R A D I O E N Z Y M A T I C D E T E R M I N A T I O N

O F ADENOSINE- 5 ' - TRIPHOSPHAT E

G . I . M e e r o v UDC 615.739.653-014.3

A s imple and re l i ab le rad ioenzymat ie method was developed for the de te rmina t ion of ATP and ADP in a p repa ra t ion of adenos ine -5 ' - t r i phospha te (ATP).

The disodium sa l t of adenos ine -5 ' - t r i phospha te (ATP-Na) is used in m u s c u l a r dystrophy, espec ia l ly when the neu romuscu la r conductivity is i m p a i r e d ; in m u s c u l a r dyst rophy resu l t ing f r o m denervat ion of the l imbs of t r auma t i c origin; s p a s m s of the v e s s e l s of the h e a r t - s tenocardia ; dys t rophic changes in the ca rd iac musc l e (s imul taneously with the use of ca rd iac remedies)~ and spas t ic s t a t e s of the pe r iphe ra l v e s s e l s - i n t e r m i t t e n t dysbasia , endar te r i t i s , etc.

We M v e developed a r ad ioenzymat ie method for de termining ATP and adenos ine -5 ' -d iphospha te (ADP) in p repa ra t ions of ATP-Na , accord ing to which ATP is or iginal ly de te rmined as a r e su l t of i ts en- zyrnatic hexokinase reac t ion with a known excess of C14-glucose. In this react ion, CU-g lucose -6 -phospha te (C14-GP) will be f o rm ed in amounts equ imola r to the ATP taken in the react ion. By prec ip i ta t ing the CI4-GP in the f o r m of the b a r i u m salt, we can ca lcu la te the amount of c i4 -GP fo rmed according to the r ad io - act ivi ty of C14-glucose remain ing in the superna tan t liquid, and f r o m this we can de te rmine the amount of ATP. If the enzyme adenylate kinase is a lso ~dded to the reac t ion mix tu re [1], then two m i c r o m o l e s of C14-GP will be fo rm ed pe r m i c r o m o l e of ATP introduced into the react ion. If ADP is p r e s e n t in the r e a c - tion medium, an additional one m i c r o m o [ e of C14-GP wilt be fo rmed per m i e r o m o l e of ADP, according to the reac t ions :

Hexokinase ATP + Cir , C14-GP + ADP, (1)

2ATP+ 2Ci4-glucose dexokinase 2Ci4_Gp+ 2ADP, (a)

Adenylate kinase 2ADP �9 ATP +AMP. (b)

When the two reactions are summed, the equation takes the following form:

ATP + 2C14-glucose , 2CI4-GP + AMP, (c)

(2)

2ADP Adenylate kinase~ ATP + AMP, (a)

,, Hexokinase _ ATP + C~-glucose , C~-GP + ADP. (b) (3)

When the two reactions are summed, the equation takes the form:

ADP + Cl4-glucose L Ct4-GP + AMPo (c)

Thus, if the preparation contains only ATP, then the ratio of the amount of Ci4-GP formed in reaction

(2) to the amount formed in reaction (i) will be equal to 2. Cases in which an excess over this value is ob-

served will indicate that an impurity of ADP is present in the preparation; this can be determined according

to the following formula: y-----x--2x,

u M. Bekhterev Psychoneurological Institute, Leningrad. Translated from Khirniko-Farmatsevti- cheskii Zhurnal, No. 2, pp. 40-42, February, 1967. Original article submitted October 29, 1966.

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TABLE 1. Content of ATP and ADP in an ATP Prepara t ion Produced by the Ivanovo Meat Combine (Series No. 19; 1965)

ATP content in preparation

laTP I taken by ATP determined weight

" (in ! micro- I o, moles I "~ micro- n moles) I M ~m

I5] 5 2,49_+0,03149,94-0,57 ] i5

ADP content in preparation zl x M+_m n [ M+_m

5,16+--0,02 ] 15 I 4,98--+0,06

ADP deter- mined _

P microJ ]moiesl 0,01 I 0,18 3,1

Other impu- rides (in %)

47,0

where y is the amount of the ADP impuri ty (in micromoles) ; z is the amount of Ct4-GP (in micromoles) formed in react ion (1).

We conducted 15 investigations of an "average" preparat ion of ATP-Na, produced by the Ivanovo Meat Combine (see Table 1). The ATP content in the prepara t ion is 49.9 �9 0.57% (M • m), while the ADP content, while s tat is t ical ly significant, is negligibly small (3.1%).

An analysis of prepara t ions of ATP-Na produced by the Hungarian f i rm "Reanal" and the Amer ican firfn "Calbiochem" indicated that the prepara t ions contained 78.1 + 1.7 and 88.7 ! 1.1% ATP, respect ively.

E X P E R I M E N T A L

The following were poured into a centrifuge tube (10 ml volume) where ATP was determined (sample Aol): 0.2 ml of 1-C14-glucose, containing 6 mic romoles of the substance with a total activity of 0,53 # Ci (exact ly!) ; 0.2 ml of pre l iminar i ly neutral ized ATP, containing 5 mic romoies of the substance (exactly!); 0.2 ml MgCI2, containing 5 mie romoles of the substance; 0.2 ml hexokinase (1 mg of enzyme in 0.2 ml of 0.2 M borate buffer with pH 7.4). The test tube was placed in a constant t empera ture bath (37 ~ for 1 h, then 1.5 ml of a 5% ZnSO 4 solution and 1.5 ml of a 0.3 N solution of Ba(OH)2 and 0.2 ml of adenylate kinase were added to it, and after 20 rain the sample was centrifuged at 3000 rpm for 13 rain. The supernatant liquid was drawn off, and applied on a target (16 mm diameter) in a volume of 0.1 ml. F rom one sample, liquid was applied on three targets, which were dried for 2 h in a thermos ta t at 75 ~ A second parallel sample was p repared analogously. The samples were p repared for the determinat ion of ATP + ADP (Ao2) in the same way as sample Aol, but adenylate kinase was added to them simultaneously with the hexokinase. Stan- dard samples were p repared in the same way as descr ibed for the experimental samples A % but they posses s a zero t ime of incubation with the enzymes; hexokinase and adenyiate kinase were added after the precipi tat ing reagents ZnSO 4 and Ba(OH)2. The radioactivi ty of the s tandard (Ac) and experimental (Ao 1 and Ao2) samples was determined for 3 rain on a T -25 -BFL end-window counter (thickness of mica i mg/cm2), connected to the B-2 rad iometer .

The amount of C14-GP (in micromoles) , formed in samples Aol, was calculated according to the formula:

Ac-- Ao,.6 A c

and in sample Ao2-according to the formula:

A c - - A o 2 . 6

Ar

1.

L I T E R A T U R E C I T E D

S. P. Colowick and N. O. Kaplan, Methods in Enzymology, New York~ 2, 598 (1955).

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