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Previous work demonstrated that the 3H label in [3',5',9-3H]folic acid exchanges in the rat (Barford & Blair, 1975) and that DEAE-cellulose chromatograms of liver extracts from rats receiving [3H]folic acid showed the anticipated peaks at high tube number, which did not appear when ['*C]folic acid was administered (Beavon, 1973). We have been able to demonstrate the presence of high-molecular-weight folates in extracts of livers and tumours by using Sephadex G-15 chromatography. DEAE-cellulose chromatography did not show peaks at high ionic strength as reported for folate polyglutamates. A folate polyglutamate would be more highly charged than a monoglutamate and should be eluted from the DEAE-cellulose column after the monoglutamates. This does not happen with the high-molecular-weightfolates obtained from livers of rats receiving oral doses of [2-'4C]folic acid. The experiments reporting the presence of folate polyglutamates in the literature have nearly all used [3',5',9-3H]folic acid as a marker for polyglutamate formation. Osborne-White & Smith (1973) reported the presence of peaks at high ionic strength on DEAE-cellulose in extracts of livers from sheep after giving large doses of [2-'*C]folic acid over several days. Lavoie et al. (1974) reported that [5-1*C]methyltetrahydrofolatedid not form polyglutamates in lymphocytes, and used the absence of peaks at high ionic strength on DEAE-cellulose chromatography to support this claim, but reported the presence of polyglutamates in lymphocytes after [3H]folicacid incubation. Our work has failed to produce conclusive evidence for the presence of folate polyglutamates in rats. We have demonstrated the presence of a high-molecular-weightform of folate in rats, but it does not seem to have the necessary charge for a polyglutamate. It is possible that the high-molecular-weight fraction contains other amino acids, resulting in a net charge that is little different from the charge on 5-methyltetrahydrofolate, and that the 'peaks at high tube number' reported by other workers are artifacts caused by using r3H]folicacid of very high specific radioactivity. Barford, P. A. & Blair, J. A. (1975) Pteridine Chem. Proc. Znt. Symp. 5th in the press Beavon, J. R.G. (1973) Ph.D. Thesis, University of Aston Blair, J. A., Pearson, A. J. & Robb, A. J. (1975)J. C&m. Soq. f i r k i n Trans. II, 18-21 Brown, J. P., Davidson, G. E. & Scott, J. M. (1974) Biochirq. Blophys. Acta 343,78-88 Lavoie, A., Tripp, E. & Hoffbrand, A. V. (1974) Clin.Sci. Mol. Med. 47,617-630 Noronha, J. M. & Silverman, M. J. (1962) J. Biol. Chem. 237, 3299-3302 Osborne-White, W. S. & Smith, R. M. (1973) Biochem. J. 136,265-278 Shin, Y.S.,Williams, M. A. & Stokstad, E. L.-R. (1972) Biochem. Biophys. Res. Commun. 47, 35-43

Hydrolytic Rupture of Ascorbate by Adenosine 3' :5'Xyclic Monophosphate Phosphodiesterase" SHERRY LEWIN Department of Postgraduate Molecdar Biology, North East London Polytechnic, London E l 5 4LZ, U.K.

Ascorbic acid is a y-lactone which on hydrolytic rupture should give the corresponding acid, thus: CHZOH

CI-I,OH

I HOCH

I

q-> 0

H OH

0-

Ascorbate

HOCH I/OH

7\ c-cI

OH /

,c=o

I

OH 0-

Delactonized ascorbate

* Not presented at the Meeting, owing to the death of Dr. Lewin. VOl. 4

72

BIOCHEMICAL SOCIETY TRANSACTIONS

Cyclic AMP and other cyclic nucleotides can be hydrolytically ruptured with specific phosphodiesterase with the formation of the corresponding acids. Certain theoretical considerations, including the similarity of the ring structures of cyclic AMP and the ascorbate anion, led me to conclude that the ascorbate anion should be capable of hydrolytic rupture by a phosphodiesterase capable of hydrolysing cyclic AMP or cyclic GMP (Lewin, 1974~). The pH-stat method was used to check this prediction. As a result I have established that phosphodiesterase preparations capable of hydrolysing cyclic AMP can also hydrolyse the ascorbate anion. Fig. 1 illustrates the uptake of NaOH by a phosphodiesterase system to which ascorbate was added. The essential conditions for demonstrating the formation of acid, by subjecting ascorbate to hydrolysis by phosphodiesterase, and avoiding anomalous results were found to include the following. (i) Absence of light. Exposure to daylight or artificial light at the preparatory stage or in the reaction mixture results in anomalous results which can be explained in terms of the formation of ascorbic free radicals and associated oxidation products such as dehydroascorbic acid. (ii) Freshly prepared solutions of ascorbate and of phosphodiesterase. Several-daysold ascorbate solution (although refrigerated at approx. 4°C) can also give rise to anomalous results. These can be understood if one considers that the ascorbate anion is under electrostatic stress and tends to interact with water at the double bond and/or to undergo delactonization by hydrolysis. Aging of phosphodiesterase solutions (although refrigerated) appears to have an adverse effect on the reproducibility of the results, often also involving considerable increase in the induction period (Lewin, 1976). The attainment of an ‘end point’ lower than the strict 1gequiv. can be understood in terms of enzyme reversibility. Deactivation/degradation of ascorbate solutions is normally retarded by the use of chelating agents. However, their use is precluded in this hydrolysis because phosphodiesteraserequires for its activity M$+, MnZ+,Znz+or Coz+(Cheung, 1970). I have suggested (Lewin, 197&,6) that ascorbate can affect physiological activity by interacting with phosphodiesterase and thereby inhibiting the hydrolysis of cyclic AMP. This proposal has been substantiated by Van Wyk & Kotze (1975) and Tisdale (1975). The significance of the hydrolysis of ascorbate by phosphodiesterase lies in its

0

I

Time (h)

Fig. 1. pH-statting of the reaction of ascorbate with bovine heart cyclic AMP phosphodiesterase Anaerobic conditions were maintained at 30°C in the absence of light; 3ml of the reaction mixture contained 0.18mg of phosphodiesterase (from a freshly prepared stock solution) supplied by Sigma, SOmM-NaC1, 0.1 mM-MgC12, 0.1mM-ascorbate (freshly prepared). Titrant was 3mM-NaOH. (l), (2) and (3) represent curves obtained at different times under stated conditions. 1976

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effect on vitamin C assay in tissue where oxidationlreduction techniques are used, since both the hydrolysed product of ascorbate (delactonized ascorbate) and ascorbate are equally reduced. On the other hand delactonized ascorbate is not biologically active. This can result in confusion with respect to vitamin C activity (Lewin, 1976). I thank Mr. R. Stoker for invaluable technical assistance and rechecks.

Cheung, W. Y. (1970) Ado. Biochem. Psychopharmacol.3, 51-65 Lewin, S. (1974~) Vitamin C: Recent Aspects of its Physiological and Technical Importance, pp. 221-252, Applied Science Publishers, Barking Lewin, S . (19746) Biochem. SOC.Trans. 2,922-924 Lewin, S . (1976) The MolecuIar BioIogy and Medical Potential of Vitamin C, Academic Press, New York and London, in the press Tisdale, M. J. (1975) Biochem. Biophys. Res. Commun. 62,877-881 Van Wyk, C. P. & Kotze, J. P.(1975) S. Afr. J. Sci. 71, 28-29

Production of the Superoxide Radical by Horseradish Peroxidase BARRY HALLIWELL and SUNIL AHLUWALIA Department of Biochemistry, King’s College London, Strand, London WC2R 2LS, U.K.

Horseradish peroxidase catalyses the oxidation of dihydroxyfumaric acid (Swedin & Theorell, 1940; Chance, 1952), and aromatic compounds can be hydroxylated by this reaction mixture (Buhler & Mason, 1961). Since peroxidase may be involved

Table 1. Effect of reagents on the oxidation of dihydroxyfumarate and the hydroxylarion of pcoumaric acid by horseradish peroxidase The reaction mixture contained, in a total volume of 1.00m1, enzyme, p-coumaric acid (2.5pmol), dihydroxyfumaric acid (30pmol), KH2P04(8.3pmol) and sufficient KOH to adjust the pH to 6. Incubations were carried out for 0.5h at 25°C and caffeic acid production was assayed as described by Halliwell(l975). 100% corresponded to a rate of 60nmol of caffeic acid formed in 0.5h. Oxidation of dihydroxyfumarate was followed by the decrease in E300 (althoughp-coumaric acid also absorbs light at 300nm, very little is used up during a 30min incubation). Samples (10~1)were removed from the reaction mixture at intervals, added to lOml of water and the absorbance recorded at once. Units of superoxide dismutase were as described by McCord & Fridovich (1969). %rate ofp-coumarate % rate of dihydroxyhydroxylation fumarate oxidation 100 100 None 10 0.02rn~-CuSO~ 0.1mM-CuS04 0 60 20 O.O5rn~-MnCl~ 0 175 0.2mwMnC12 45 1 unit of superoxide dismutase lounits of superoxide dismutase 0 80 89 95 *lounits of heated superoxide dismutase 103units of catalase 20 20 1 . 5 103units ~ of catalase 0 14 0.1 M-mannitol 60 100 0.1Methanol 54 100 0.1 M-Tris 46 92 5Om~-sodiumformate 8 76 * Heated at 100°C for 30min and cooled before use. Reagent added

Vol. 4

Hydrolytic rupture of ascorbate by adenosine 3':5'-cyclic monophosphate phosphodiesterase.

560th MEETING, OXFORD 71 Previous work demonstrated that the 3H label in [3',5',9-3H]folic acid exchanges in the rat (Barford & Blair, 1975) and tha...
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