Absence of galactose enzyme activities in axenic Drosophila melanogaster

Insect Biochem., Vol. 1 I, No. 2, pp. 221-224, 1981. Printed in Great Britain.

0020-1790/81/0202214)4502.00/0 Pergamon Press Ltd.

A B S E N C E OF G A L A C T O S E E N Z Y M E ACTIVITIES IN AXENIC DROSOPHILA MELANOGASTER H. N. KAMEROW, C. R. MERRIL* and W. G. NASHt National Institute of Alcohol Abuse, * Laboratory of General and Comparative Biochemistry, National Institute of Mental Health and t Laboratory of Viral Carcinogenesis, National Cancer Institute, Bethesda, MD 20205, U.S.A. (Received 3 July 1980; revised 4 September 1980)

Abstract Four developmental stages of Drosophila melanogaster were surveyed for the enzymes of the Leloir pathway and no enzyme activity was detected with the exception of trace amounts of 5-uridine diphosphogalactose-4-epimeraseactivity in the egg. Despite the lack of detectable enzyme activity, the flies were able to convert 14C-labelled galactose to 14CO2, suggesting low-level Leloir enzyme activities, below the level of detection, or the presence of an alternate pathway for galactose metabolism. Non-axenic flies displayed variable amounts of enzyme activities, probably due to contaminating organisms. Key Word Index: Drosophila melanogaster, egg, larva, pupa, adult, Leloir pathway, galactose oxidation

tissue culture flasks, containing sterile fly media ( 10% glucose (w/v), 2% Baker's yeast (w/v, autoclaved) and 1.4°o bactoCONVERSION o f galactose to 5'-uridine d i p h o s p h o - agar), were inoculated with the axenic eggs and plugged with cotton. Radiochemical experiments were performed as glucose (UDP-glu) by the Leloir p a t h w a y is a m a j o r route for galactose metabolism in most organisms described except that [l-~4C]-D-galactose (New England (LELOIR, 1951; KALCKAR, 1958). The Leloir Nuclear, 31.82 Ci/mole, 0.1 MCi/ml of media) or [UL-14C] p a t h w a y effects this conversion by utilizing D-glucose (ICN, 240 Ci/mole, 0.1 #Ci/ml of media) was added to the media before the agar hardened. Radioactive galactokinase (E.C. 2.7.1.6), g a l a c t o s e - l - p h o s p h a t e flasks were plugged with a rubber stopper and CO 2 uridyl transferase (E.C. 2.7.7.12) and 5'-uridine samples were collected during fly maturation using a 10 ml diphosphogalactose-4-epimerase (E.C. 5.1.3.2). The glass syringe and a 19 gauge needle. The carbon dioxide pathway has been extensively studied in bacteria was monitored for radioactivity after absorption in (ADHYA and SHAPIRO, 1969), yeast (DOUGLAS and phenethylamine (LAPOLLA el al., 1975). The radioactive sample was counted in dry mix (Mallinckrodt) on a Beckman HAWTHORN, 1966) and man (KALCKAR et al., scintillation counter (model LS250). The counting efficiency 1956). A few organisms utilize other p a t h w a y s as a for 14C was 93°,,'0. major metabolic route for galactose metabolism. One to three weeks after inoculations, non-radioactive Staphylococcus aureus metabolizes galactose by flasks were broken open and flies in various developmental converting it directly to galactose-6-phosphate, an stages were collected. Samples were homogenized manually intermediate in the tagatose p a t h w a y (BISSETT and in 1.5 ml capacity Eppendorf tubes with a sintered glass ANDERSON, 1973). pestle (Kontes Glass Co., Duall 20) in phosphate-buffered We have recently completed a survey for the saline (NaCI 8 g/l, KCI 0.2 g/I, Na2HPO 4 1.15 g/l, enzymes o f the Leloir pathway in D. melanogaster. KHEPO4 0.2 g/l, MgCI 2 6H20 and CaC1z 2H20 at 0.1 g/l). After freeze-thawing 3 times with alternate immersion in an The organism's ability to metabolize galactose was alcohol and dry ice bath and water bath at 21°C, samples also studied. were centrifuged for 3 min at 12,500 g in an Eppendorf centrifuge to pellet cellular debris. The supernatants were saved and kept at 4°C until they were assayed. Assays were MATERIALS AND METHODS always performed within 2 hr after cell lysis. E. coli fSA758 Wild type stock fORE-r) of D. melangaster (obtained and SA500, gift of Sankar Adhya, NIH, Bethesda, MD, U.S.A.) was colony purified and grown in M56 media from Dr. William Nash of the National Cancer Institute, NIH, Bethesda, MD, U.S.A.) were raised in 250 ml culture (a minimal media) which was supplemented with 0.Y'~ flasks at 25°C. Culture flasks contained 3% Bacto-agar galactose (w/v), 0.3,% fructose (w/v), 0.1% casein amino acids (w/v), 0.013{, histidine (w/v) and 0.0025°;; vitamin (DIFCO), 1~,~propionic acid, 10°/,,glucose and 7.5% Baker's yeast (autoclaved). A paste consisting of 10 g of yeast mixed B 1 {w/v). E. coil was grown to log phase and then with 15 ml of water was placed on the agar surface. The paste concentrated by centrifuging in a Sorval centrifuge at 14,612 g at 4°C for 15 min. The supernatant was discarded (the fruit flies' nutriment) contained less than 0.05 mg/ml of following centrifugation. The E. coli pellet was resuspended galactose. Tissue paper was placed in the flask to provide a moisture-free area for the adults. Flies were transferred into and lysed by freeze-thawing 3 times in appropriate buffer solutions. The buffer for the transferase and epimerase new flasks every 2 days. Eggs of non-axenic D. melanogaster (ORE-r) were isolated assays was 0.1 M glycine (pH 8.7) while 0.1 M Tris-HCl (pH 7.8) was used for the kinase assay. E. coli strain SA500 by allowing females to lay eggs in 250 ml Ehrlenmeyer flasks is a wild type E. coli KI2 strain with a normal galactose containing soft agar. The eggs were collected and sterilized operon, while strain SA758 contains a deletion of the with 43~, sodium hypochlorite and ethanol, and placed on nitex screens (from the NAZ DAR Company). Sterile 250 ml galactose operon and served as a null control. Galactokinase 221 INTRODUCTION

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H . N . KAMEROW, C. R. MERRIL AND W. G. NASH

Table 1 Enzyme activities in axenic Drosophila melanogaster Kinase Adults Pupae Larvae Eggs E. coli(SA758) E. coli(SA500)

<0.5 <0.5 <0.5 <0.5 <0.5 9.3 3.6

+ 0.14 + 0.20 +_ 0.14 + 0.043 +_ 0.19 x 103+_ x 103

Transferase <0.1 <0.1 <0.1 <0.1 <0.1 2.8 7.9

+_ 0.04 +_ 0.02 + 0.02 +_ 0.04 + 0.02 x 103+_ x 102

Epimerase <0.1 <0.1 <0.1 <0.91 <0.1 4.8 3.5

+_ 0 + 0 + 0 + 0.11 _+ 0.13 × 10"~+ × 10~"

Enzyme activities are expressed in nmoles/mg/hr. The limits of detection for these assays are 0.5, 0.1 and 0.1 nmoles/mg/hr for the kinase, transferase and epimerase assays respectively. Enzyme assays were repeated 3 times for each sample and the results are reported as the mean + S.D. was assayed by the method of CHACKO et al. (1972). Galactose-l-phosphate uridyl transferase was assayed as described by MERRXL et al. (1971). 5'-uridine diphosphogalactose-4-epimerase activity was determined by the method of MERRIL et al. (1976). Protein determinations were by the method of LowRy et al. (1951). Sterility tests were performed at the beginning and end of each experiment utilizing axenic flies. These sterility tests were performed by inoculating media from the axenic egg flasks onto tryptone plates. These plates were kept at 25°C for 2 days to assay for yeast contaminants and for an additional 2 days at 37°C to assay for bacterial contaminants. Sterility tests were also performed whenever flasks were broken open and flies removed. The glucose concentration in the yeast extract was determined with a Beckman glucose analyzer and the galactose concentration was determined with a lactose/galactose food analysis kit (Boehringer Mannheim). The glucose concentration was 0.3 mg/ml and the galactose concentration was less than 0.05 mg/ml (the limits of the assay). The dilution effect of the non-radioactive glucose on the metabolism of the radioactive glucose is considered in the radiochemical calculations.

RESULTS N o enzyme activities of the keloir p a t h w a y were detected in the egg, larval, pupal or adult forms of Drosophila melanogaster ( m a i n t a i n e d axenically) with the exception of a trace a m o u n t of cold-labile 5'-uridine diphosphogalactose-4-epimerase in the egg (Table 1). A l t h o u g h all four stages of D. melanogaster exhibited deficiencies o f g a l a c t o k i n a s e a n d galactose- 1phosphate uridyl transferase, axenic flies d e m o n s t r a t e d the ability to metabolize radioactive [l-14C]-Dgalactose, converting it to radioactive c a r b o n dioxide. Germ-free flies (all stages) metabolized radioactive galactose at a rate of 5.3 x 10 ' m m o l e s / o r g a n i s m / d a y while control flies metabolized radioactive [UL-14C]-D-glucose at a rate of 2.9 × 10 -3 m m o l e s / o r g a n i s m / d a y . Flasks c o n t a i n i n g [1-~4C]-D-galactose (0.1 ~Ci/ml, 31.82 Ci/mole) without addition of flies failed to evolve ~4CO2. The c o n c e n t r a t i o n o f galactose in the media was 3.4 × 10 4 mg/ml while the c o n c e n t r a t i o n of glucose was 100.23 mg/ml. Enzyme assays were performed on fly extracts mixed with E. coli extracts to determine whether the lack of enzyme activity in the fly might be due to the presence of enzyme inhibitors in the fly extracts. N o inhibition was observed for galactokinase or 5'-

uridine diphosphogalactose-4-epimerase from all stages of fly extracts. G a l a c t o s e - l - p h o s p h a t e uridyl transferase from E. coli extracts was not inhibited by fly extracts from eggs, but was inhibited by extracts from adults, p u p a e and larvae. These three extracts inhibited transferase enzyme activity by 50°;.

DISCUSSION The ability of flies to metabolize galactose may be due to the presence of an alternate pathway(s) or to extremely low-level activities of the Leloir enzymes. Kinase activity below 0.5 n m o l e s / m g / h r and transferase and epimerase activities below 0.1 n m o l e s / m g / h o u r could not be detected by the radiochemical assays. The observed rate of [1-14C]-D galactose conversion into radioactive 14CO2 could have been due to Leloir enzymes' activities below the assay limits. In fact, activities just below the radiochemical assay limits could account for as m u c h as 10 times the observed rate of [1-14C]-D-galactose conversion to 14CO2. On the other h a n d there is the possibility that alternate routes of galactose metabolism which have been proposed to account for metabolic events in h u m a n studies could also account for some o f this galactose metabolism in Drosphila melanogaster. M o s t of these alternate pathways require a p h o s p h o r y l a t e d galactose. No galactokinase activity was observed in D. melanogaster. However, three pathways which do not require p h o s p h o r y l a t i o n have also been proposed. The first utilizes galactose dehydrogenase (CAUTRECASAS a n d SEGAL, 1966) but this p a t h w a y was seriously questioned by STRIVASTAVA and BEUVLER (1969) w h o failed to detect the formation of galactonic acid from uniformly labelled [lgC]-galactose in rat liver s u p e r n a t a n t fractions. The second uses aldose reductase to metabolize galactose. This reaction usually results in the accumulation o f galactitol which c a n n o t be further metabolized in most organisms (KINDSHITA, 1974). No galactitol could be detected in axenic flies raised in the presence of radioactive [l-~4C]-D-galactose (New England Nuclear, 31.82 Ci/mole), at a c o n c e n t r a t i o n of 3.4 x 10 4 mg/ml. A thin layer c h r o m a t o g r a p h y separation described by MERRIL et al. (1971) was utilized to assay for galactitol. This m e t h o d used P.E.I. thin layer plates, (Brinkman, 20 x 20 cm) with a one m o l a r sodium formate (pH 3.5) solvent system.

Galactose in D. melanogaster

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Table 2. Enzyme activities in non-axenic Drosophila melanogaster Kinase Adults Pupae Larvae Eggs

0.63 <0.5 <0.05 1.17

+ ± ± ±

0.09 0.25 0.12 0.12

Transferase <0.1 <0.1 <0.1 <0.1

± + ± ±

0.01 0.07 0.01 0.04

Epimerase 4.63 0,19 0,73 3,06

± ± ± +

0.17 0.05 0.17 0.39

Enzyme activities are expressed in nmoles/mg/hr. D. melanogaster from non-axenic cultures were collected and processed for enzyme assays as described for axenic flies. Each assay was repeated 3 times and the results are reported as the mean ± S.D. Authentic [14C]-galactitol (7 Ci/mole, Amersham) was used as a marker. No radioactivity above background (50 cpm) was observed at the RFValue of the authentic galactitol marker in the fly lysates. The third possibility might involve the metabolism of galactose to an intermediate of the glycolytic sequence by a pathway similar to the tagatose pathway found in Staphylococcus aureus. Further experiments will be required to determine whether the observed galactose metabolism is due to low levels of Leloir pathway enzymes or whether some alternate pathway(s) are functioning in D. melanogaster. The absence of galactokinase and galactose-1phosphate uridyl transferase in D. melanogaster does not set a precedent in eukaryotes. STEPHENS et al. (1974) found grey kangaroos which were missing both of these enzymes. The presence of detectable 5'-uridine diphosphogalactose-4-epimerase activity in the eggs of the flies may be explained by the need for galactose in the membrane glycoproteins of developing flies. This explanation favours galactose synthesis from glucose by the epimerase enzyme rather than galactose catabolism in D. melanogaster. Although Leloir enzymes could not be detected in Drosophila melanogaster, galactose metabolism did occur. The Leloir enzymes may not be present. Thus, galactose metabolism may be due to an alternate pathway. If the Leloir enzymes are present in D. melanogaster, three different hypotheses may explain the findings. The Leloir enzymes in the fly extracts may have requirements which are not compatible with the enzyme assays which were employed. These assays have been used in systems ranging from bacteria to mammals, thus it is unlikely the enzyme assays are inadequate to detect the Leloir enzymes in D. melanogaster. Endogenous substances may be inhibiting the Leloir enzymes in the fly extracts. Since no Leloir enzymes were found in the fly extracts except for a trace amount of epimerase activity in the eggs, this possibility could not be tested. As an alternative, E. col±Leloir enzymes were used to test for inhibitors in the fly extracts. No inhibition of E. col±galactokinase or epimerase was found and there was only partial inhibition of E. col± transferase by adult, pupal and larval extracts. None of the E. col± Leloir enzymes were inhibited by the egg extract. This stressed assay for inhibition does not rule out the possibility of endogenous inhibitors of the Leloir enzymes in D. melanogaster. Finally, the fruit flies may maintain low levels of the Leloir enzymes which could not be detected. The enzyme assays would have to be 10 times as sensitive to rule out the possibility that l_eloir enzymes may be converting galactose to CO:.

The assays employed for the Leloir enzymes are among the most sensitive assays in the literature. Failure to detect the Leloir enzymes under one set of assay conditions cannot be taken as proof that the structural genes are absent in Drosophila melanogaster. The enzyme hypoxanthine-guaninephosphoribosyl transferase (HGPRT) is not detectable in extracts of D. melanogaster larvae or adults (BECKER, 1974). Furthermore BECKER (1978) found that cultured cells established from D. melanogaster embryos possessed no H G P R T activity, and only traces of 5'-nucleotidase activity. Upon addition of purine bases, nucleosides, orotate, glutamate, azaserine or antifolates to the culture media, de novo purine biosynthesis was inhibited and H G P R T activity appeared. 5'-Nucleotidase activity also increased after addition of these inducing compounds to the culture media. Another enzyme which is inducible in D. melanogaster cell cultures is fl-galactosidase (BEST-BELLPOMMEet al., 1978). This enzyme is induced by ecdysterone (fl-ecdysone). Attempts to induce the Leloir pathway by the addition of exogenous galactose (10°% w/v galactose) to the media of axenic flies failed; no significant levels of enzymes were detected. The galactose-fed flies showed low viability and fecundity, but enough organisms were obtained for the enzyme assays, Leloir pathway enzymes were also assayed in flies grown non-axenically. The data in Table 2 show that even when flies were grown in the presence of bacteria and yeast only very low levels of kinase and epimerase activity appeared. High levels of Leloir pathway enzymes might have been expected due to the contaminating organisms. However, since the Leloir pathway enzymes are usually regulated in bacteria, such as E. col±,the contaminating organisms may not have been induced. However, the use of axenic cultures for studies of intermediate metabolism in D. melanogaster is strongly urged, as the presence of even trace amounts of enzymes from contaminating organisms can seriously confuse attempts to ascertain metabolic pathways.

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