Purine salvage pathways in the Australian termite, Nasutitermes walkeri hill

In.wct Biochem. Molec. Biol. Vol. 22, No. 2, pp. 175-179,1992 Printed in Great Britain. All rights reserved

0965-1748/92$5.00+ 0.00 Copyright 0 1992Pcrgamon Press plc

PURINE SALVAGE PATHWAYS TERMITE, NASUTITERMES

IN THE AUSTRALIAN WALKERI HILL

D. J. CHAPPELL and M. SLAYTOR* Department of Biochemistry, The University of Sydney, Sydney, NSW 2006, Australia (Received 2 January 1991; revised and accepted 12 September

1991)

Abstract-Extracts of the degutted body of the termite Nasutitermes walkeri contained adenine phosphoribosyltransferaae (APRT) (0.277 f 0.014 nmol/min/mg protein) but neither hypoxanthine-guanine phosphoribosyltransferase (HGPRT) nor inosine kinase indicating that the synthesis of inosine is the committed step in the catabolism of IMP and AMP to mate. The presence of a low activity of adenosine kinase (0.025 f 0.002 nmol/min/mg protein) may be required to prevent depletion of the adenylate pool. Bacterial extracts from the gut by contrast contained the full range of salvage enzymes including HGPRT (0.117 + 0.003 nmol/min/mg protein), APRT (0.371 f 0.015 nmol/min/mg protein), adenosine kinase (0.068 f 0.025 nmol/min/mg protein) and inosine kinase (0.032 rt 0.002 nmol/min/mg protein). The inability of N. walkeri to salvage inosine and hypoxanthine suggests a mechanism for the apparently uncontrolled synthesis of urate, by termites, during laboratory storage. Key Word Index: purine salvage enzymes; urate; termites; Isoptera; adenine phosphoribosyltransferase; hypoxanthine-guanine phosphoribosyltransferase; adenosine kinase; inosine kinase; Nasutitermes walkeri

INTRODUCTION

The importance of the salvage enzymes, adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), in the control of purine synthesis is well established in vertebrates (Palella and Fox, 1989) but the siguificance of these enzymes in the control of purine synthesis in insects is unknown. The presence of the phosphoribosyltransferases APRT and HGPRT in insects has been demonstrated only in Drosophila (Becker, 1978; Le Menn et al., 1987; melanogaster Silber and Becker, 1981) in a number of cell lines and in several strains of adult flies. Adenosine kinase has also been detected (Becker, 1974a). Wild type cell lines of the mosquito Aedes albopictus, by contrast, are deficient in HGPRT activity (Abidi et al., 1987) but are capable, like adult and larval Aedes aegypti, of phosphoribose. transfer to xanthine (Swerdel and Fallon, 1987). Evidence for the presence of salvage enzymes in other species comes only from the incorporation of [i4C]purines into nucleotides or nucleic acids. In Musca domesticus (Miller and Collins, 1973) the high incorporation of adenine contrasted with the inability to utilise hypoxanthine and guanine suggesting that there was no HGPRT activity in this species. This pattern was repeated in Anopheles a/himanus (Miller, 1980) with the exception that the moderate incorporation of guanine suggested a weak HGPRT activity. Additionally, both dipterans were capable of incorporating selected nucleosides raising the possibility that nucleoside kinases may be present. M. domestica efficiently utilised guanosine (Miller and Collins, 1973) while A. albimanus salvaged adenosine and inosine (Miller, 1980). Information on the salvage capabilities of the exopterygote insects is *Author for correspondence.

even more fragmentary. Short term in vitro incorporation experiments, using fat body from the termite Nasutitermes walkeri, have shown an inability to salvage hypoxanthine and inosine while a trace amount of guanosine was incorporated into nucleotides (Chappell and Slaytor, 1991). In this paper we present methods of assaying the purine phosphoribosyltransferases and the purine nucleoside kinases in the presence of non-specific phosphatases and S-nucleotidase. These assays have enabled the documentation of the purine salvage pathways in the termite N. walkeri, the major finding being the lack of an HGPRT activity. MATERIALS

AND METHODS

Radioactive substrates

[8%]Adenosine, [8%]hypoxanthine, [U%]adenosine, [U~14C&anosine and [8-‘4C]inosine were all obtained from Amersham (Buckinehamshire. U.K.). 18-‘4C1Guanine was obtained from New England Nuclear (Boston, Mass.). [2J4C]Xanthine was obtained from Schwarz Bioresearch (Orangeburg, N.Y.). Preparation of termite extracts Nasutitermes walkeri Hill were collected as previously described (Chappell and Slaytor, 1991) and used within 2 h of collection. Termite guts or degutted bodies were homogenised in a Ten Broeck homogeniser in 2.0 ml of 0.1 M Tris-HCl buffer pH 7.5 containing 1 mM EDTA, 5 mM MgCl, and 25 mM KCl. Cellular debris was removed by centrifugation at lO,ooOg for 15 min. The supematant was centrifuged at 100,000g for 2 h to remove membranes then simultaneously desalted and concentrated using Centricon 30 microconcentrators (Amicon, Danvers, Mass.) prior to analysis. Deproteinised extracts were prepared by heating at 100°C for 5 min followed by centrifugation at lO,OOOgfor 15 min. Alternatively the extracts were adjusted to 0.4 M HClO, and centrifuged for 15 min at lO,OOOg.After neutralisation with

175

D.J. CHAPPELLand M. SLAYTOR

176

2.5 M KOH, buffered with 0.6 M KHCO,, the centrifugation step was repeated to remove KClO,. Each extract was derived from 50 to 100 mature worker caste termites selected on the basis of size and cranial pigmentation (McMahan and Watson, 1975). All operations were carried out at &4”C. Protein was estimated by the method of Lowry et al. (1951). Inhibition studies on S-nucleotidase

The assay mixture for ti’nucleotidase contained in 1 ml: Tris-HCl, pH 7.5, 100 pmol; MgCl,, 20 pmol; IMP, 1 pmol; potassium phosphate, 2 pmol; glycerol-Zphosphate, 100 pmol; nucleoside phosphorylase, 0.1 units (U)*; xanthine oxidase, 0.1 U. The mixture was assayed at 37°C by the increase in absorbance at 292 nm. For inhibition studies, the concentration of a$-methyleneadenosine-5’diphosphate (Sigma, St Louis, MO) ranged from O-O.5 mM and for concanavalin A (Sigma, St Louis, MO) from (r56 PM. The molecular weight of concanavalin A is 71,000 (Olsen and Liener, 1967). IMP concentrations were varied over the range 0.5 to 5.0mM. Kinetic data was evaluated using the Lineweaver-Burk or Hanes-Woolf linear transformations (Segel, 1975). Phosphoribosyltransferase

assays

Assays for hypoxanthine-guanine phosphoribosyltransferase activity were carried out using the procedure of Smithers and O’Sullivan (1985) with the exception that the concentration of hypoxanthine was increased (0.4 mM; 9 mCi/mmol). Alternatively guanine (0.1 mM; 12.5 mCi/mmol) was used as substrate. Dephosphorylation of the product was prevented by the addition of the phosphatase inhibitor glycerol-2-phosphate (100 mM) (Behield and Goldberg, 1970) and the inclusion of a 5’-nucleotidase inhibitor, either a$-methyleneadenosine-5’diphosphate (0.5 mM) or concanavalin A (60 FM). Adenine (0.4 mM; 9 mCi/mmol) replaced hypoxanthine in assays of adenine phosphoribosyltransferase and xanthine (0.2 mM; 9 mCi/mmol) was used in assays of xanthine phospho-

ribosyltransferase. 5-Phosphorylribose-1-pyrophosphate (PRPP) was prepared on the day of use after assay by the method of Zalkin (1985). The effect of termite extracts and phosphatase or 5’-nucleotidase inhibitors on phosphoribosyltransferase activity was examined using yeast HGPRT (Sigma, St Louis, MO) as standard. Radioactive products were counted as previously described (Chappell and Slaytor, 1991). Nucleoside kinuse assays

The 1 ml reaction mixture contained Tris-HCl, pH 7.5, (0.1 M), MgCl, (lOmM), adenosine, guanosine or inosine (0.12 mM; 15 mCi/mmol) and ATP (2.6mM). In addition glycerol-2-phosphate (100 mM) and concanavalin A (60pM) or a$-methyleneadenosine-Ydiphosphate (0.5mM) were added to inhibit non-specific phosphatases and 5’nucleotidase. Assays were begun by the addition of extract and were terminated by application a 10 ~1 aliquot to the origin of PEI-cellulose TLC strips (6.5 x 1.5 cm) prepared and developed according to the method of Smithers and O’Sullivan (1985). Statistical analyses

The standard deviation for the regression coefficient was calculated, after least squares analysis, using the method described by Bryant (1966). Inhibitor constants were derived from secondary plots of the slope and intercept of the Lineweaver-Burk plots (Segel, 1975) and uncertainties were represented as the standard error (S,). All other errors were represented by the standard error of the mean. *One unit of enzyme activity is defined as the amount of protein producing 1 pmol of product in 1 min.

Table 1. Inhibition of yeast hypoxanthine-guanine phosphoribosyltransferase activity by desalted termite extracts Extract

Whole termite

Treatment

boiled HClO,

Body Gut

-

Protein It Si (ma/ml) ~

Inhibition f St (%)

13.25 * 5.0 1.75 f 0.3 0.85 f 0.1

99.2 + 0.8 0 0 99.7 + 0.3 92.5 + 1.5

“.

I

.

Assays were carried out as described in the text but contained, in addition, yeast HGPRT (1 x IO-‘U) and termite extract as indicated in the table. Rates were calculated from the initial rate of IMP synthesis. Each value is the result of three determinations.

RESULTS

Preliminary experiments with both phosphoribosyltransferases showed no activity in either gut or body extracts. Commercial yeast HGPRT was completely inhibited by desalted extracts of whole termites and degutted bodies but was not affected by acid precipitated or boiled extracts (Table 1). Desalted gut extracts appeared less inhibitory causing 92.5 + 7.5% inhibition though this marginal outcome was not significant (P > 0.10) and may only reflect the lower concentration of gut protein in these assays. Presumed interference by a cytosolic 5’-nucleotidase initiated a study of inhibitors of this enzyme. Thymidine triphosphate (100 ~1M) caused only 13.0 f 0.29% inhibition and adenosine diphosphate (100 PM) and adenosine triphosphate (100 PM) inhibited at 56.7 + 1.34% and 42.4 + 1.00% respectively and were unsatisfactory for assays of APRT activity as they were potential inhibitors. The adenosine diphosphate analogue a$-methyleneadenosine-5’-diphosphate was a potent inhibitor of termite 5’nucleotidase. Inhibition was mixed with competitive (Ki, = 0.008 + 0.002 mM; n = 5) and uncompetitive effects (KU = 0.006 + 0.006 mM; n = 4) and a minor allosteric effect was also demonstrated. The Hill coefficient of 0.833 &-0.019 (n = 5) was significantly different from 1 (P < 0.001) suggesting negative cooperativity in the binding of this inhibitor. This differs from previous reports, for human placenta (Madrid-Marina and Fox, 1986) and Torpedo electric organ (Grondal and Zimmermann, 1987), where purely competitive behaviour, with no allosteric effects, was exhibited. A concentration of 0.5 mM, which caused 94.7 f 1.8% (n = 3) inhibition of 5’-nucleotidase but only marginally affected HGPRT activity (Fig. l), was chosen for use in assays of HGPRT activity. At this inhibitor concentration the recovery of yeast HGPRT activity, in the presence of termite extracts, was 82.1 f 2.7% (n = 3). This inhibitor concentration was also used in assays of inosine and guanosine kinase. The presumed inhibition of APRT activity by this adenosine diphosphate analogue, in early trials, led to the use of concanavalin A as an alternative for the assay of this enzyme. The inhibition of termite 5’-nucleotidase by concanavalin A, though showing uncompetitive characteristics, was complex, Inhibition was biphasic over the range 0-6OpM, the initial phase quickly reaching 66.2 + 0.08% (n = 3) inhibition by 0.6 PM. Additional increments in the inhibitor concentration were less effective leading to 94.6 f 0.01% (n = 3)

Purine salvage pathways in N. walkeri

12 29 E a6 z

3 0

L

0

6

3

9

12

Time (min)

Fig. 1. Recovery of yeast hypoxanthine-guanine phosphoribosyltransferase activity with a,/?-methyleneadenosine-5’diphosphate. Assays were carried out as described in the text but contained in addition yeast HGPRT (1 x IO-’ U). (@), yeast HGPRT control; (O), termite extract (3.6 f 1.6 mg/ml protein); (A), 0.5 mM a,/?-methyleneadenosine-5’-diphosphate; (A), 0.5 mM a$-methyleneadenosine-5’-diphosphate and termite. extract (3.6 + 1.6 mg/ml protein). Rates were calculated from the initial rate of IMP synthesis. Each point is the mean of three determinations. Error bars indicate the standard deviation of the mean.

inhibition at 60pM, the concentration which was chosen for use in assays of APRT and adenosine kinase activities. Under these conditions the activities of the termite phosphoribosyltransferases and kinases are shown in Table 2. To ensure that the extraction or assay procedures were not at fault the bacterial activities of the gut were employed as a control. APRT was readily detected in both the bacterial and the termite extracts, but no phosphorylribose transfer to either hypoxanthine or guanine was found in termite extracts. This is despite the addition of the stabilising substrate PRPP (0.1 mM) or the anti-oxidant dithiothreitol (1 mM) to the extraction buffer. The gut extract, by contrast, utilised both substrates with hypoxanthine the most rapidly salvaged. Xanthine was not utilised by either system. Kinase activities for both adenosine and inosine were found in the gut extracts but only adenosine was phosphorylated by termite extracts. DISCUSSION

The sensitivity of HGPRT to inhibition by endogenous nucleotides and the interference by phosphatases and nucleotidases cast doubts on reports of its absence. Becker (1974b) did not detect activity in D. melanogaster extracts but in subsequent work was able to restore activity after dialysis of extracts or

171

after treatment with activated charcoal (Becker, 1978). Radioassays of the phosphoribosyltransferases and nucleoside kinases in crude extracts, as a consequence of S-nucleotidase and phosphatase interference, have generally used the total label in both nucleotide and nucleoside products for calculation of activity (Miller et al., 1979; Silber and Becker, 1981). This has the undesirable effect of including nucleoside produced by the synthetic activity of nucleoside phosphorylase. Though this enzyme is believed to function largely in nucleoside catabolism in vivo, the equilibrium of the reaction favours synthesis. The use of S-nucleotidase and phosphatase inhibitors, in extracts desalted to remove endogenous inhibitors, is an effective means of determining the activity of nucleotide-producing enzymes in crude extracts. Results indicate that a consistent and high recovery of HGPRT activity from insect extracts is possible. Interference, probably from general esterases, inflicts only a minor loss of activity and is preferable to discrepancies introduced through the inclusion of presumed nucleoside catabolites in the calculation of activity. HGPRT activity is completely inhibited by desalted termite extracts making necessary the development of the regimen of S-nucleotidase and phosphatase inhibitors described. Despite these precautions, however, HGPRT was shown to be absent from termite body extracts, supporting earlier work which demonstrated that termite fat body was unable to incorporate [‘4C]hypoxanthine into nucleotides (Chappell and Slaytor, 1991). This parallels the situation in Aedes albopictus (Abidi et al., 1987) where there is no HGPRT but a significant APRT activity, indicated by a rapid incorporation of [14C]adenine into nucleotides. In the bacterial extract from the termite gut, by contrast, both enzymes are present and in addition there are minor activities of both adenosine and inosine kinase. This is not surprising as the hindgut of N. walkeri contains a large variety of bacterial species (Hogan et al., 1988) which would be expected to provide an adequate source of enzymes for control assays. The paunch of the closely related species N. exitiosus, for example, contains at least 27 morphotypes (Czolij et al., 1985). These bacteria, however, in common with the termite, did not contain a xanthine phosphoribosyltransferase activity, an alternative salvage enzyme found in Aedes sp. (Swerdel and Fallon, 1987). The absence of HGPRT activity has major sigof the control of nificance in our understanding purine synthesis in termites. HGPRT in mammals, through the maintenance of cell mononucleotide

Table 2. Specific activity of the purine salvage enzymes in termite tissues *Rate (nmol/min/mg protein) * Si Enzyme

Substrate

Termite

Bacteria

Phosphoribosyltransferase

Adcnine Guanine Hypoxanthine Xanthine

0.277 5 0.014 0 0 0

0.371 * 0.015 0.021 + 0.002 0.117+0.003 0

Nucleosidekinase

Adenosine Guanosine Inosine

0.025 * 0.002 0 0

0.068 + 0.025 0 0.032 + 0.002

*Each value is the nsult of three determinations.

178

D. J. CHAPPELL and M. SLAYTOR

concentrations, causes feedback inhibition of de nova synthesis. Furthermore the associated consumption of PRPP lowers the steady state concentration reducing the rate of amidophosphoribosyltransferase activity and hence de nouo synthesis. In termites, which are uricotelic organisms, purine biosynthesis has an additional function in the disposal of excess nitrogen. Inosine monophosphate (IMP) is located at a critical branch point in the pathway which must allow the flow of excess nitrogen to urate whilst maintaining adequate nucleotide concentrations for nucleic acid synthesis and energy metabolism. It is possible that regulation is effected by a more rigorous control of IMP catabolism such as the pattern described in mouse liver where IMP for nucleic acid synthesis is spared by the high K,,, of 5’nucleotidase for this substrate. The K,,, values of inosinate dehydrogenase and adenylate synthetase are typically > 10 fold lower and closer to cellular IMP concentrations (Wyngaarden and Kelley, 1983). In these circumstances, and in the absence of an inosine kinase, dephosphorylation of IMP in N. walkeri commits the purine ring to urate synthesis. APRT functions in the recycling of adenine generated from 5’-methyl thioadenosine, a byproduct of polyamine synthesis (Simmonds et al., 1989). In mammals, adenosine is not a good substrate for nucleoside phosphorylase and consequently significant quantities of adenine are not generated by phosphorolytic cleavage of adenosine (Divikar, 1976). Instead hypoxanthine is produced through the action of adenosine deaminase followed by nucleoside phosphorylase with the result that HGPRT is responsible for the salvage of purities produced through the catabolism of AMP. This is the route of adenosine catabolism in D. melanogaster (Becker, 1974a; Hodge and Glassman, 1967) and if it is also the pathway utilised by termites then the moderate activity of adenosine kinase may be responsible for the maintenance of the adenylate pool. The inability to salvage hypoxanthine or inosine may explain the well documented deposition of urate in termite fat body where accumulation, during laboratory storage, can reach 45% of the dry weight (Potrikus and Breznak, 1980). Urate in freshly collected N. walkeri is only 6.2% of the dry weight but doubles within 14days of laboratory storage (Lovelock et al., 1985). This increase has been shown in ReticulitermesfEavipes to be partly at the expense

of endogenous nitrogen (Potrikus and Breznak, 1980) supplementing a low dietary intake from wood which contains only 0.034.15% nitrogen (Cowling and Merrill, 1966). The increased de nouo synthesis of IMP required to effect this increase, under conditions which reflect a general imbalance in nitrogen metabolism, may reflect the absence of feedback inhibition on the committed step in the pathway, amidophosphoribosyltransferase.

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Acknowledgements-This

investigation was supported by the award of a Commonwealth Postgraduate Studentship to one of us (D. J. Chappell) and a grant from the Australian Research Council. We thank Professor W. J. O’Sullivan for his generous gift of [8-‘Qguanidine and [2-‘4C]xanthine.

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