Biochem. J. (1982) 203, 707-715 707 Printed in Great Britain The effect of tetrahydrofolate on the reduction of electron transfer flavoprotein by sarcosine and dimethylglycine dehydrogenases Daniel J. STEENKAMP and Mazhar HUSAIN Molecular Biology Division, Veterans Administration Medical Centre, San Francisco, CA 94121 and Department ofBiochemistry and Biophysics, University ofCalifornia, San Francisce, CA 94143, U.S.A. (Received 7 December 1981/Accepted 23 February 1982) Pig liver electron transfer flavoprotein (ETF) is rapidly reduced by sarcosine and dimethylglycine dehydrogenases to the anionic semiquinone form, the subsequent formation of the flavoquinol form being a much slower process. In the presence of tetrahydrofolate the yield of anionic semiquinone at the end of the rapid phase of reduction of ETF is only about 10% less than without tetrahydrofolate, as judged by e.p.r. spectroscopy. Tetrahydrofolate does not alter the rate of reduction of ETF by either sarcosine or dimethylglycine dehydrogenase. Nevertheless, it was clearly demon- strated that tetrahydrofolate is a substrate for both sarcosine and dimethylglycine dehydrogenases and is converted to N',10-methylenetetrahydrofolate. Sarcosine and dimethylglycine dehydrogenases creased incorporation of radioactivity from the (EC 1.5.99.1 and 1.5.99.2) are flavoproteins which N-methyl group of sarcosine into serine, sarcosine catalyse the oxidative N-demethylation of sarcosine oxidase activity was unaffected (Dac & Wriston, and dimethylglycine (MacKenzie & Abeles, 1956; 1958), indicating that H4PteGlu did not participate MacKenzie & Frisell, 1958). Although oxidative directly in the oxidation of sarcosine. More recently, N-demethylations are of general occurrence in however, the identification of dimethylglycine and biochemistry, the immediate fate of the methyl sarcosine dehydrogenases as folate-binding proteins groups which are oxidized to the oxidation level of in rat liver mitochondria by Wittwer & Wagner formaldehyde is not unequivocally understood (1980, 198 la,b) led to the proposal that these because of facile addition reactions of formaldehyde enzymes use H4PteGlu as a co-substrate in the direct with nucleophiles. It is not always apparent whether synthesis of 5,10-CH2-H4PteGlu from sarcosine and adduct formation of formaldehyde with biologically dimethylglycine. However, since formaldehyde con- important nucleophiles such as N-5 of H4PteGlu or denses non-enzymically with H4PteGlu to form thiol groups, as in glutathione, is enzyme-catalysed 5,10-CH2-H4PteGlu, positive identification of 5,10- or not. The sarcosine and dimethylglycine de- CH2-H4PteGlu as the immediate reaction product in hydrogenases provide interesting cases in point. The the oxidative N-demethylation of dimethylglycine rapid incorporation of radioactive carbon from the was not possible (Wittwer & Wagner, 198 lb). methyl group of sarcosine, but not from free form- If indeed H4PteGlu is a co-substrate in the aldehyde, into serine was reported by MacKenzie oxidative demethylation of sarcosine or dimethyl- (1955) and Lewis et al. (1978), who postulated that glycine it may be expected that H4PteGlu should 'active' rather than free formaldehyde is formed as stimulate the turnover rate of the sarcosine and a product in the oxidation of sarcosine to glycine. dimethylglycine dehydrogenases markedly, by The nature of 'active formaldehyde', presumably an analogy with the rate-enhancing effect of H4PteGlu adduct, was unclear. While liver mitochondria on the interconversion of formaldehyde and glycine isolated from folate-deficient rats showed a de- to serine by serine hydroxymethyltransferase (Chen & Schirch, 1973). Since H4PteGlu reacts rapidly Abbreviations used: ETF, electron transfer flavo- with the dyes commonly used as electron acceptors protein; H4PteGlu, tetrahydrofolate; 5,10-CH2-H4PteGlu, in assays of flavoprotein dehydrogenases in vitro, N5"0-methylenetetrahydrofolate; 10-CHO-H4PteGlu, the effect of H4PteGlu on the reaction kinetics of 10-formyltetrahydrofolate; SDS, sodium dodecyl these enzymes cannot be ascertained in such assays. sulphate. In this study the effect of H4PteGlu on the rate of Vol. 203 0306-3275/82/060707-09$01.50/1 (© 1982 The Biochemical Society 708 D. J. Steenkamp and M. Husain reduction of the physiological oxidant, the electron (Schnaitman & Greenawalt, 1968) and subjected to transfer flavoprotein (ETF), by purified preparations one freeze-thaw cycle followed by osmotic lysis and of dimethylglycine and sarcosine dehydrogenases sonication (Hoskins & MacKenzie, 1961) to release from pig liver mitochondria is examined. Evidence soluble enzymes. Further fractionation involved confirming the earlier proposals of Wittwer & (NH4)2SO4 precipitation and chromatography on Wagner (1980, 1981b) that 5,10-CH2-H4PteGlu is a DEAE-cellulose. Dimethylglycine and sarcosine direct reaction product of the oxidative N- dehydrogenases were further purified by chromato- demethylation of sarcosine and dimethylglycine is graphy on Sephadex G-150, hydroxylapatite, and a presented. second DEAE-cellulose column. Alternatively, the enzymes obtained from the gel chromatography step Materials and methods were subjected to affinity chromatography as described (Wittwer & Wagner, 198 lb). Chemicals ETF was eluted from DEAE-cellulose chrom- Folinic acid, folic acid, sodium borohydride, atography of the (NH4)2SO4 precipitate at low ionic 5,10-CH2-H4PteGlu dehydrogenase (EC 1.5.1.5), strength and was further purified by chromatography formaldehyde dehydrogenase (Ando et al., 1979) on hydroxylapatite and CM-Sephadex. and c-aminohexyl-agarose were obtained from Sarcosine and dimethylglycine dehydrogenases Sigma and were used without further purification. were assayed as described (Wittwer & Wagner, Molecular weight markers used in polyacrylamide- 1980). Protein was determined by the Lowry (Lowry gel electrophoresis were obtained from BDH. et al., 1951) and biuret (Gornall et al., 1949) H4PteGlu and 5,10-CH2-H4PteGlu were synthesized methods. Polyacrylamide-gel electrophoresis was by modification of established procedures performed in the system of Davis (1964) and with (Scrimgeour & Vitols, 1966; Blair & Saunders, SDS and fi-mercaptoethanol as described (Weber & 1970). Folic acid (20mg) was reduced using 60mg of Osborn, 1969). Sarcosine dehydrogenase was detec- NaBH4 in a final volume of 1.5 ml. After 45 min the ted on gels run under non-denaturing conditions by borohydride was destroyed by acidification with using an activity stain (Wittwer & Wagner, 198lb). glacial acetic acid or 6 M-HCI and the pH was Further experimental details are given in the adjusted to approx. 7.5 with KOH. Untreated folate Figure legends. and dihydrofolate were removed anaerobically under argon by selective batch adsorption to DEAE- Results and discussion cellulose acetate or chloride under conditions where tetrahydrofolate is not adsorbed. In the preparation Properties ofpurified dimethylglycine and sarcosine of H4PteGlu for kinetic experiments it was essential dehydrogenases and ofETF to use chloride as the anion, because sarcosine and Sarcosine and dimethylglycine dehydrogenases, dimethylglycine dehydrogenases are competitively purified by affinity chromatography, migrated as inhibited by acetate (MacKenzie, 1955). 5,10-CH2- single bands with subunit Mr values of 91 000 and H4PteGlu was synthesized by treating H4PteGlu 93 000, respectively, on SDS/polyacrylamide-gel with a two-fold excess of formaldehyde. The electrophoresis. Yellow fluorescence co-migrated concentrations of H4PteGlu and of 5,10-CH2-H4- with the protein in each case, indicating the presence PteGlu were determined from their absorption of covalently bound flavin. The number of FAD coefficients of 29 mm-l . cm-l at 297 nm and residues in the two enzymes was estimated by assum- 33mml cm-l at 292nm, respectively (Kallen, ing an absorption coefficient of 11.3mm-l cm- 1971). The concentration of 5,10-CH2-H4PteGlu at 459nm and 457nm for dimethylglycine dehydro- determined by means of the NADP-linked conver- genase and sarcosine dehydrogenase, respectively, sion to 10-HCO-H4PteGlu (Ramasastri & Blakley, and that the Lowry procedure with bovine serum 1964) in the presence of 2mM-f-mercaptoethanol albumin as a standard accurately reflects the protein was generally approx. 90% of the value calculated content of the enzymes. Based on these assump- from the absorbance at 292nm. tions, dimethylglycine dehydrogenase and sarcosine dehydrogenase contained 0.93 and 1.19 mol of Purification ofenzymes and ofETF FAD, respectively, per mol of subunit. The specific The purification and characterization of sarco- activity of purified sarcosine dehydrogenase was sine and dimethylglycine dehydrogenases and of 263nmol min-'-mg-1 and that of dimethylglycine ETF by modifications of existing procedures dehydrogenase was 157nmol * min-l mg-1. These (Hoskins & MacKenzie, 1961; Frisell & MacKenzie, activities are about 500-fold less than reported by 1962, 1970; Wittwer & Wagner, 1981a) will be Wittwer & Wagner (1980), but our specific activities described in detail in a separate communication and agree more closely with that reported by other are only briefly summarized here. Pig liver mito- workers (Frisell & MacKenzie, 1962; Hoskins & chondria were prepared essentially as described Bjur, 1964) at corresponding stages of purification. 1982 Effect of tetrahydrofolate on oxidative N-demethylation 709 The reason for the discrepancy is uncertain, par- ticularly since the data presented in Table