QUANTITATIVE RADIOENZYMATIC

AD ENOSIN EDIP HOSP HORI C ACID

G. I . M e e r o v

DETERMINATION OF

UDC 615.739.653-0.14.3:[543.52+577.15.087

Adenosinediphosphoric acid (ADP) preparat ions are widely used in basic and applied medical bio- chemical r e sea r ch . Our repor t concerns a rapid, simple and rel iable radioenzymatic method for quanti- tative ADP determinat ion in such prepara t ions ;* the method is based on the use of two conjugated enzymatic react ions: that of adenylate kinase (ATP myokinase: AMP-phosphotransferase ; KP 2.7.4.3. [1]) and hexo- kinase (ATP: D-giucose-6-phosphot ransferase ; KP 2.7.1.1. [1]), and a known excess of C 14 glucose. As a resul t of the reaction, an equimolecular amount of C14-glucose-6-phosphate (C14-G6P) is formed which is removed from the medium as the bar ium salt. Since the radioact ivi ty of the C 14 glucose solution before the react ion is known and radioactivi ty of C14-G6P can be determined after its precipitation, the unknown ADP amount can be determined.

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

Analysis

A centrifugal tube (i0 ml) is filled with 0.2 ml Cl4-glucose,~ containing 6 ~moles of the substance

with a total activity of 0.53 # Ci (accurate !); 0.2 ml of previously neutralized ATP containing 5 #moles sub- stance (accurate !); 0.2 ml MgCl 2 containing 5 gmoles substance; 0.2 ml adenylatekinase,~ and 0.2 mlhexo-

kinase** (0.5 mg enzyme in 0.2 ml of a 0.2 M borate buffer with pH 7.4. The test tube was placed in a water thermostat (37 ~ for an hour. Then 1.5 ml of a 5% ZnSO 4 solution and 1.5 ml of a 0.3 N Ba(OH)2 solu- tion were added; the test tube was then left to rest for 20 minutes, after which it was centrifuged at 3000 r.p.m, for 15 rain. The supernatant fluid was syphoned off and 0.i ml was introduced into the target (di- ameter 16 ram). Three targets were filled with the fluid from each tube, and dried at 75 ~ for 2 hours in the thermostated cell. Another parallel test was prepared in the same way. Standard samples were prepared like the experimental specimens, except for zero time incubation with hexokinase (hexokinase was added after the preeipitants ZnSO 4 and Ba(OH)2 ). Radioactivity (in pulses per minute) was determined both for standards (Ac) and the experimental (Ao) samples with an endwindow counter T-25-BFL for 3 rain (thick- ness of the mica 1 mg/cm2); the counter was connected with a radiometer B-2.

The percentile content of ADP in the preparation was calculated according to the equation

Q __ (Ac-- Ao).lO0 Ar ' (1)

6

where Q is the ADP content in the preparat ion (%); (Ac-Ao) is the radioactivi ty of the precipitate c o r r e s - ponding to the unknown ADP amomlt; 100 is the percent i le expression; Ac-5 /6 is the radioactivi ty per 5 mic romoles of 100% ADP.

* The paper was presented at the meeting "All-Union Communications on Methods for Obtention andAnalysis of Biochemical Prepara t ions , " Riga, September 16, 1966, and also at the 507th combined sess ion of the Leningrad Division of the All-Union Biochemical Society and the Biochemis t ry Section of the Leningrad Society of Physiologis ts , Biochemists , and Pharmacologis t s , September 17, 1966.

~We used 1 C14-glucose. SThe adenylatekinase (myokinase) preparat ion, without any other enzymatic activity, was prepared accord -

ing to Colowick and Ka l ' ka r ' s method [2]. **In our studies we used a crys ta l l ine hexokinase preparat ion from the f irm Cyclo, USA.

V. M. Bekhterev Leningrad Psychoneurologic Institute. Translated from Khimiko-Farmatsevtiches- kii Zhurnal, No. 6, pp. 45-47, June, 1967. Original article submitted December 17, 1966.

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3500

�9 3000

�9 ~ .~ zsoo .o.E

o o 2000

~,soo "~ ~ t000

5-00

i i i i I ,

1 z a 4 5 ADP content in sample (p moles)

Fig. 1. Functional r e l a - t ionship between ADP amount in the sample and radioac t iv i ty of the p r e -

After t rans format ion , Eq. (1) a s s u m e s a s imp le r form;

120 (Ac -- Ao) Q = Ar (la)

V e r i f i c a t i o n o f M e t h o d a n d R e s u l t s o f S t u d i e s

The studies de te rmined that under these exper imenta l conditions the re is a l inear re la t ionship between the amount of ADP in the sample and the radioac t iv i ty of the prec ip i ta te (Ac-Ao) of C14-G6PBa (see Fig. 1).

This radioenzymat ic method also se rved for de terminat ion of ADP content in two p repara t ions of the ADP sodium sal t supplied by the Hun- gar ian f i rm "Reanal."*

P repa ra t ion 1. The following percent i le contents of ADP were found (14 tests) : 70.5; 70.4; 70.0; 70.2; 70.4; 70.5; 71.4; 71.7; 71.4; 70.6; 68.1; 68.4; 67.6;66.4 . A r i t h m e t i e m e a n : 69.8, Mean square: ~1.5.

cipi tate (C14-G6PBa) P repa ra t ion 2. The following percen t i l e contents of ADP were found

(27 tests) : 74.6; 75.8; 76.3; 72.3; 72.4; 70.4; 70.5; 69; 70.1; 69.9; 73.3; 73; 72.6; 69.7; 69.8; 69.3; 70.4; 73.2; 73.5; 74.9; 75.8; 74.4; 70.4; 68.0; 68.1; 67.9; 67.5. Ar i thmet ic mean: 71.6. Mean square: :~2.6.

The method desc r ibed for ADP determinat ion can be used only for ch romatograph ica l ly pure ADP p repa ra t ions . In the p re sence of any ATP admixture (which is improbable) , a control tes t mus t be con- ducted for the quantitat ive de te rmina t ion of ATP. This t es t is the same as the exper imenta l ADP test , except for ze ro incubation t ime with adenylatekinase . Then C14-G6P will be formed in the sample contain- ing adenylatekinase and hexokinase; the C14-G6P amount will be

Z = y + 2x (2)

where y is the m i c r o m o l e amount of CI4-G6P equimolecular to the ADP content in the sample; 2x is the m i c r o m o l e amount of C14-G6P formed by x ATP m i c r o m o l e s . The sample containing hexokinase~ will con- tain C14-G6P in anequimolecu la r amount to x m i c r o m o l e s ATP. Since Z and x a r e known, fo rmula (2) s e r v e s to find the ADP content in the prepara t ion , f i r s t in m i c r o m o l e s (y), then in percent .

LITERATURE CITED

1. A . E . Braunshtein (editor), Enzyme Classif icat ion and Nomencla ture [in Russian], Moscow (1962). 2. S . P . Colowick, in the book: S. P . Colowick and N. O. Kaplan, Methods in Enzymology, Vol. 2, New York

(1955), p. 598.

*According to data supplied by the company, these ADP prepa ra t ions a r e ch romatograph ica l ly pure. t in this s ample adenylatekinase has zero incubator t ime .

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