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Purine catabolism in Drosophila melanogaster
335
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95755
P U R I N E CATABOLISM IN D R O S O P H I L A M E L A N O G A S T E R I. REACTIONS LEADING TO X A N T H I N E D E H Y D R O G E N A S E
L. D. HODGE* AND E. GLASSMAN
Department of Biochemistry and the Genetics Curriculum University of North Carolina, Chapel Hill, N.C. (U.S.A.) (Received May 29th, 1967) (Revised manuscript received July 27th , 1967)
SUMMARY
I. The pathway of purine catabolism in larval extracts of Drosophila melano-
gaster has been investigated. 2. The enzymes prior to xanthine dehydrogenase in the pathway appear to be adenosine deaminase, inosine phosphorylase and guanine deaminase. 3. The evidence indicates that adenine deaminase and urate oxidase are not present in extracts of larvae. 4. A Canton-S wild-type stock strain did not metabolize [14C]guanine, suggesting a possible mutation affecting guanine deaminase. 5. Some conversion of [14Clxanthine to uric acid was noted in extracts of the xanthine dehydrogenase mutants, ma-l and ry.
INTRODUCTION
One of the enzymes in purine catabolism in Drosophila melanogaster, xanthine dehydrogenase, has been extensively studied genetically and biochemically1. 3 loci exert control over this enzyme. These are maroon-like (ma-l) at 64~- on the X chromosome, rosy (ry) at 52± on the third chromosome and low xanthine dehydrogenase (lxd) at 33 ± on the third chromosome 1. The ma-l and. ry mutants lack xanthine dehydrogenase, while lxd flies have 20 ~/o normal xanthine dehydrogenase activity (I). At least 4 electrophoretie variants have been mapped at the ry locus indicating that this is the structural gene for xanthine dehydrogenase2; the role of lxd+ and ma-l+ is not clear. To date only minimal evidence has been presented concerning this pathway Abbreviations: P R P P , phosphoribosylpyrophosphate; Tris-albumin, Tris containing 1 mg per ml of crystalline bovine plasma albumin; CS, Canton-S wild-type stock; Pac, Pacific wildtype stock. Present address: D e p a r t m e n t of Cell Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y. 1o461, U.S.A.
Biochim. Biophys. Acta, I49 (1967) 335-343
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L. D. HODGE, E. GLASSMAN
in Drosophila. The disappearance of adenosine in larval extracts of Drosophila has been reported s but it is not known if it is converted directl?,7 to inosine, to adenine or to adenosine 5'-phosphate before deamination. Two reports of guanine deaminase activity in Drosophila have appeared4, 5. Uric acid can be converted to allantoin e, although large quantities of uric acid are excreted b y adult flies. This lack of information was one reason for studying the pathway of purine catabolism. A second reason was the differences in viability between the Pacific wild-type stock (Pac) and lxd strains when grown on the purine analogue, 2,6diaminopurine 7. Both Pac and lxd strains were equally susceptible to 2,6-diaminopurine. However, adenine partially reversed the harmful effect of 2,6-diaminopurine on the Pac strain and augmented the effect on lxd. In contrast guanine partially reversed the effect of 2,6-diaminopurine on lxd strain and augmented the effect on Pac. Extracts of these two strains metabolized 2,6-E14C~diaminopurine in the same manner, probably to uric acid. In the presence of 2,6-diaminopurine and phosphoribosylpyrophosphate (PRPP) both extracts formed a nucleotide 7. Therefore the purpose of this investigation was to obtain a more complete picture of the sequence of reactions in purine catabolism in Drosophila b y studying the metabolic fate of ~4C-labeled adenine, guanine, adenosine, xanthine, hypoxanthine and uric acid in cell free extracts of larvae of various strains. This pathway was studied with the aid of 2 mutants of xanthine dehydrogenase, ma-l and ry. Since these mutants lack significant xanthine dehydrogenase activity, substrates of xanthine dehydrogenase accumulated in the reaction mixtures and this greatly facilitated the elucidation of the pathway.
MATERIALS AND METHODS
Materials [lac]- and [12C]purines were purchased from California Corporation for Biochemical Research or Schwarz Bioresearch. Glacial acetic acid, NH4OH, HC1, isoamyl alcohol and N a O H were obtained from Allied Chemical. Formic acid, methanol and urea were obtained from Baker Chemical. Third instar larvae of D. melanogaster were used. Larvae were raised on standard media s at 2 5 ° + 1 °. The strains utilized in these investigations wereg: ru Ixd by which contained roughoid eye (3--0 ±), low xanthine dehydrogenase (3--33 ±) and blistery wing (3--48±); st ry which contained scarlet eye (3--44 ±) and rosy eye (3--52± ); v / B x 3 ma-l which contained vermillion eye color (I--33 :~ ), forked bristles (I--56± ), Beadex wing ( I - - 5 9 ± ) and maroon-like eye color ( I - - 6 4 ± ); and a Canton-S wild-type stock (CS) containing the st (3--44 ± ) gene. This CS stock was a gift of ]3. W. GEER and is reported to be a RNA nutritional m u t a n t 1°. The Pacific wildtype stock (Pac) was used as our standard strain.
Preparation o/extract Extracts were prepared at less than 5 °. 1. 5 g of larvae per ml of o.i M Tris (pH 7.5) were homogenized for approx. I rain. Homogellates were centrifuged at 34 ooo × g for 20 min in a Servall RC-2 centrifuge. The supernatant solution beneath Biochim. Biophys. Acta, 149 (1967) 335-343
D. melanogaster PURINE CATABOLISM. I
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the overlying fatty layer was removed and filtered through glasswool. Norite A (50 mg per ml) was added to this supernatant solution and the charcoal suspension was kept on ice for I h with occasional stirring. To remove the charcoal, the extract was centrifuged twice at 34 ooo ×g for 20 min and filtered through glasswool. This extract is referred to as the crude charcoal-treated extract. Extracts were also prepared by adjusting the supernatant solution prior to charcoal treatment described above to pH 5.1 with I.O M acetic acid and immediately centrifuging the resulting solution at 34 ooo ×g for 5 min. After adjusting the supernatant solution to pH 8.0 with I.O M KOH, this extract was treated with 50 mg Norite-A per ml for 60 min on ice with occasional stirring. The charcoal was removed by 2 centrifugations at 34 ooo ×g for 20 min. This supernatant solution is called the pH 5-charcoal-treated extract. Subcellular fractionation was carried out on larvae homogenized (I g per 1. 5 ml) in o.I M Tris (pH 7.0) containing 0.05 M KC1, O.Ol M MgC12 and 0.25 M sucrose (Buffer A) according to the method of SCHNEIDER, CLAUDE AND HOGEBOOM11. Precipitates after centrifugation at 770 xg, io ooo ×g, lO5 ooo ×g and the supernatant solution following centrifugation at lO5 ooo ×g were prepared. These precipitates were homogenized for 30 sec in a glass tissue grinder in a volume of o. i M Tris (pH 7.0) equal to one-half the original volume of the homogenate (usually 6 to 7 ml). These suspensions were sonicated in ice for I min with setting No. 7 on a Branson Sonifier model S i i o using a flat-tip probe. When the metabolism of 14C-labelled uric acid was investigated, the pH of the o.I M Tris was 8.6.
Paper chromatography and autoradiography 2-dimensional paper partition chromatography using 8 inch × 8 inch No. 3 MM Whatman chromatography paper was performed in Shandon chromatotanks TM. In the first dimension the solvent was n-propanol i % NH4OH (2:1, by vol.) and in the second dimension n-butanol-conc, glacial acetic acid-water (12:3:5, b y vol.) was used. Using the completed chromatogram, autoradiography was performed with X-ray film (Kodak Blue Brand io" × 12") TM. In these experiments an exposure time of 4 days was adequate but occasionally io days was necessary. The purity of the commercial [14Clpurines was checked by routine chromatography and autoradiography in 3 solvent systems: propanol-NH4OH, butanolacetic acid and 5% aq. ammonium acetate satd. with isoamyl alcohol. Although [14C]hypoxanthine, [14C]uric acid and [14Clguanine contained trace amounts of impurities, these impurities did not interfere with the investigations. The identity of the reaction products of the [l*C~purines was established by paper chromatography. Chemical identity was assumed if the unknown migrated similarly to the known compound in 5 solvent systems propanol-NHiOH, butanolacetic acid, ammonium acetate-isoamyl alcohol, 5% aq. monobasic sodium phosphate satd. with isoamyl alcohol, n-butanol-isobutyric acid-25 % NH4OH-water (75:73.5:2.5:25, by vol.). The amount of the [14C]purine metabolites present on the paper was measured b y a modification of the method published b y WANG AND JONESis. A standard curve to convert counts/min to amount was prepared by chromatographing known quantities of [14C]purine and determining the radioactivity of the spots on the paper. Biochim. Biophys. Acta, 149 (1967) 335-343
338
L. D. I-lODGE, E. GLASSMAN
RESULTS
Fig. I shows the relative migration of various purines using 2-dimensional paper-partition chromatography employing propanol-NH,OH, and butanol-acetic
~/M
T
Hvooxe,Tth/nG
a
~tNt~
F E 0 I
URIC ACID
origin Q_
Butanol-acetic acid
Fig. I. Migration of p u r i n e s on 2-dimensional p a p e r c h r o m a t o g r a m s , o.i ml of i mM solutions of p u r i n e s were s p o t t e d s e p a r a t e l y a n d in various c o m b i n a t i o n s on 8" × 8" c h r o m a t o g r a m s . These c h r o m a t o g r a m s were r u n in p r o p a n o l - N H 4 O H in the first dimension and b u t a n o l - a c e t i c acid in t h e second dimension and viewed u n d e r 26o-m/, light. F r o m these c h r o m a t o g r a m s this composite of the relative m i g r a t i o n of p u r i n e s w a s developed. DAP, 2,6-diaminopurine.
acid in the first and second dimension, respectively. Nucleotides are clustered about the origin. Purine bases and purine nucleosides are separated fairly well, although some overlapping does occur. Fig. 2 shows the result of a 3-h incubation of larval extracts with [8-14C]hypoxanthine. As expected xanthine and uric acid appeared when extracts of the
Fig. 2. 3 h i n c u b a t i o n of Pac and ma-1 e x t r a c t s w i t h [8-14C]hypoxanthine. 0. 5 ml of crude charcoal-treated e x t r a c t s p r e p a r e d w i t h o.I M Tris (pH 7.5) were i n c u b a t e d at 3 °0 w i t h o.i/*C of [8-1*C]hypoxanthyine. Before and after a 3 h incubation, o. i ml of the reaction m i x t u r e was applied to 8 inch × 8 inch W h a t m a n 3 MM paper. C h r o m a t o g r a p h y and a u t o r a d i o g r a p h y w i t h an e x p o s u r e t i m e of 4 d a y s were p e r f o r m e d as described in MATERIALS AND METHODS. The origin of the c h r o m a t o g r a m s is at the lower left corner. I. Pac extract, after 3 h incubation; I I . ma-l extract, after 3 h incubation.
Biockim. Biophys. Acta, 149 (1967) 335-343
D. melanogaster PURINE .CATABOLISM. I
339
Pac or the CS strains were used. When extracts of the ma-l or ry larvea were used (these lack xanthine dehydrogenase), only inosine was detected. The autoradiographic data obtained when larval extracts were incubated with other [14C]purines are presented below in tabular form. When [8-14C]adenine was incubated with crude charcoal-treated extracts for 3 h, inosine, xanthine, hypoxanthine and uric acid were formed. To determine whether adenine is converted directly to hypoxanthine, aliquots were removed from the reaction mixture at various times. Table I shows that at 5 min the nucleotides, AMP TABLE I SEQUENCE OF CONVERSIONS IN [8-14C]ADENINE CATABOLISM A Chax-crude e x t r a c t of P a c s t r a i n w a s p r e p a r e d w i t h o.I M Tri s (pH 7-5). o.5 m l of t h i s e x t r a c t w a s i n c u b a t e d a t 3 °0 w i t k o . i pC of [8-14C~adenine. A f t e r 5-, 2o-, 35- a n d 5 o - m i n i n c u b a t i o n , o. i - m l a l i q u o t s w e r e r e m o v e d a n d c h r o m a t o g r a p h y a n d a u t o r a d i o g r a p t x y p e r f o r m e d as d e s c r i b e d in MATERIALS AND METHODS. P l u s e s i n d i c a t e t h e r e l a t i v e a m o u n t s of a m e t a b o l i t e .
Sampling time (rain)
Reaction products AMP
1MP
Inosine
5
++
++
.
20 35 50
++ ++ ++
++ ++ ++
+ ++ ++
Hypoxanthine Xanthine .
. 4+ ++
Uric acid
. -4+
--+
and IMP, are present. Hypoxanthine and xanthine are noticeable at 35 min, but are not readily apparent until after 5° min of incubation. Thus, the order of appearance of metabolites derived from adenine seems to be adenine ~ nucleotides--~ inosine --~ hypoxanthine. In addition, no evidence for the direct conversion of adenine to hypoxanthine by an adenine deaminase was found in the 77 ° ×g precipitate, the IO ooo x g precipitate or the lO5 ooo ×g precipitate before or after sonication or in the lO5 ooo ×g supernatant solution which readily metabolized adenine to nucleotides. Table I I shows the result of incubating [8-14Cladenosine, [8-14Clguanine and [8-14Cluric acid with various larvel extracts. [8-14C]Adenosine is converted readily to inosine, hypoxanthine, xanthine and uric acid except in the xanthine-dehydrogenase mutant ma-1. When aliquots were removed from the reaction mixture at shorter times during the incubation, autoradiograms did not show metabolites in the proximity of the origin where nucleotides migrate. Thus, it appears that unlike adenine, it is not necessary for adenosine to be converted to a nucleotide for catabolism. When [8-1*C~guanine is incubated with crude charcoal-treated extracts for 3 h, the new metabolite formed in extracts of Pac was shown to be uric acid. None of the solvent systems employed in these investigations was capable of separating guanine and xantkine. Uric acid did not form in extracts of the CS strain although trace amounts of uric acid were noted if the exposure time was doubled or if twice the usual amount of radioactive guanine was added to the reaction mixture. Since the metabolism of hypoxanthine to uric acid was normal in extracts of the CS strain, a defect in the conversion of guanine to xanthine was suspected. Biochim. Biophys. Acta, 149 (1967) 335-343
340
L . D . HODGE, E. GLASSMAN
TABLE II INCUBATION OF LARVAL EXTRACTS WITH ~8-14C~ADENOSINE, [8-14C~GUANINE AND [8-14C]uRIC ACID
A. o. 5 m l of c r u d e c h a r c o a l - t r e a t e d e x t r a c t s of v a r i o u s s t r a i n s p r e p a r e d w i t h o.i M Tris (pH 7.5) were i n c u b a t e d a t 3 °0 w i t h o.I # C of [8-14C]adenosine a n d [8-14C]guanine. B e fore a n d a f t e r a 3-h i n c u b a t i o n o. I m l of t h e r e a c t i o n m i x t u r e w a s a p p l i e d t o 8 i nc h × 8 i n c h W h a t m a n 3 MM p a p e r . C h r o m a t o g r a p h y a n d a u t o r a d i o g r a p h y were p e r f o r m e d as d e s c r i b e d i n MATERIALS AND METHODS. T h e r e l a t i v e a m o u n t of m e t a b o l i t e n o t e d on t h e a u t o r a d i o g r a m is i n d i c a t e d b y pluses. B. IO g of P a c l a r v a e w e r e h o m o g e n i z e d w i t h I5 m l of B u f f e r A. B y m e a n s of d i f f e r e n t i a l c e n t r i f u g a t i o n a 77 ° × g p r e c i p i t a t e , i o ooo × g p r e c i p i t a t e , lO 5 ooo × g p r e c i p i t a t e a n d a lO 5 ooo × g s upe rn a t a n t s o l u t i o n w e r e o b t a i n e d . The t h r e e p r e c i p i t a t e s w e r e r e s u s p e n d e d in 6 m l of o.I IV[ Tri s ( pH 8.6) b u f f e r a n d s o n i c a t e d for 60 sec. 0. 5 ml of each s o n i c a t e w a s i n c u b a t e d w i t h o.I/ *C of [8-x4C]uric acid. Before a n d a f t e r a 3-h i n c u b a t i o n a t 3 o°, o . i ml a l i q u o t s of t h e r e a c t i o n m i x t u r e s were used for c h r o m a t o g r a p h y a n d a u t o r a d i o g r a p h y . The r e s u l t s w h i c h are s h o w n are o n l y for t h e p a r t i c u l a t e f r a c t i o n p r e p a r e d b y IO ooo × g c e n t r i f u g a t i o n .
Strain
l*C-Metabolite
Reaction products Inosine
Hypoxanthine
Xanthine
Uric acid
Allantoin
A. A d e n o s i n e
+ + + +
+ + + +
-+ +
-+
--
Pac CS
A. G u a n i n e
---
---
? ?
+ + --
---
Pac
B. Ur ic acid
.
ma-l Pac
.
.
.
±
T h e o n l y evidence t h a t [14C]uric acid was c o n v e r t e d to o t h e r m e t a b o l i t e s was o b t a i n e d w i t h sonicates of t h e IO ooo × g p r e c i p i t a t e . A f t e r a 3 h i n c u b a t i o n a new m e t a b o l i t e d i d a p p e a r w i t h this fraction b u t it was n o t clear w h e t h e r this was a p r o d u c t f o r m e d from uric a c i d or from one of the impurities. I t m i g h t h a v e been allantoin, b u t it was n o t possible to o b t a i n sufficient q u a n t i t i e s for identification. Since u r a t e oxidase a c t i v i t y , if present, was at low levels, this was n o t p u r s u e d
TABLE CONVERSION
III OF XANTHINE
TO
URIC
ACID
IN X A N T H I N E - D E H Y D R O G E N A S E
MUTANTS
L a r v a e a n d flies were c h e c k e d for c o n t a m i n a t i o n before p r e p a r a t i o n of e x t r a c t s . 3.0 g of l a r v a e a n d 3.0 g of flies f r o m Pac, ma-1, a n d ry w e r e h o m o g e n i z e d s e p a r a t e l y i n 4.5 m l of o.I M Tri s ( p H 7.5). F r o m e a c h c r u d e h o m o g e n a t e a c r u d e c h a r c o M - t r e a t e d e x t r a c t a n d a p H 5-chaxcoait r e a t e d e x t r a c t w e r e p r e p a r e d . 0. 5 m l of e a c h of t h e s e 8 e x t r a c t s w a s i n c u b a t e d w i t h o.I #C (8-14C]-xanthine a n d o . i m l of I m M N A D . A f t e r a 3 o - m i n i n c u b a t i o n a t 3 o°, o . i - m l a l i q u o t s w e re c h r o m a t o g r a p h e d as d e s c r i b e d in MATERIALS AND METHODS. E x p o s u r e t i m e for a u t o r a d i o g r a p h y w a s I o d a y s . T h e r a d i o a c t i v i t y of t h e s p o t s c o n t a i n i n g u r i c a c i d w a s d e t e r m i n e d a n d t h e d a t a c o n v e r t e d to m/tg.
Strain
ml~g o[ uric acid [ormed per reaction mixture Larvae
ma-1 ry Pac
Adults
Crude charcoaltreated extract
p H 5-charcoaltreated extract
Crude charcoaltreated extract
pH5-chamoaltreated extract
199 389 4426
2o2 696 4733
168 333 3169
16o 749 3843
Biochim. Biophys. Acta, 149 (1967) 335-343
D. melanogaster PURINE CATABOLISM. I
341
further. There was no Change when E14C]uric acid was incubated with sonicates of the 6o0 x g or the lO5 o o o × g precipitates, the lO5 o o o × g supernatant solution, the crude charcoal-treated extracts, or the pH 5-charcoal-treated extract. Data in these investigations indicated that if autoradiograms of ma-l and ry extracts were exposed for 2.5 times the usual exposure time, low levels of xanthine dehydrogenase activity were detectable. To confirm this observation crude and pH 5-charcoal-treated extracts of strains Pac, ry and ma-1 were incubated with [8-14C]xanthine and NAD, the electron acceptor for xanthine dehydrogenase. Table III shows that cell-free extracts of both larvae and adults do convert some xanthine to uric acid. More xanthine is converted to uric acid by the pH 5-charcoal-treated extracts of all strains than b y the crude charcoal-treated extracts.
DISCUSSION
Purine catabolism was studied in D. melanogaster using extracts of third instar larvae. Since active cell division and growth occur in this developmental stage, it was thought that the enzymes involved in these reactions would be present. Some aspects of the pathway of purine catabolism were established by incubating [14C]purines with these extracts and by separating the radioactive products of these reactions by chromatography and autoradiography. The reactions shown in Fig. 3
Adenine
LN o~/J-~>
HOCHA~ OH Adeno eine-SJ monophosphate
I
~ 1 II ~" Hypoxonthine
HOCH=-~ / j Inosine OH ."
I
Inosine-5" / monophosphate
N
N
I ") HIN-1,~_.,,~'
N neH Guani
.
~--",> Xanthine
J
OH
HO N~
ON
Uric Acid Allantoin Fig. 3. P u r i n e catabolism in larval e x t r a c t s of Drosophila. This figure is a s u m m a r y of t h e in vitro reactions involving p u r i n e c a t a b o l i s m in t h i r d i n s t a r larvae of D. melanogaster. D a s h e d lines are conversions which need m o r e w o r k for confirmation.
Biochim. Biophys. Acta, i49 (1967) 335-343
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L. D. HODGE, E. GLASSMAN
were elucidated. The only exception to this scheme was tile result obtained with extracts of the CS strain where the data indicate a possible defect in the conversion of guanine to xanthine. Of interest is that these conversions of purines indicated that the enzymes which produce substrates for xanthine dehydrogenase are adenosine deaminase, inosine phosphorylase and guanine deaminase. When [8-14Cladenine was used the order of appearance of metabolites was the nucleotides, AMP and IMP, then inosine, hypoxanthine, xanthine and uric acid. The method b y which adenine is converted to nucleotides is not clear. I t is consistent with a pyrophosphorylase reaction as found in Escherichia coli, yeast, avian and mammalian tissue 14. The AMP could be hydxolyzed to adenosine or deaminated to IMP, as reported in microorganisms and mammals la. By removal of the phosphate, IMP and AMP could be converted to inosine and adenosine, respectively 14. This suggestion was supported b y incomplete data in which [8-1aC]AMP was converted to IMP, adenosine, inosine, hypoxanthine and uric acid. I t is also possible that AMP could have arisen b y the transribosidation of adenine to adenosine followed b y an adenosine kinase reaction. However, adenosine was not one of the reaction products as would be expected. This evidence combined with the data on the catabolism of adenosine indicates that adenine itself is not deaminated; only adenosine and AMP are deaminated. This result is consistent with reports that adenine deaminase is present in microorganismslS, le but not in higher organisms. Urate oxidase, the enzyme responsible for catabolizing uric acid to allantoin has been reported in a number of insects: Popillia iaponica (Japanese beetle) 17, Periplaneta americana linnaeus (American cockroach) 18 and Musca domestica (house fly) ~9. AuF DER MAULs reports uricase activity in adults of various mutants of D. melanogaster. Since we were unable to find significant conversion of uric acid to allantoin in extracts of third instar larvae, it is possible that urate oxidase is present only in pupae and adults. Many other conversions involving purines, nucleosides and nucleotides could be established in D. melanogaster b y appropriate experiments. Since a complete picture of purine catabolism would describe m a n y biochemical phenotypes, continued study should be of interest to the Drosophila geneticist. Hopefully, mutants affecting purine metabolism can be found b y assaying established strains or b y some selection procedure. Where alternate pathways exist for the synthesis or breakdown of a purine, or where such reactions are not absolutely necessary, as for example those catalyzed b y xanthine dehydxogenase, viable mutants can probably be obtained. An interesting finding was the conversion of xanthine to uric acid in the extracts of ma-1 and ry. Other work in this laboratory using an assay based on the increase in fluorescense when 2-amino-4-liydroxy-pteridine is converted to isoxanthopterin indicated that extracts of ma-l and ry have about 0.25 % of the xanthine dehydrogenase activity of wild type (E. GLASSMAN, unpublished). However, ry and ma-l flies and larvae do not contain detectable amounts of uric acid or isoxanthopterin 2°, indicating that the in vivo activity of xanthine dehydrogenase is very low, if present at all. The xanthine dehydrogenase activity which we observed in ma-l and ry is not due to a contamination of the ry and ma-l stocks with wild type. Based on the amount of xanthine dehydrogenase in a single wild-type larvae, we calculate that about 5 ° contaminant larvae per g of ma-l or IOO contaminants Biochim. Biophys. Acta, 149 (1967) 335-343
D. melanogaster PURINE
CATABOLISM. I
343
per g of ry larvae would be necessary to produce the amount of conversions observed here and contamination of this size would be easily noted. Since these experiments were done in the presence of excess amounts of xanthine dehydrogenase, there probably was not proportionality between the amount of xanthine converted and xanthine dehydrogenase activity and it is difficult to determine the actual amount of xanthine dehydrogenase activity present in the ma-1 and ry extracts. Whether this activity in ma-l or ry is due to xanthine dehydrogenase or to other enzymes with a low specificity for this reaction is not clear.
ACKNOWLEDGEMENTS
Part of the work reported here was supported b y Public Health Service Grant GM-o82o2. L.D.H. was supported by Genetics Training Grant, 5TI GH-685, from The Public Health Service. E.G. was supported b y a Research Career Development Award, 2-K3-GM-I4,9II, from the Public Health Service. REFERENCES I 2 3 4 5 6 7
GLASSMAN, Federation Proc., 24 (1965) 1243, Suppls. 14, 15. T. T. YEN AND E. GLASSMAN,Genetics, 52 (1965) 977. P. WAGNER AND H. K. MITC:-IELL, Arch. Biochem., 17 (1948) 87. SEECOF, Genetics, 46 (1961) 605. MORITA, Japan J. Genetics, 39 (1964) 222. AUF DER MAUL, Z. Vererbungslehre, 92 (1961) 42. HODGE, Thesis, p r e s e n t e d to t h e D e p a r t m e n t of B i o c h e m i s t r y a t Univ. of N o r t h Carolina p a r t i a l fulfillment for t h e P h . D . degree. GLASSMAN AND J. MCCLEAN, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1712. B. BRIDGES AND K. S. BREHME, The mutants o/ D rosophila melanogaster, Carnegie Inst. Wash. Publ., 552 (1944) 257. B. W. GEER, Genetics, 49 (1964) 787 • W. C. SCHNEIDER, A. CLAUDE AND G. H. HOGEBOOM, J. Biol. Chem., 172 (1948) 451. I. SMITH, Chromatographic and Electrophoretic Techniques, Vol. I, P i t m a n Press, Bath, 1963, p. 491 • C. H. WANG AND D. E. JONES, Biochem. Biophys. Res. Commun., I (1959) 2o3. A. G. MOAT AND H. FRIEDMAN, Bacteriol. Rev., 24 (196o) 309 . A. L. HEPPEL, J. HURWlTZ AND B. L. HORECKER, J. Am. Chem. Soc., 79 (1957) 63 o. A. H. RAUSH, Arch. Biochem. Biophys., 5 ° (1954) 5 lo. D. J. R o s s , Physiol. Zool., 32 (1959) 239. S. M. CORDERO AND D. LUDWIG, J. N. Y. Entomol. Soc., 71 (1963) 66. F. W. NELSON, Cutup. Biochem. Physiol., 12 (1964) 37. J. L. HUBBY AND H. S. FORREST, Genetics 45 (196o) 211.
E. T. R. R. T. P. L. in 8 E. 9 C. io ii 12 13 14 15 16 17 18 19 20
Biochim. Biophys. Acta, 149 (1967) 335-343
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95755
P U R I N E CATABOLISM IN D R O S O P H I L A M E L A N O G A S T E R I. REACTIONS LEADING TO X A N T H I N E D E H Y D R O G E N A S E
L. D. HODGE* AND E. GLASSMAN
Department of Biochemistry and the Genetics Curriculum University of North Carolina, Chapel Hill, N.C. (U.S.A.) (Received May 29th, 1967) (Revised manuscript received July 27th , 1967)
SUMMARY
I. The pathway of purine catabolism in larval extracts of Drosophila melano-
gaster has been investigated. 2. The enzymes prior to xanthine dehydrogenase in the pathway appear to be adenosine deaminase, inosine phosphorylase and guanine deaminase. 3. The evidence indicates that adenine deaminase and urate oxidase are not present in extracts of larvae. 4. A Canton-S wild-type stock strain did not metabolize [14C]guanine, suggesting a possible mutation affecting guanine deaminase. 5. Some conversion of [14Clxanthine to uric acid was noted in extracts of the xanthine dehydrogenase mutants, ma-l and ry.
INTRODUCTION
One of the enzymes in purine catabolism in Drosophila melanogaster, xanthine dehydrogenase, has been extensively studied genetically and biochemically1. 3 loci exert control over this enzyme. These are maroon-like (ma-l) at 64~- on the X chromosome, rosy (ry) at 52± on the third chromosome and low xanthine dehydrogenase (lxd) at 33 ± on the third chromosome 1. The ma-l and. ry mutants lack xanthine dehydrogenase, while lxd flies have 20 ~/o normal xanthine dehydrogenase activity (I). At least 4 electrophoretie variants have been mapped at the ry locus indicating that this is the structural gene for xanthine dehydrogenase2; the role of lxd+ and ma-l+ is not clear. To date only minimal evidence has been presented concerning this pathway Abbreviations: P R P P , phosphoribosylpyrophosphate; Tris-albumin, Tris containing 1 mg per ml of crystalline bovine plasma albumin; CS, Canton-S wild-type stock; Pac, Pacific wildtype stock. Present address: D e p a r t m e n t of Cell Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y. 1o461, U.S.A.
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L. D. HODGE, E. GLASSMAN
in Drosophila. The disappearance of adenosine in larval extracts of Drosophila has been reported s but it is not known if it is converted directl?,7 to inosine, to adenine or to adenosine 5'-phosphate before deamination. Two reports of guanine deaminase activity in Drosophila have appeared4, 5. Uric acid can be converted to allantoin e, although large quantities of uric acid are excreted b y adult flies. This lack of information was one reason for studying the pathway of purine catabolism. A second reason was the differences in viability between the Pacific wild-type stock (Pac) and lxd strains when grown on the purine analogue, 2,6diaminopurine 7. Both Pac and lxd strains were equally susceptible to 2,6-diaminopurine. However, adenine partially reversed the harmful effect of 2,6-diaminopurine on the Pac strain and augmented the effect on lxd. In contrast guanine partially reversed the effect of 2,6-diaminopurine on lxd strain and augmented the effect on Pac. Extracts of these two strains metabolized 2,6-E14C~diaminopurine in the same manner, probably to uric acid. In the presence of 2,6-diaminopurine and phosphoribosylpyrophosphate (PRPP) both extracts formed a nucleotide 7. Therefore the purpose of this investigation was to obtain a more complete picture of the sequence of reactions in purine catabolism in Drosophila b y studying the metabolic fate of ~4C-labeled adenine, guanine, adenosine, xanthine, hypoxanthine and uric acid in cell free extracts of larvae of various strains. This pathway was studied with the aid of 2 mutants of xanthine dehydrogenase, ma-l and ry. Since these mutants lack significant xanthine dehydrogenase activity, substrates of xanthine dehydrogenase accumulated in the reaction mixtures and this greatly facilitated the elucidation of the pathway.
MATERIALS AND METHODS
Materials [lac]- and [12C]purines were purchased from California Corporation for Biochemical Research or Schwarz Bioresearch. Glacial acetic acid, NH4OH, HC1, isoamyl alcohol and N a O H were obtained from Allied Chemical. Formic acid, methanol and urea were obtained from Baker Chemical. Third instar larvae of D. melanogaster were used. Larvae were raised on standard media s at 2 5 ° + 1 °. The strains utilized in these investigations wereg: ru Ixd by which contained roughoid eye (3--0 ±), low xanthine dehydrogenase (3--33 ±) and blistery wing (3--48±); st ry which contained scarlet eye (3--44 ±) and rosy eye (3--52± ); v / B x 3 ma-l which contained vermillion eye color (I--33 :~ ), forked bristles (I--56± ), Beadex wing ( I - - 5 9 ± ) and maroon-like eye color ( I - - 6 4 ± ); and a Canton-S wild-type stock (CS) containing the st (3--44 ± ) gene. This CS stock was a gift of ]3. W. GEER and is reported to be a RNA nutritional m u t a n t 1°. The Pacific wildtype stock (Pac) was used as our standard strain.
Preparation o/extract Extracts were prepared at less than 5 °. 1. 5 g of larvae per ml of o.i M Tris (pH 7.5) were homogenized for approx. I rain. Homogellates were centrifuged at 34 ooo × g for 20 min in a Servall RC-2 centrifuge. The supernatant solution beneath Biochim. Biophys. Acta, 149 (1967) 335-343
D. melanogaster PURINE CATABOLISM. I
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the overlying fatty layer was removed and filtered through glasswool. Norite A (50 mg per ml) was added to this supernatant solution and the charcoal suspension was kept on ice for I h with occasional stirring. To remove the charcoal, the extract was centrifuged twice at 34 ooo ×g for 20 min and filtered through glasswool. This extract is referred to as the crude charcoal-treated extract. Extracts were also prepared by adjusting the supernatant solution prior to charcoal treatment described above to pH 5.1 with I.O M acetic acid and immediately centrifuging the resulting solution at 34 ooo ×g for 5 min. After adjusting the supernatant solution to pH 8.0 with I.O M KOH, this extract was treated with 50 mg Norite-A per ml for 60 min on ice with occasional stirring. The charcoal was removed by 2 centrifugations at 34 ooo ×g for 20 min. This supernatant solution is called the pH 5-charcoal-treated extract. Subcellular fractionation was carried out on larvae homogenized (I g per 1. 5 ml) in o.I M Tris (pH 7.0) containing 0.05 M KC1, O.Ol M MgC12 and 0.25 M sucrose (Buffer A) according to the method of SCHNEIDER, CLAUDE AND HOGEBOOM11. Precipitates after centrifugation at 770 xg, io ooo ×g, lO5 ooo ×g and the supernatant solution following centrifugation at lO5 ooo ×g were prepared. These precipitates were homogenized for 30 sec in a glass tissue grinder in a volume of o. i M Tris (pH 7.0) equal to one-half the original volume of the homogenate (usually 6 to 7 ml). These suspensions were sonicated in ice for I min with setting No. 7 on a Branson Sonifier model S i i o using a flat-tip probe. When the metabolism of 14C-labelled uric acid was investigated, the pH of the o.I M Tris was 8.6.
Paper chromatography and autoradiography 2-dimensional paper partition chromatography using 8 inch × 8 inch No. 3 MM Whatman chromatography paper was performed in Shandon chromatotanks TM. In the first dimension the solvent was n-propanol i % NH4OH (2:1, by vol.) and in the second dimension n-butanol-conc, glacial acetic acid-water (12:3:5, b y vol.) was used. Using the completed chromatogram, autoradiography was performed with X-ray film (Kodak Blue Brand io" × 12") TM. In these experiments an exposure time of 4 days was adequate but occasionally io days was necessary. The purity of the commercial [14Clpurines was checked by routine chromatography and autoradiography in 3 solvent systems: propanol-NH4OH, butanolacetic acid and 5% aq. ammonium acetate satd. with isoamyl alcohol. Although [14C]hypoxanthine, [14C]uric acid and [14Clguanine contained trace amounts of impurities, these impurities did not interfere with the investigations. The identity of the reaction products of the [l*C~purines was established by paper chromatography. Chemical identity was assumed if the unknown migrated similarly to the known compound in 5 solvent systems propanol-NHiOH, butanolacetic acid, ammonium acetate-isoamyl alcohol, 5% aq. monobasic sodium phosphate satd. with isoamyl alcohol, n-butanol-isobutyric acid-25 % NH4OH-water (75:73.5:2.5:25, by vol.). The amount of the [14C]purine metabolites present on the paper was measured b y a modification of the method published b y WANG AND JONESis. A standard curve to convert counts/min to amount was prepared by chromatographing known quantities of [14C]purine and determining the radioactivity of the spots on the paper. Biochim. Biophys. Acta, 149 (1967) 335-343
338
L. D. I-lODGE, E. GLASSMAN
RESULTS
Fig. I shows the relative migration of various purines using 2-dimensional paper-partition chromatography employing propanol-NH,OH, and butanol-acetic
~/M
T
Hvooxe,Tth/nG
a
~tNt~
F E 0 I
URIC ACID
origin Q_
Butanol-acetic acid
Fig. I. Migration of p u r i n e s on 2-dimensional p a p e r c h r o m a t o g r a m s , o.i ml of i mM solutions of p u r i n e s were s p o t t e d s e p a r a t e l y a n d in various c o m b i n a t i o n s on 8" × 8" c h r o m a t o g r a m s . These c h r o m a t o g r a m s were r u n in p r o p a n o l - N H 4 O H in the first dimension and b u t a n o l - a c e t i c acid in t h e second dimension and viewed u n d e r 26o-m/, light. F r o m these c h r o m a t o g r a m s this composite of the relative m i g r a t i o n of p u r i n e s w a s developed. DAP, 2,6-diaminopurine.
acid in the first and second dimension, respectively. Nucleotides are clustered about the origin. Purine bases and purine nucleosides are separated fairly well, although some overlapping does occur. Fig. 2 shows the result of a 3-h incubation of larval extracts with [8-14C]hypoxanthine. As expected xanthine and uric acid appeared when extracts of the
Fig. 2. 3 h i n c u b a t i o n of Pac and ma-1 e x t r a c t s w i t h [8-14C]hypoxanthine. 0. 5 ml of crude charcoal-treated e x t r a c t s p r e p a r e d w i t h o.I M Tris (pH 7.5) were i n c u b a t e d at 3 °0 w i t h o.i/*C of [8-1*C]hypoxanthyine. Before and after a 3 h incubation, o. i ml of the reaction m i x t u r e was applied to 8 inch × 8 inch W h a t m a n 3 MM paper. C h r o m a t o g r a p h y and a u t o r a d i o g r a p h y w i t h an e x p o s u r e t i m e of 4 d a y s were p e r f o r m e d as described in MATERIALS AND METHODS. The origin of the c h r o m a t o g r a m s is at the lower left corner. I. Pac extract, after 3 h incubation; I I . ma-l extract, after 3 h incubation.
Biockim. Biophys. Acta, 149 (1967) 335-343
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Pac or the CS strains were used. When extracts of the ma-l or ry larvea were used (these lack xanthine dehydrogenase), only inosine was detected. The autoradiographic data obtained when larval extracts were incubated with other [14C]purines are presented below in tabular form. When [8-14C]adenine was incubated with crude charcoal-treated extracts for 3 h, inosine, xanthine, hypoxanthine and uric acid were formed. To determine whether adenine is converted directly to hypoxanthine, aliquots were removed from the reaction mixture at various times. Table I shows that at 5 min the nucleotides, AMP TABLE I SEQUENCE OF CONVERSIONS IN [8-14C]ADENINE CATABOLISM A Chax-crude e x t r a c t of P a c s t r a i n w a s p r e p a r e d w i t h o.I M Tri s (pH 7-5). o.5 m l of t h i s e x t r a c t w a s i n c u b a t e d a t 3 °0 w i t k o . i pC of [8-14C~adenine. A f t e r 5-, 2o-, 35- a n d 5 o - m i n i n c u b a t i o n , o. i - m l a l i q u o t s w e r e r e m o v e d a n d c h r o m a t o g r a p h y a n d a u t o r a d i o g r a p t x y p e r f o r m e d as d e s c r i b e d in MATERIALS AND METHODS. P l u s e s i n d i c a t e t h e r e l a t i v e a m o u n t s of a m e t a b o l i t e .
Sampling time (rain)
Reaction products AMP
1MP
Inosine
5
++
++
.
20 35 50
++ ++ ++
++ ++ ++
+ ++ ++
Hypoxanthine Xanthine .
. 4+ ++
Uric acid
. -4+
--+
and IMP, are present. Hypoxanthine and xanthine are noticeable at 35 min, but are not readily apparent until after 5° min of incubation. Thus, the order of appearance of metabolites derived from adenine seems to be adenine ~ nucleotides--~ inosine --~ hypoxanthine. In addition, no evidence for the direct conversion of adenine to hypoxanthine by an adenine deaminase was found in the 77 ° ×g precipitate, the IO ooo x g precipitate or the lO5 ooo ×g precipitate before or after sonication or in the lO5 ooo ×g supernatant solution which readily metabolized adenine to nucleotides. Table I I shows the result of incubating [8-14Cladenosine, [8-14Clguanine and [8-14Cluric acid with various larvel extracts. [8-14C]Adenosine is converted readily to inosine, hypoxanthine, xanthine and uric acid except in the xanthine-dehydrogenase mutant ma-1. When aliquots were removed from the reaction mixture at shorter times during the incubation, autoradiograms did not show metabolites in the proximity of the origin where nucleotides migrate. Thus, it appears that unlike adenine, it is not necessary for adenosine to be converted to a nucleotide for catabolism. When [8-1*C~guanine is incubated with crude charcoal-treated extracts for 3 h, the new metabolite formed in extracts of Pac was shown to be uric acid. None of the solvent systems employed in these investigations was capable of separating guanine and xantkine. Uric acid did not form in extracts of the CS strain although trace amounts of uric acid were noted if the exposure time was doubled or if twice the usual amount of radioactive guanine was added to the reaction mixture. Since the metabolism of hypoxanthine to uric acid was normal in extracts of the CS strain, a defect in the conversion of guanine to xanthine was suspected. Biochim. Biophys. Acta, 149 (1967) 335-343
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L . D . HODGE, E. GLASSMAN
TABLE II INCUBATION OF LARVAL EXTRACTS WITH ~8-14C~ADENOSINE, [8-14C~GUANINE AND [8-14C]uRIC ACID
A. o. 5 m l of c r u d e c h a r c o a l - t r e a t e d e x t r a c t s of v a r i o u s s t r a i n s p r e p a r e d w i t h o.i M Tris (pH 7.5) were i n c u b a t e d a t 3 °0 w i t h o.I # C of [8-14C]adenosine a n d [8-14C]guanine. B e fore a n d a f t e r a 3-h i n c u b a t i o n o. I m l of t h e r e a c t i o n m i x t u r e w a s a p p l i e d t o 8 i nc h × 8 i n c h W h a t m a n 3 MM p a p e r . C h r o m a t o g r a p h y a n d a u t o r a d i o g r a p h y were p e r f o r m e d as d e s c r i b e d i n MATERIALS AND METHODS. T h e r e l a t i v e a m o u n t of m e t a b o l i t e n o t e d on t h e a u t o r a d i o g r a m is i n d i c a t e d b y pluses. B. IO g of P a c l a r v a e w e r e h o m o g e n i z e d w i t h I5 m l of B u f f e r A. B y m e a n s of d i f f e r e n t i a l c e n t r i f u g a t i o n a 77 ° × g p r e c i p i t a t e , i o ooo × g p r e c i p i t a t e , lO 5 ooo × g p r e c i p i t a t e a n d a lO 5 ooo × g s upe rn a t a n t s o l u t i o n w e r e o b t a i n e d . The t h r e e p r e c i p i t a t e s w e r e r e s u s p e n d e d in 6 m l of o.I IV[ Tri s ( pH 8.6) b u f f e r a n d s o n i c a t e d for 60 sec. 0. 5 ml of each s o n i c a t e w a s i n c u b a t e d w i t h o.I/ *C of [8-x4C]uric acid. Before a n d a f t e r a 3-h i n c u b a t i o n a t 3 o°, o . i ml a l i q u o t s of t h e r e a c t i o n m i x t u r e s were used for c h r o m a t o g r a p h y a n d a u t o r a d i o g r a p h y . The r e s u l t s w h i c h are s h o w n are o n l y for t h e p a r t i c u l a t e f r a c t i o n p r e p a r e d b y IO ooo × g c e n t r i f u g a t i o n .
Strain
l*C-Metabolite
Reaction products Inosine
Hypoxanthine
Xanthine
Uric acid
Allantoin
A. A d e n o s i n e
+ + + +
+ + + +
-+ +
-+
--
Pac CS
A. G u a n i n e
---
---
? ?
+ + --
---
Pac
B. Ur ic acid
.
ma-l Pac
.
.
.
±
T h e o n l y evidence t h a t [14C]uric acid was c o n v e r t e d to o t h e r m e t a b o l i t e s was o b t a i n e d w i t h sonicates of t h e IO ooo × g p r e c i p i t a t e . A f t e r a 3 h i n c u b a t i o n a new m e t a b o l i t e d i d a p p e a r w i t h this fraction b u t it was n o t clear w h e t h e r this was a p r o d u c t f o r m e d from uric a c i d or from one of the impurities. I t m i g h t h a v e been allantoin, b u t it was n o t possible to o b t a i n sufficient q u a n t i t i e s for identification. Since u r a t e oxidase a c t i v i t y , if present, was at low levels, this was n o t p u r s u e d
TABLE CONVERSION
III OF XANTHINE
TO
URIC
ACID
IN X A N T H I N E - D E H Y D R O G E N A S E
MUTANTS
L a r v a e a n d flies were c h e c k e d for c o n t a m i n a t i o n before p r e p a r a t i o n of e x t r a c t s . 3.0 g of l a r v a e a n d 3.0 g of flies f r o m Pac, ma-1, a n d ry w e r e h o m o g e n i z e d s e p a r a t e l y i n 4.5 m l of o.I M Tri s ( p H 7.5). F r o m e a c h c r u d e h o m o g e n a t e a c r u d e c h a r c o M - t r e a t e d e x t r a c t a n d a p H 5-chaxcoait r e a t e d e x t r a c t w e r e p r e p a r e d . 0. 5 m l of e a c h of t h e s e 8 e x t r a c t s w a s i n c u b a t e d w i t h o.I #C (8-14C]-xanthine a n d o . i m l of I m M N A D . A f t e r a 3 o - m i n i n c u b a t i o n a t 3 o°, o . i - m l a l i q u o t s w e re c h r o m a t o g r a p h e d as d e s c r i b e d in MATERIALS AND METHODS. E x p o s u r e t i m e for a u t o r a d i o g r a p h y w a s I o d a y s . T h e r a d i o a c t i v i t y of t h e s p o t s c o n t a i n i n g u r i c a c i d w a s d e t e r m i n e d a n d t h e d a t a c o n v e r t e d to m/tg.
Strain
ml~g o[ uric acid [ormed per reaction mixture Larvae
ma-1 ry Pac
Adults
Crude charcoaltreated extract
p H 5-charcoaltreated extract
Crude charcoaltreated extract
pH5-chamoaltreated extract
199 389 4426
2o2 696 4733
168 333 3169
16o 749 3843
Biochim. Biophys. Acta, 149 (1967) 335-343
D. melanogaster PURINE CATABOLISM. I
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further. There was no Change when E14C]uric acid was incubated with sonicates of the 6o0 x g or the lO5 o o o × g precipitates, the lO5 o o o × g supernatant solution, the crude charcoal-treated extracts, or the pH 5-charcoal-treated extract. Data in these investigations indicated that if autoradiograms of ma-l and ry extracts were exposed for 2.5 times the usual exposure time, low levels of xanthine dehydrogenase activity were detectable. To confirm this observation crude and pH 5-charcoal-treated extracts of strains Pac, ry and ma-1 were incubated with [8-14C]xanthine and NAD, the electron acceptor for xanthine dehydrogenase. Table III shows that cell-free extracts of both larvae and adults do convert some xanthine to uric acid. More xanthine is converted to uric acid by the pH 5-charcoal-treated extracts of all strains than b y the crude charcoal-treated extracts.
DISCUSSION
Purine catabolism was studied in D. melanogaster using extracts of third instar larvae. Since active cell division and growth occur in this developmental stage, it was thought that the enzymes involved in these reactions would be present. Some aspects of the pathway of purine catabolism were established by incubating [14C]purines with these extracts and by separating the radioactive products of these reactions by chromatography and autoradiography. The reactions shown in Fig. 3
Adenine
LN o~/J-~>
HOCHA~ OH Adeno eine-SJ monophosphate
I
~ 1 II ~" Hypoxonthine
HOCH=-~ / j Inosine OH ."
I
Inosine-5" / monophosphate
N
N
I ") HIN-1,~_.,,~'
N neH Guani
.
~--",> Xanthine
J
OH
HO N~
ON
Uric Acid Allantoin Fig. 3. P u r i n e catabolism in larval e x t r a c t s of Drosophila. This figure is a s u m m a r y of t h e in vitro reactions involving p u r i n e c a t a b o l i s m in t h i r d i n s t a r larvae of D. melanogaster. D a s h e d lines are conversions which need m o r e w o r k for confirmation.
Biochim. Biophys. Acta, i49 (1967) 335-343
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L. D. HODGE, E. GLASSMAN
were elucidated. The only exception to this scheme was tile result obtained with extracts of the CS strain where the data indicate a possible defect in the conversion of guanine to xanthine. Of interest is that these conversions of purines indicated that the enzymes which produce substrates for xanthine dehydrogenase are adenosine deaminase, inosine phosphorylase and guanine deaminase. When [8-14Cladenine was used the order of appearance of metabolites was the nucleotides, AMP and IMP, then inosine, hypoxanthine, xanthine and uric acid. The method b y which adenine is converted to nucleotides is not clear. I t is consistent with a pyrophosphorylase reaction as found in Escherichia coli, yeast, avian and mammalian tissue 14. The AMP could be hydxolyzed to adenosine or deaminated to IMP, as reported in microorganisms and mammals la. By removal of the phosphate, IMP and AMP could be converted to inosine and adenosine, respectively 14. This suggestion was supported b y incomplete data in which [8-1aC]AMP was converted to IMP, adenosine, inosine, hypoxanthine and uric acid. I t is also possible that AMP could have arisen b y the transribosidation of adenine to adenosine followed b y an adenosine kinase reaction. However, adenosine was not one of the reaction products as would be expected. This evidence combined with the data on the catabolism of adenosine indicates that adenine itself is not deaminated; only adenosine and AMP are deaminated. This result is consistent with reports that adenine deaminase is present in microorganismslS, le but not in higher organisms. Urate oxidase, the enzyme responsible for catabolizing uric acid to allantoin has been reported in a number of insects: Popillia iaponica (Japanese beetle) 17, Periplaneta americana linnaeus (American cockroach) 18 and Musca domestica (house fly) ~9. AuF DER MAULs reports uricase activity in adults of various mutants of D. melanogaster. Since we were unable to find significant conversion of uric acid to allantoin in extracts of third instar larvae, it is possible that urate oxidase is present only in pupae and adults. Many other conversions involving purines, nucleosides and nucleotides could be established in D. melanogaster b y appropriate experiments. Since a complete picture of purine catabolism would describe m a n y biochemical phenotypes, continued study should be of interest to the Drosophila geneticist. Hopefully, mutants affecting purine metabolism can be found b y assaying established strains or b y some selection procedure. Where alternate pathways exist for the synthesis or breakdown of a purine, or where such reactions are not absolutely necessary, as for example those catalyzed b y xanthine dehydxogenase, viable mutants can probably be obtained. An interesting finding was the conversion of xanthine to uric acid in the extracts of ma-1 and ry. Other work in this laboratory using an assay based on the increase in fluorescense when 2-amino-4-liydroxy-pteridine is converted to isoxanthopterin indicated that extracts of ma-l and ry have about 0.25 % of the xanthine dehydrogenase activity of wild type (E. GLASSMAN, unpublished). However, ry and ma-l flies and larvae do not contain detectable amounts of uric acid or isoxanthopterin 2°, indicating that the in vivo activity of xanthine dehydrogenase is very low, if present at all. The xanthine dehydrogenase activity which we observed in ma-l and ry is not due to a contamination of the ry and ma-l stocks with wild type. Based on the amount of xanthine dehydrogenase in a single wild-type larvae, we calculate that about 5 ° contaminant larvae per g of ma-l or IOO contaminants Biochim. Biophys. Acta, 149 (1967) 335-343
D. melanogaster PURINE
CATABOLISM. I
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per g of ry larvae would be necessary to produce the amount of conversions observed here and contamination of this size would be easily noted. Since these experiments were done in the presence of excess amounts of xanthine dehydrogenase, there probably was not proportionality between the amount of xanthine converted and xanthine dehydrogenase activity and it is difficult to determine the actual amount of xanthine dehydrogenase activity present in the ma-1 and ry extracts. Whether this activity in ma-l or ry is due to xanthine dehydrogenase or to other enzymes with a low specificity for this reaction is not clear.
ACKNOWLEDGEMENTS
Part of the work reported here was supported b y Public Health Service Grant GM-o82o2. L.D.H. was supported by Genetics Training Grant, 5TI GH-685, from The Public Health Service. E.G. was supported b y a Research Career Development Award, 2-K3-GM-I4,9II, from the Public Health Service. REFERENCES I 2 3 4 5 6 7
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