... the enzymes which catalyse the interchange between PG-ATP and free .... Wulff, K. & Doppen, W. (1985) Method of Enzymatic Analysis. (Bergmeyer, H. U., ed.) ...
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BIOCHEMICAL SOCIETY TRANSACTIONS
The metabolism of an entirely new cellular nucleotide derivative: phosphoglyceroyl-ATP BRINDA PATEL and JOHN MOWBRAY Department of Biochemistry, University College London, Cower Street, London WCIE 6BT, U . K . Langendorff-perfused rat hearts have been found to show rapid and extensive oscillations in total soluble adenine nucleotide content over the first hour of perfusion. The observed variations could not be explained as metabolism of any known precursor, derivatives or polymer, and it was proposed that some hitherto unrecognized species of adenine nucleotide must exist 11I. Specific labelling of heart nucleotides with 1814C]adenosinerevealed a labile highly phosphorylated oligomeric species at specific radioactivity equilibrium with ATP [ 21. A combination of selective digestion, chromatography, ” P n.m.r. and fast-atom-bombardment mass spectrometry suggested that this species is a linear oligomer of ATP and 3-phosphoglycerate of the form ppp( S’A3’p3-glyceroyll-ppp),,S’A3’p3-glycerate[3]. We have provisionally named this oligophosphoglyceroyl-ATP (PG-ATP) and work is currently in progress to confirm its structure by chemical synthesis. Identical species have been found in rat kidney [4] and liver (B. Patel, C. Cove & W. S. Hutchinson, unpublished word). The very labile mixed anhydride bond in PG-ATP can be stabilized by Mn2+or Mg?’ ions and provided Mg2+/ Mn2’ concentration in the buffer is above 2 mM, PG-ATP is not spontaneously degraded. The rapid attainment of specific radioactivity equilibrium with soluble nucleotides, presumably resulting from a fast and complete turnover of PG-ATP, suggests a dynamic role for this oligomer. It seems likely that PG-ATP has a major role to play in ATP homoeostasis and that the enzymes which catalyse the interchange between PG-ATP and free nucleotide are potentially important regulatory steps in nucleotide metabolism. This report describes the partial purification of an enzyme from rat liver mitochondria which specifically hydrolyses PG-ATP oligomer to the monomer unit. A crude assay used to survey cell fractions for their ability to degrade PG-ATP depends on the fact that the [‘“CIPGATP oligomer is readily precipitated from 50% (v/v) cold aqueous ethanol and can be trapped on a millipore filter (0.45 p m ) and assayed for radioactivity. The breakdown products remain in solution and pass through the filter. A purified rat liver mitochondrial fraction was separated from other vesicle fractions by combined differential and Percoll density gradient centrifugation [ 5 ] .The findings from this (not shown) were that the major part of the PG-ATP cleavage activity was found along with a succinoxidase peak in the light mitochondrial fraction separated from the main lysosomal and endoplasmic reticular fractions marked by acid phosphatase and glucose-6-phosphatase activities, respectively. The PG-ATP degradation activity was latent until the vesicles were disrupted by freeze thawing and sonication. This suggests that the enzyme is either intramitochondrial or present within the intermembrane space. This activity has been further purified from soluble extracts of rat liver mitochondria by 60% saturated (NH&SO, precipitation followed by f.p.1.c. on a Mono Q anion-exchange column using a linear 0-1 M-NaCl gradient in 20 mM-Bis-Tris propane at pH 6.6. The soluble product(s) of PG-ATP cleavage by the partially purified activity were subjected to two-dimensional thin-layer chromatography on PEI-cellulose in (NH,),BO,, pH 7.0, and LiCI/HCOONa pH 2.0 [3].This showed that the only purine-containing product co-chromatographed with Abbreviation used: PG-ATP, oligophosphoglyceroyl-ATP.
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Fig. 1. Lineweuver-Hitrk p l o ~of tlir enzyrnic. hreukdown of 1’G-A TI’ Between 7 and 40 nmol (adenosine equivalents) of [“CIPGATP in 10 mM-Tris/HCI buffer, pH 7.4, and 1 0 mM-MgCI, was incubated with the partially purified enzyme (440 pg of protein) at 37°C for 2 min. The reaction was terminated by the addition of 50% ( v / v ) cold aqueous ethanol and the samples were left at - 20°C for 2 h before being filtered and assayed for radioactivity as described in the text.
authentic ATP. This identification was, however, not confirmed by either the firefly bioluminescent assay 161 nor the hexokinase/glucose-6-phosphatedehydrogenase assay [ 7 1. Moreover, this product had a phosphate/purine ratio of greater than 3 : 1. Examination of the solubilized cleavage product in acid conditions by t.1.c. on silica gel in ethyl acetate/formic acid/water ( 3: 1 : 1, v/v) showed that one component co-migrates with authentic glyceric acid. The observation that the purine breakdown product migrated with ATP in (NH,),BO, suggests that both the 2’ and 3’ O H groups of the ribose are free [8]. Hence, a tentative proposal for the identity o f the hydrolysis produce would be: 3 phosphoglyceroyl 5 ‘-triphosphoadenosine (3pGripppS’A). Under conditions where the breakdown o f AG-ATP was linear, the apparent K , for the degradation o f oligomeric PG-ATP by this partially purified activity was estimated from Lineweaver-Burk plots to be about 35 p~ (Fig. 1). The relatively low V,,,,,, ( 6 nmol min- I mg- I ) found suggests loss of activity has taken place during purification and that activating factors may be required, given the very rapid interconversion of PG-ATP with soluble nucleotides in intact cells. We are grateful to the M.R.C. and to the British Heart Foundation for project grant support.
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633rd MEETING, LONDON I . Bates, D. J., Perrett, D. & Mowbray, J. (1978)Biochem. J. 176,
485-493 2. Mowbray, J., Hutchinson. W. L., Tibbs, G. R. & Morns, P. G. (1984) Biochern. J. 223,627-632 3 Hutchinson, W. L., Morris, P. G. & Mowbray, J. ( 1 986) Biochem. J. 234.623-627 4. Hutchinson. W. L., Ratcliffe, P. J. & Mowbray, J. (1986) Biochern. J. 240,597-599 5. Blume. H. (1979)Archiv. Pharmazie. 312,561-572
6. Wulff, K. & Doppen, W. (1985) Method of Enzymatic Analysis (Bergmeyer, H. U., ed.), 3rd edn., vol. VIII, pp. 357-360. Academic Press, NY 7. Lamprecht, W. & Trautschold, I. (1974) Methods of Enzymatic Analysis, 2nd edn., vol. IV, pp. 2 10 1-2 1 10 8. Neuhard, J., Randerath, E. & Randerath, K. ( 1 965) Anal. Hiochem.
13,211-222
Received 23 November 1989
Diagnosis of respiratory chain defects using 'H n.m.r. spectroscopy in vitro JOAN E. M. RAFTER,* RONALD A. CHALMERS,t ANDREW JOHNSON$ and RICHARD A. ILES* *Medical Uiiit, The London Hospital Medical College, Whitechapel, London E l IRB, U.K.; ?Department of Child Health, St George's Hospital Medical School, London SWI 7 ORE, U.K . and $Department of Clinical Biochemistry, Queen Elizabeth Hospital, London E2 8PS, U.K. Lactic acidosis is a frequently occurring cause of metabolic acidosis in the neonatal period and since it may arise as a result of a diversity of acquired and inherited disorders differential diagnosis is of critical importance [I].However, such diversity makes diagnosis extremely difficult, particularly as many of both the clinical and biochemical indications are equivocal. Initial biochemical investigations often involve measurement o f plasma lactate, followed by gas-liquid chromatography/mass spectrometry of urine. We have been using 'H n.m.r. spectroscopy to investigate several inherited disorders of organic acid metabolism [2-41. The advantages of the
technique are first that it is extremely rapid as untreated urine is used and a spectrum can often be obtained within 10 min of n.m.r. analysis. Secondly, whereas classical analytical techniques are usually restricted to one class of compounds, 'H n.m.r. spectroscopy detects not only organic acids, but also neutral and basic components which may provide additional metabolic information [3, 51. We have analysed urine samples from a number of neonates with disorders of lactate metabolism including cytochrome b and cytochrome c deficiency, pyruvate dehydrogenase deficiencies, and disorders for which no diagnosis was available. The respiratory chain and pyruvate dehydrogenase deficiency disorders were diagnosed by tissue biopsy of muscle or liver. Random, diagnostic urines which had been stored at - 20°C were used for n.m.r. analysis. Aliquots of urine (0.5 ml) were placed in a 5 mm n.m.r. tube to which 50 pI of ? H 2 0 and 20 p1 of 500 m~-3-trimethykilyl2,2,3,3-tetradeuteropropionate (TSPd4) were added to lock the magnetic field and to act as a chemical shift reference standard, respectively. The samples were run at room temperature
.ac Cr
Ala I
-
OHB
!
Cit
3.0
2.8
2.6
OHB
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
6 (p.p.m.) Fig. 1. ' H i1,m.r. spectra of urine from (a) u patient with a deficiency in the cytochrome-c oxiduse complex and (b) a patient with a pyruvate dehydrogeriuse (E,,) deficiency The spectra were accumulated at 250 MHz. Ac, acetate; Ala, alanine; Cit, citrate; Cr, creatine; Crn, creatinine; DMA, dimethylamine; DMG, dimethylglycine; Lac, lactate; OHB, 3-hydroxybutyrate; OHV, 3-hydroxyisovalerate; PD, propan- 1,2-diol; Sar, sarcosine; Succ, succinate; TMAO trimethylamine-N-oxide; Val, valine.
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