ANALYTICAL BIOCHEMISTRY 90, 829-83 1 (1978)

Specific Enzymatic Determination of Adenosine Triphosphate

The well-known soluble kinases are not specific for ATP (1). All these enzymes convert ATP as well as GTP, ITP, CTP, and UTP. although at different rates. The only exception is adenylate kinase (1). However, with this enzyme, a direct determination of ATP in tissue extracts which contain both the di- and mononucleotides is not possible.

Phosphoglycerate kinase from various sources is specific for ATP, GTP, and ITP and does not react with the pyrimidine nucleotides (2). Now, however, it was found that phosphoglycerate kinase from the blue alga Spirulina platensis does not convert GTP and ITP. With this enzyme, therefore, it is possible to specifically determine ATP in tissue extracts or in mixtures of nucleotides. In the same test, GTP and ITP can be determined by adding phosphoglycerate kinase from yeast or from other sources (2).

MATERIALS AND METHODS

ATP, GTP, and ITP were determined in 100 mM triethanolamine-HCl (pH 7.5) containing 8 mM MgSO,, 0.3 mM NADH. 10 mM 3-phosphoglycerate, glyceraldehyde phosphate dehydrogenase (5 U/ml), and phosphoglycerate kinase (4 U/ml). The measurements were performed at 365 nm and 25°C. The extinction coefficient used for NADH was 3.4 x IO3 rv-‘cm-’ (4). Activity of phosphoglycerate kinase (30 mu/ml) in the presence of different nucleotides was tested with the same reaction mixture, the concentration of ATP or other nucleotides being 2.7 mM. Activity with ATP was taken as 100%.

The blue alga Spirulincr platensis was cultivated for 14 days (5) and collected by centrifugation at 28001: for 30 min. The cells were then disrupted by sonication with three bursts of full power for 60 s (Branson Model S75 sonifier), and debris was removed by centrifugation at 300,OOOg for 2 h. Phosphoglycerate kinase was isolated from the supernatant by affinity chromatography as described previously for phosphoglycerate kinases from other sources (3).

All enzymes, substrates. and buffers were purchased from Boehringer Mannheim GmbH.

RESULTS AND DISCUSSION

Since enzymatic determinations are convenient in that they are simple and time-saving, in contrast to chromatographic and other techniques, a

829 0003.2697/78/0902-0829$02.00/O Copyright 10 1978 by Academic Press. Inc. All rights of reproduction m any form reserved.

830 SHORT COMMUNICATIONS

TABLE I

NUCLEOTIDE TRIPHOSPHATE SPECIFICITY OF SPIRULINA PLATENSIS AND YEAST PHOSPHOGLYCERATE KINASE

Phosphoglycerate kinase

Nucleotides

ATP dATP GTP dGTP ITP dITP CTP dCTP UTP dUTP

Spirulinu Yeast (2) (% of activity)

100 100 4 20 0.4 55 0 35 0.1 70 0 40 0 0 0 0 0 1 0 0

specific enzymatic evaluation of ATP seems desirable. The known and commercially available soluble kinases are not specific for ATP. In a screening of nucleotide specificity of phosphoglycerate kinase from bacteria, plants, and animals, the enzyme of the blue alga Spirulina was found to be the only one with a high specificity for ATP. Among all the nucleotides tested in the enzyme assay, only deoxy-ATP was utilized in an appreciable time with a rate of 4% of ATP. For GTP and ITP, the relative

0.8 -

PGK

-1

0.4 ,1,

0 1 3 5 7 9 11 min

FIG. 1. A typical nucleotide assay with phosphoglycerate kinase (PGK) from Spiruha and yeast. (A) The assay mixture was composed as stated under Materials and Methods, and contained 32 pM ATP. GTP. and ITP. The reaction was started with PGK from Spirulina. (B) After 4 min. PGK from yeast was added. (C)The sam,e assay mixture was used as in A. but the reaction was started immediately with PGK from yeast. Absorbance was followed at 365 nm. reflecting a decrease in NADH.

SHORTCOMMUNICATIONS 831

rate was significantly lower than 1%. No other nucleotides were utilized for the phosphorylation of 3-phosphoglycerate (Table 1).

In a mixture of ATP, GTP, and ITP in equimolar concentrations, only ATP was quantitatively determined by phosphoglycerate kinase from Spinllina. After adding the yeast enzyme, GTP and ITP reacted too (Fig. 1).

ACKNOWLEDGMENTS

The authors are grateful to Prof. Dr. H. v. Witsch and Dr. A. Muntz (Technische Universitlt Miinchen, Lehrstuhl fiir Botanik) for the cultivation of Spitdim. Special thanks is extended to Dr. G. Falkner (Institut fur Limnologie der Akademie Wien, Gsterreich) for liberally providing us with information about Spirulincl. Thanks also to I. U. Freier for expert technical assistance. M. L. Everett is thanked for proof-reading and preparing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft, Sonder- forschungsbereich 51.

REFERENCES

I. ,Bergmeyer, H. U. (1974) Methoden derenzymatischen Analyse. p. 2142. Verlag Chemie. WeinheimiBergstrasse.

2. Krietsch, W. K. G.. and B&her. Th. (1970) Eltr. J. Biochrtn. 17, 568-580. 3. Kuntz, G. W. K.. Eber. S.. Kessler. W.. Krietsch, H.. and Krietsch. W. K. G. (1978)&r.

J. Biochem. 85, 493-501. 4. Ziegenhorn. J.. Senn. M., and Biicher, Th. (1976) C/in. C/wtn. 22, 151-160.

5. Runkel. H. A.. Heussler. P.. and v. Witsch. H. (1970) Ber. D. Bat. Cr.\. 63, 567-577.

WOLFGANG K. G. KRIETSCH GUNTER W. K. KUNTZ