereomicroscope in a r o o m with constant t e m p e r a t u r e (21°C) and relative hum i di t y (40%). These samples were then weighed on a quartz-fiber balance [5], the weights being in the range 0.1--6.4 gg. In one experimental group enzymatic digestion was utilized to isolate cell groups. Islets of Langerhans were prepared by a p r o c e dur e [6] which was based on a collagenase digestion technique developed for studies of the endocrine part of the pancreas [71. The digested tissue was washed in Hank's balanced salt solution and the liberated islets were identified under a stereomicroscope. The islets collected in this way were freeze-dried and treated in the same way as the other specimens.

A naly tical procedure Th e activity of the adenylate kinase was determined photokinetically using ATP f o r m e d f r om ADP as the measured product, and firefly luciferase as light yielder. The samples were inserted into microtubes (i.d. 3 ram; length 50 mm) containing 2.0 pl 10 mM NaOIt and 5 mM EDTA. The tubes were k e p t on the ice until incubated for 30 min at 38°C, after addition of 10 pl 50 mM Tris buffer, pi t 8.0, supplemented with 7.2 mM ADP, 3 mM MgC12 and 0.03 mg/ml bovine plasma albumin. Following incubation, the tubes were transferred to ice and their cont ent s diluted 32 times with 10 mM NaOH/ 5 mM EDTA to stop the reaction. Blanks and standards were carried through the entire procedure together simultaneously with the samples. F r o m each tube a sample of 2.0 pl was taken and injected into a reaction cell containing 25 pl of a light-yielding fb.efly luciferase solution. This was prepared by dissolving 50 mg firefly luciferase extract in 5 ml 0.005 M potassium arsenate/0.02 M MgSO4, pH 7.4. After centrifugation, 2.7 mg apyrase was added to the solution in order to decrease light emission caused b y contaminating ATP [8,9]. The light emission was elicited in a reaction cell placed in f r o n t of an EMI photomultiplier tube with reduced cat hode area and chilled to --20°C in order to diminish electronic noise. The photomultiplier was operated at 900 V and the pulses building up the signal were stored in a multichannel scaler (Canberra 8100) as described previously [10]. T h e time profile of the counts representing the p h o t o n emission was displayed on its screen for selection of appropriate channels and the subsequent read-out o f their integral. In our analyses a procedure was adopt ed where the integrals of the first 30 channels, each set at a collection time of 50 ms, were displayed and utilized as seen in Fig. 1.

166 T

TT

~

..

b

I

..'I

Fig. 1. P h o t o n emissions displayed o n t h e screen o f the m u l t i - c h a n n e l scaler f r o m four recordings. T h e height o f each d o t r e p r e s e n t s the p h o t o n c o u n t s during 20 ms. Due to the versatility o f the i n s t r u m e n t the curves o f each f o u r t h p a r t can be e x p a n d e d tO fill the w h o l e screen and, if desired, e x p a n d e d further. It is also possibie to s u p e r i m p o s e t w o curves for c o m p a r i s o n , a. The four r e c o r d s s h o w t h e p h o t o n emission f r o m a blank (I), a s a m p l e (II), a s t a n d a r d o f 68 p m o l ATP (III) and a n o t h e r blank (IV). b. R e c o r d s II and IV displayed t o g e t h e r on the w h o l e screen, c. F r o m the records s h o w n in b the first 30 c h a n n e l s f o l l o w i n g t h e light f o r m a t i o n are isolated and f u r t h e r e x p a n d e d . The integrals o f t h e s e c h a n n e l s are used for the calculations.

RESULTS AND DISCUSSION

The signal obtained from the tissue samples consisted of two components. The first phase of the photon emission emanated from ATP formed in the preceding adenylate kinase reaction, whereas a second strong emission was caused by the conversion of residual ADP. This was due to adenylate kinase present as contaminant in the ]uciferase solution. The versatility of the scaler made it easy to recognize the start of the initial ATP-induced emission,

167 nanomot

converted substrate/30min

20

10

2

4

6

~Jg dry wt

Fig. 2. The relationship between ATP formation and the weights of liver samples from mice in the assay for adenylate kinase. Each symbol represents the mean and standard error of six determinations whereas the curve gives the regression as calculated from the single observations.

t o limit t h e c o u n t i n g , a n d t o select the n u m b e r o f c h a n n e l s e m p l o y e d . Since t h e b l a n k s c o n t a i n e d o n l y m i n u t e a m o u n t s of A T P t h e first emission p h a s e o f t h e s e was r a t h e r w e a k b u t t h e c h a n n e l r e p r e s e n t i n g its s t a r t c o u l d be r e c o g n i z e d b y having a higher c o u n t t h a n the p r e c e d i n g o n e s which contained o n l y t h e c o u n t of b a c k g r o u n d emission w h i c h was n o t quite elimin a t e d f r o m t h e luciferase solution. T h e r e l a t i o n s h i p b e t w e e n s a m p l e w e i g h t and m e a s u r e d activity o f a d e n y late kinase was e v a l u a t e d , using f r e e z e - d r i e d sections o f t h e liver f r o m t w o m i c e (Fig. 2). A g o o d linearity was o b t a i n e d up to 6 pg d r y w e i g h t b u t was lost w h e n weights o f 10 p g was a p p r o a c h e d . T h e latter w e i g h t s w e r e r e a c h e d b y p o o l i n g o f samples. I t was also c h e c k e d t h a t t h e c o n v e r s i o n r a t e was p r o p o r t i o n a l to t h e i n c u b a t i o n t i m e u p t o 45 m i n w h e n small s a m p l e s w e r e used. A c o n s i d e r a b l e c o n v e r s i o n m a y t h u s be allowed, b u t is s e e m s advisable t o k e e p t h e s u b s t r a t e c o n s u m p t i o n b e l o w 10% b y limiting t h e s a m p l e weights and/or the incubation time. F o r this a p p l i c a t i o n of t h e b i o l u m i n e s c e n c e m e t h o d , m i c e w e r e u s e d of the s a m e strain and age as t h o s e in p r e v i o u s f l u o r o m e t r i c m e a s u r e m e n t s in o u r l a b o r a t o r y [1,2]. T h e results o b t a i n e d w i t h t h e t w o m e t h o d s were c o m p a r e d a n d t h e values f o u n d t o be v e r y close to each o t h e r , a s s h o w n in T a b l e 1. T h e o n l y e x c e p t i o n was w h e n d i f f e r e n t t e c h n i q u e s w e r e used f o r t h e s a m p l e p r e p a r a t i o n resulting in l o w e r a c t i v i t y in o n e g r o u p w i t h heavier samples. In studies o f a d e n y l a t e kinase b o t h m e t h o d s can be u s e d f o r assay

168 TABLE 1 T H E A D E N Y L A T E K I N A S E A C T I V I T Y IN V A R I O U S O R G A N S O B T A I N E D IN F L U O R O M E T R I C A N D P H O T O K I N E T I C A N A L Y S E S

OF

MICE

AS

T h e m e a n s values -+ S.E. r e p r e s e n t 4--6 d e t e r m i n a t i o n s for each animal. N u m b e r s o f animals are given in p a r e n t h e s e s . T h e f l u o r o m e t r i c m e a s u r e m e n t s were carried o u t using A D P as s u b s t r a t c [2]. Specimen

Liver Kidney P a n c r e a t i c islets

E n z y m e activity (mol c o n v e r t e d s u b s t r a t e • h - l

. kg dry w e i g h t - l )

F l u o r e o m e t r i c assay

P h o t o k i n e t i c assay

Lean mice

Obese mice

L e a n mice

Obese mice

6.3-+0.4(13) 16.3 -+ 1.9 (4) 15.4 ± 0.8 (13)

6.2+0.4(12) 15.6 +-+-2.1 (3) 17.4 -+ 0.9 (12)

6.4-+0.5(5) 12.8 ± 0.5 (3) 12.5 a -+ 1.5 (3)

6.7-+0.8(3) 1.3.6 -+ 2.8 (3) 18.7 -+ 1.2 (4)

a This value r e p r e s e n t s w h o l e islets isolated w i t h a collagenase m e t h o d a n d with greater w e i g h t s t h a n all t h e o t h e r s p e c i m e n s w h i c h were dissected f r o m freeze-dried sections.

of cell samples much smaller than with fine needle biopsies but in addition to the convenience and high sensitivity of bioluminescence analyses, our new photokinetic method also offers the advantage of direct measurements of the ATP formation. The fluorometric technique requires coupled enzyme reactions for this. It may be surmised that use of a purified firefly preparation [11,12] would permit a simplified recording in case of successful purification, since removal of contaminating adenylate kinase from the light yielding solution would obviate the second phase of photon emission. Purified luciferase preparations have become commercially available and further improvements m a y be achieved. The suitability for adenylate kinase assay in small samples remains to be settled. When as in this study an only partly purified preparation is used, it is necessary to distinguish the true light signals of ATP from the non-specific photon emission by limiting the time of the light measurement. S I M P L I F I E D D E S C R I P T I O N O F T H E M E T H O D A N D ITS A P P L I C A T I O N S In t h e assay o f a d e n y l a t e kinase w i t h c o m m e r c i a l firefly p r e p a r a t i o n s , A T P is g e n e r a t e d in 12 /ll o f i n c u b a t i o n m e d i u m c o n t a i n i n g 6 m M ADP and 2.5 m M MgCl2. T h e r e a c t i o n is s t o p p e d b y a 32-fold d i l u t i o n w i t h l 0 m M N a O t t / 5 mM E D T A p e r f o r m e d b e f o r e 10% o f t h e A D P has b e e n c o n s u m e d . A f t e r rapid m i x i n g w i t h a light-yielding solut i o n o f firefly luciferase, t h e a m o u n t s of A T P f o r m e d are d e t e r m i n e d . T h e light is meas u r e d for a p e r i o d l i m i t e d to 1.5 s w i t h c u s t o m - b u i l t e q u i p m e n t w h i c h can be replaced b y c o m m e r c i a l l y available i n s t r u m e n t s , such as b i o l u m i n e s c e n c e a n a l y z e r s or specially a d a p t e d l i q u i d - s c i n t i l l a t i o n c o u n t e r s . T h e m a i n a d v a n t a g e o f t h e m e t h o d w h e n comp a r e d t o usual f l u o r o m e t r i c t e c h n i q u e s , is t h e simplified analytical p e r f o r m a n c e , facilitating assay of small liquid a n d cell samples o f physiological i m p o r t a n c e . I t is a p p l i c a b l e iN b i o e n e r g e t i c studies o f e n d o c r i n e cells a n d is of p o t e n t i a l value for analyses of fine n e e d l e biopsies.

169 ACKNOWLEDGEMENTS F i n a n c i a l s u p p o r t was g e n e r o u s l y given b y t h e S w e d i s h D i a b e t e s Association, the Medical Faculty of the University of Uppsala and the Swedish Medical Research Council (Project B79-12X-535). REFERENCES 1 Borglund, E., Brolin, S.E. and Agren, A. (1978) Upsala J. Med. Sci. 83, 81--84 2 Borglund, E., Brolin, S.E. and Ag~'en, A. (1978) J. Histochem. Cytochem. 26, 127-130 3 Grodsky, G.M., Batts, A.A., Bennett, L.L., Vcella, C., MeWilliams, N.B. and Smith, D.F. (1963) Am. J. Physiol. 205,638--644 4 Westman, S. (1968) Diabetologia 4, 141--149 5 Lowry, O.H. (1953) J. Histochem. Cytochem. 1,420--428. 6 Howell, S.L. and Taylor, K.W. (1968) Biochem. J. 108, 17--24 7 Asplund, K. and Hellerstr~im, C. (1972) Horm. Metab. Res. 4, 159--163 8 Lyman, G.E. and De Vineenzo, J.P. (1.967) Anal. Biochem. 21,435----443 9 Wettermark, G., Tegn6r, L., Brolin, S.E. and Borglund, E. (1970) in The Structure and Metabolism of the Pancreatic Islets (Falkmer, S., Hellman, B. artd T~/ljedal, I.-B., eds.), pp. 275--282. Pergamon Press, Oxford 10 Wettermark, G., Stymne, H., Brolin, S.E. and Petersson, B. (1975) Anal. Biochem. 63, 293--307 11 Nielsen, R. and Rasmussen, H. (1968) Acta Chem. Scand. 22, 1757--1762 12 Rasmussen, H.N. (1978) in Methods in Enzymology (DeLuea, M.A., ed.), Vol. 57, pp. 28--36. Academic Press, New York

Photokinetic microassay of adenylate kinase using the firefly luciferase reaction.

Journal of Biochemical and Biophysical Methods, 1 (1979) 1.63--1 69 © Elsevier/North-Itolland Biomedical Press 163 PHOTOKINETIC MICROASSAY OF ADENYL...
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