Journal of Neurochemisrry. 1977. Vol. 29. pp. 1147-1 149. Pergamon
Praa. Printed in Great Britain.
SHORT COMMUNICATION ~~~
Mammalian brain dihydrofolate reductase' (Received 25 April 1977 Accepted 13 June 1977) .
I
MAKULUet at. (1973) have recently reported that mammalian brain lacks dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP' oxidoreductase; EC 1.5.1.3). Since brain has an active uptake system for tetrahydrofolate and its derivatives (LEVITTet a/., 1971; SPECTOR& LORENZO, 1975), presumably the absence of dihydrofolate reductase from brain would be no particular handicap to brain folate metabolism. On the other hand, the absence of this enzyme from brain would limit the in uiuo usefulness of some of the concepts of tetrahydrobiopterin metabolism derived from in uitro work (CRAINEet a/., 1972; LOVENBERG et al., 1967). A reduced pteridine cofactor, presumably tetrahydrobiopterin, is an obligatory cofactor of the initial enzymes in the pathways of catecholamine and serotonin synthesis: phenylalanine hydroxylase (L-phenylalanine, dihydrobiopterin : oxygen oxidoreductase [4-hydroxylating] ; EC 1.14.3.l), tyrosine hydroxylase (L-tyrosine, tetrahydropteridine: oxygen oxidoreductase [3-hydroxylating] ; EC 1.14.3.a).and tryptophan hydroxylase (L-tryptophan, tetrahydropteridine: oxygen oxidoreductase [5-hydroxylating] ; EC 1.14.16.4). During the course of these reactions the tetrahydrobiopterin is oxidized to quinoid-dihydrobiopterin and cyclically re-reduced to tetrahydrobiopterin through the action of quinoid-dihydrobiopterin reductase (reduced NADP: 6,7-dihydropteridine oxidoreductase; EC 1.6.99.7)(Reaction 1) (CRAINE et a/., 1972): quinoid-dihydrobiopterin + NAD(P)H +tetrahydrobiopterin
+ NAD(P)+.
(1)
These systems in uitro, however, are complicated by two additional reactions. Quinoid-dihydrobiopterin is extremely unstable and spontaneously and irreversibly rearranges (Reaction 2) to 7$-dihydrobiopterin which is no longer a substrate for quinoid-dihydrobiopterinreductase. 7,8-Dihydrobiopterin, however, can be salvaged by being reduced back to tetrahydrobiopterin through the action of dihydrofolate reductase (Reaction 3) (KAUFMAN, 1967; et al., 1972): CRAINE quinoid-dihydrobiopterin 4 7&dihydrobiopterin, 7,8-dihydrobiopterin NADPH -+ tetrahydrobiopterin + NADP'.
+
(2) (3)
I n uitro, therefore, dihydrofolate reductase is an essential component of phenylalanine, tyrosine and tryptophan hydroxylating systems (KAUFMAN, 1967; CRAINEet a/., 1972). Whether or not Reactions 2 and 3 play an important role in uiuo is not yet known. However because of the potential importance of dihydrofolate reductase to tetrahydrobiop'Supported by Grant 5 R 0 1 NS-12153 from the National Institute of Neurological and Communicative Disorders and Stroke.
terin metabolism and therefore to biogenic amine synthesis (KAUFMAN, 1967), we have undertaken to re-examine the question of whether or not mammalian brain contains this enzyme. In contrast to the previous investigators (MAKULU et al., 1973). we find that brain does, in fact, contain significant amounts of dihydrofolate reductase; and we have partially purified the enzyme from calf brain. MATERIALS AND METHODS Fresh calf brain was obtained from a slaughter house. Rat brain and liver were obtained from male animals of the Wistar strain (300-400 9). All tissues were homogenized in 3 vol of ice-cold potassium phosphate buffer (0.1 M) pH 6.8 containing 1 mM-EDTA either for 2 min using a PotterElvehjem tissue homogenizer or for 1 min in a Waring blender. The homogenate was centrifuged at 25,000g for 30min at 0°C and the resultant supernatant was either assayed directly or subjected to partial purification. For partial purification of calf brain dihydrofolate reductase, the supernatant from the crude homogenate was treated with 0.2 vol of 2% protomine sulfate. The mixture was stirred at 0°C for 30min and centrifuged at 20,OOOg for 20 min. The resulting supernatant was fractionated with solid (NH&SO, (50-75%). The mixture was stirred gently for 30min after each addition of (NH&SO, and centrifuged at 20,000 g (0°C) for 20 min. The pellet obtained from the last centrifugation was dissolved in a minimum volume of potassium phosphate buffer (50 mM) pH 6.8 containing 1 mM-EDTA and 1 p~ NADP. After brief centrifugation to remove insoluble material, 0.3-0.5 g protein was applied directly to a G-100 Sephadex column (1.9 x 55cm) previously equilibrated with the same buffer. The enzyme was eluted at a rate of 1 ml/min, 3 ml fractions being collected. For the partial purification of rat tissue, only the (NH&S04 fractionations were performed. Dihydrofolate reductase was assayed essentially as outlined by MAKULU et nl. (1973) except that the reaction was followed at 25°C. Reaction mixtures contained 100 mhi-Tris-HC1 pH 7.5, 50mM-KCI; 0.1 mM-NADPH (Sigma Chemical Co., St. Louis, MO) and 0.05 mM-dihydrofolate. Blanks omitting either dihydrofolate or NADPH were included with each assay. The rates observed in both blanks were subtracted from the rate in the complete reaction mixture. The reaction was initiated by addition of the enzyme and followed at 340nm at 30s intervals for up to 10min on a Zeiss PMQ 111 spectrophotometer. An extinction coefficient of 11.65 (KAUFMAN & GARDINER,1966) was taken for the dihydrofolate reductase reaction. Dihydrofolate was prepared according to the method of WILMANNS (1974) and contained 0.1 pmol mercaptoethanol/pmoI dihydrofolate. Protein was determined by the method of LOWRYet a/.
1147 KC %+a
Short communication PURIFICATION TABLE1. PARTIAL
Dihydrofolate reductase activity (pmollmg protein/h) Calf brain Rat brain Rat liver
Purification step Homogenate supernatant Protamine sulfate treatment * and (NH4),S04 fractionation Sephadex (3-100
OF DIHYDROFOLATE REDUCTASE
0.02 f 0.00 (100%) 0.02 0.048 (66%)t 0.56 (30%)t
0.00 (100%) 0.34 f 0.02(100%)
0.085 + 0.01 (48%)
1.7 f 0.3 (52%) ~
~
~~
Enzyme activity was determined as described under Methods. Each value is expressed as the mean k S.E.M. (n = 3-5). Recovery of activity at each step is given in parentheses, assuming the homogenate supernatant to be 100%. * Calf brain only. t From pool of 5 brains. (1951). The specific activity of the enzyme is expressed as pmol dihydrofolate reduced/mg protein/h.
& KENKARE, 1964). (Our 8% of the liver value (BRAGANCA own value for rat brain dihydrofolate reductase is 6% of the liver activity [Table 13). We find, therefore, the inability of MAKULUet a / . (1973) to demonstrate the enzyme in RESULTS AND DISCUSSION brain difficult to explain. The same assay system as the Even when the supernatant of the crude brain homo- previous investigators has been used and when we comgenate was assayed by the method outlined, a very low pared our extraction media, we could find no essential difbut consistent net rate in the complete assay system was ference. It is possible, however, that the relatively proalways found. Assay of the crude material was difficult, longed extraction period of MAKULU et al. (1973) may have however, because of high blanks caused by non-specific been detrimental to the low levels of enzyme found in oxidation of NADPH (Table 1). However, simple purifica- brain. tion measures such as ammonium sulfate fractionation sufR. LYNN ficiently reduced the blanks to allow the activity of brain Department of Psychiatry, M. E. RUETER dihydrofolate reductase to be easily demonstrated (Table The University of Texas Medical R. W. GUY" 2). In the case of the calf brain enzyme, the partial purificaSchool at Houston, tion was carried through a Sephadex G-100 filtration step and the University of Texas Health, to achieve a 28-fold purification (Table 1) and a reduction Science Center at Houston. of the blanks to