Glucosamine metabolism in Drosophila salivary glands separation of metabolites and some characteristics of three enzymes involved

245

Biochimica et Biophysica Acta, 544 (1978) 245--261

© Elsevier/North-Holland Biomedical Press

BBA 28706

GLUCOSAMINE METABOLISM IN D R O S O P H I L A SALIVARY GLANDS SEPARATION OF METABOLITES AND SOME CHARACTERISTICS OF THREE ENZYMES INVOLVED

ERICH ENGHOFER *, HORST KRESS ** and BERNT LINZEN Zoologisches Institu t der Universith't, Luisenstr. 14, D-8000 Miinchen 2 (F.R.G.)

(Received June 6th, 1978)

Summary The conversion of fructose 6-phosphate to mucopolysaccharide precursors was studied in extracts of Drosophila virilis salivary glands. 1. Methods for chromatography of sugar phosphates were adapted and modified to allow routine separation and quantitation of radioactivity of the metabolites from milligram amounts of tissue. Anion exchange chromatography was performed on Dowex 1-X8 employing steps of increasing ammonium formate. Final isolation of each compound was achieved by various thin-layer chromatographic systems. 2. Data obtained by isotope incorporation into glucosamine 6-phosphate compare well with results of the Morgan-Elson colorimetric assay for aminosugars. 3. Glucosaminephosphate isomerase (glutamine-forming) (EC 5.3.1.19) in gland extracts has a Km of 0.35 mM for fructose 6-phosphate, and of 0.25 mM for glutamine. The enzyme is inhibited at glutamine concentrations exceeding 1 mM and by UDP-N-acetylglucosamine (50% at 0.6 mM). Feedback inhibition by UDP-N-acetylglucosamine is enhanced by AMP and by glucose 6-phosphate. 4. Glucosaminephosphate isomerase (EC 5.3.1.10) has a twenty-fold lower affinity towards fructose 6-phosphate (K m = 6.0 mM) compared to the glutamine-forming isomerase. Km(NH~) is 7.4 mM. In the presence of 20 mM glu-

D e d i c a t e d to Professor P. Karlson on the o c c a s i o n o f his 60 t h birthday. * Present address: T h e Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, F o x Chase, Philadelphia, Pa. 19111, U.S.A. ** A u t h o r to w h o m requests for reprints should be sent. Abbreviations: Fru-6-P, fructose 6-phosphate; Glc-6-P, glucose 6-phosphate; NGIc-6-P, glucosamine 6-phosphate; AcNGte-I-P, N-acetylglucosamine 1-phosphate; AcNGIc-6-P, N-acetylglucosainine 6-phosphate; UDPGIcNAc, UDP-N-acetylglucosamine; UDPHxNAc, UDP-N-acetylhexosamine.

246 cose 6-phosphate, the pH optimum is shifted from 6.6 to 7.4, and V increased by a factor of 2.5. Furthermore, the affinity is approximately doubled for both substrates. 5. Glucosamine acetyltransferase (EC 2.3.1.3) has a K m of 2 mM for glucosamine 6-phosphate. Its activity is n o t rate-limiting in salivary glands. Since N-acetylglucosamine 6-phosphate and 1-phosphate were found near equilibrium concentrations, acetylglucosamine phosphomutase (EC 2.7.5.2) must also be present in the extracts.

Introduction During the last larval instar the salivary glands of Drosophila produce and store a mutoprotein [1--4] which is released shortly after puparium formation and which serves to attach the pupae to their substrate [5]. In Drosophila virilis the volume of the secretion protein granules has been estimated to be approx. 36% of total gland tissue volume [6], a value which agrees well with the direct determination of secretion protein content in Drosophila melanogaster salivary glands, for which values of 30--35% have been reported [7,8]. These findings suggest that the synthesis of mucoprotein is the major biochemical activity of the gland cells during the last 20--30 h of larval life. It was of interest, therefore, to find that in salivary glands from D. virilis larvae the specific activity of glucosaminephosphate isomerase (glutamineforming) (EC 5.3.1.19), hereafter referred to as the aminotransferase, the first enzyme in the metabolic sequence leading from fructose 6-phosphate to the immediate mucopolysaccharide precursors, is more than twenty times the specific activity in the fat b o d y and more than seventy times the activity in rat liver. It was also shown that the activity time-course of this enzyme, as measured in vitro, is strongly correlated with the production and release of the secretion [ 9]. It is within this sphere that we have initiated a more detailed study of hexosamine metabolism in D. virilis salivary glands. Whereas in vitro enzyme activity can serve only as a relative measure of maximal capacity to perform a given metabolic step, the true rate in vivo is subject to many variables such as changing substrate levels or the variable presence of effectors. To obtain a more realistic view of mucoprotein synthesis in the salivary glands, we worked out a m e t h o d which allows to identify and to quantitate (relative to each other) the metabolites along the fructose 6-phosphate to UDP-N-acetylhexosamine pathway following incubation of salivary gland extracts of small groups of isolated salivary glands with labelled precursors (Enghofer, E. and Kress, H., unpublished). In the present paper the synthesis of labelled metabolites by extracts in vitro is described. In addition, three enzymes opening the pathway to mucopolysaccharide synthesis are examined for substrate affinity, pH dependence, and for effects of certain modifiers. In a subsequent paper (Enghofer, E. and Kress, H., unpublished) the ontogenetic changes of hexosamine metabolism in vitro and in explanted salivary glands will be described.

247 Materials and Methods

Animals. All experiments were carried o u t on female larvae of a D. virilis wild-type stock. The larvae were reared and timed as described by Kress [6]. Chemicals, syntheses. Non-radioactive chemicals were purchased from: Boehringer, Mannheim; Merck, Darmstadt; Serva, Heidelberg and Sigma, Munich. ~-Ecdysone was a gift of Dr. P. Hocks, Schering AG, Berlin. Twicequartz
248 For thin-layer chromatography (TLC) the fractions of each peak were pooled and dried. A m m o n i u m formate-containing fractions were dissolved in 500 ~l H20, brought to pH 6 with 1 M NaOH and rechromatographed on Dowex 50-X8 (H÷), 200--400 mesh, to convert the salt to HCOOH. The length of the columns (5 mm diameter) varied, depending on the amount of NH~ present in the samples and resin capacity. Elution was with H20. The purified samples were lyophilized. Quantitative thin-layer chromatography. After drying in the desiccator or lyophilizing, the fractions were redissolved in 100--150 td of ethanol (80%). 25 , l were spotted onto thin-layer plates. The following systems were routinely employed: A, acetone/acetonitrile/1 N HC1 (64 : 26 : 10, v/v), run twice in the first dimension, on cellulose plates (Merck, Darmstadt) [13]; B, 18.2 g citric acid/10.4 g NaOH/4.7 ml HC1 (conc.), with demineralized H20 1 l; running time on Ionex 25 SA-Na ÷ resin-coated chromatoplates (Macherey-Nagel and Co., Diiren) was shortened and the R F value increased by using a wedge-shaped stencil [14]; C, 1-propanol/NH4OH (1 : 1, v/v) on silica gel G (Merck, Darmstadt) [15]; D, ethanol/1 M ammonium acetate, pH 3.8 (5 : 2, v/v) on cellulose powder (Macherey-Nagel and Co.) [16]. Nucleoside diphosphate sugars were identified under ultraviolet light. Sugar phosphates were detected by spraying ammonium molybdate [17], and aminosugar (phosphates) by ninhydrin (Merck, Darmstadt, No. 6758). AcNGlc was localized with anilinphthalate (Merck, Darmstadt, No. 1266). Areas containing 14C were detected by aid of a Berthold scanner or by exposure to Kodak Royal X-Omat-film (3--5 days of exposure). The radioactive spots were then transferred into counting vials, 500 pl HEO was added and radioactivity was counted in 10 ml toluene/Triton X-100 scintillant (4 : 1, v/v). The toluene contained 0.4% (w/v) PPO and 0.1% POPOP [16]. Preparation of enzyme extracts. For the study of enzyme kinetics, salivary glands from 15--20 larvae were dissected in buffer I, pH 7.1, containing 50 mM Tris, 4 mM EDTA and 5 mM reduced glutathione [18] and supplemented with 0.16 M sucrose (buffer II). The glands were collected at 4°C in 0.4-ml centrifuge tubes containing 10 td of buffer II. After addition of 50 , l buffer I, the glands were homogenized, the extract made up to 100 pl with buffer I and centrifuged for 15 min at 15 000 × g. Enzyme assays. 100 td of the 15 000 }( g supernatant were mixed with 400 td buffer I containing the appropriate substrates (final concentrations): (a) Glucosaminephosphate isomerase (glutamine-forming) (the aminotransferase): 20 mM Fru-6-P, 3 mM glutamine; pH 6.82. If [14C]Fru-6-P was used as substrate, the concentration of [12C]Fru-6-P was reduced to 2 mM to reduce dilution of radioactivity. (b) Glucosaminephosphate isomerase: 20 mM Fru-6-P, 20 mM NH4C1, 20 mM Glc-6-P (as activator); pH 7.4. (c) Glucosamine acetyltransferase: 23 mM NGlc-6-P, 1 mM acetyl CoA; pH 6.6. The pH of the substrate solutions was adjusted at 39°C prior to the addition of the supernatant. Incubations were carried out at 39°C, and two 100-pl samples were taken at zero time and at 45 min, respectively. The reaction was stopped by heating at 95°C for 3 min.

249 The samples were assayed for NGlc-6-P by means of the Morgan-Elson reaction [ 19] as detailed by Levvy and McAllan [20], but introducing some modifications. We modified the Morgan-Elson procedure mainly by using higher concentrations of the p-dimethylaminobenzaldehyde reagent and by adding ethylcellosolve as an absorbance enhancing and stabilizing reagent (Enghofer, E., unpublished). AcNGIc and AcNGlc-6-P were determined by the same assay, however, omitting the acetylation reaction. AcNGlc-I-P, which is not detected by this procedure, was assayed as AcNGlc after hydrolysis in 0.1 N HC1 at 100°C for 10 min [21]. Fru-6-P and Glc-6-P were determined enzymatically [12]. Results

Ion exchange chromatography of labelled metabolites in salivary gland extracts Anion exchange chromatography on Dowex lx8 (formate) has been successfully used for the separation of sugar phosphates [22--26]. For our own purpose elution conditions were worked out by which glucosamine metabolites (see scheme in Hughes, [27] ), in particular NGlc-6-P, AcNGlc-6-P, AcNGlc-I-P, UDP-N-acetylhexosamine, but also UDPhexoses, Fru-6-P and Glc-6-P could be separated either directly or after supplementary TLC. To identify the labelled metabolites after incubation of gland extracts with [14C]Fru-6-P, carrier substances were added, and the coincidence of radioactivity with peaks identified by colorimetric or enzymatic analysis established. For quantities up to 40 ~g of each component, a column of 2 mm in diameter and at least 600 mm in length was adequate. A typical elution pattern is shown in Fig. la. The positions of the peak fractions deviated at most by one fraction between different experiments. The individual compounds are considered in the following: (a) NGlc-6-P (fractions 20--23). Elution of this compound is more sensitive to changes in pH and temperature than is the case for other metabolites. To ascertain its separation from glucosamine and glucose which are eluted with the front fractions, the column must be equilibrated at a pH no lower than 4.25 at a temperature of 25°C. Labelled NGlc-6-P was identified furthermore by TLC in systems B and C. After hydrolysis by acid phosphatase (Tris/maleate buffer, pH 5.1, 2 h at 37°C; [28]) glucosamine was detected in the same TLC system. (b) Radioactivity in fractions 29--33. This is almost certainly due to lactate and pyruvate formed by glycolysis of labelled hexose phosphate. Authentic lactate and pyruvate are eluted at the same positions, but no further attempts was made to confirm the identity. (c)N-Acetylglucosamine phosphate (fractions 38--45). These were only partly resolved by TLC in system D (no separation in other systems). To quantitate the incorporation of isotope exactly, these fractions were pooled and hydrolyzed with 0.1 N HC1 for 10 min at boiling temperature. After TLC in system D two separate spots were obtained, which corresponded to AcNGlc6-P (RE = 0.42) and AcNGlc (formed quantitatively from AcNGlc-I-P), which moved near the front (R F = 0 . 7 5 ) . If Glc-6-P was still contaminating this fraction after column chromatography, a third spot ( R F = 0.30) was observed.

250 OF,

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Fig. 1. C h r o m a t o g r a p h y of substrates and metabolites of the mucopolysaccharide synthesis on Dowex l - X 8 ( f o r m a t e ) . T h e s t e p s i n t h e e l u t i o n m e d i u m are: A, H 2 0 ; B, 0 . 0 0 5 M H C O O H ; C, 3.0 M H C O O H ; D, 4.0 M H C O O H ; E, 4 M H C O O H + 0 . 1 2 M t - I C O O N H 4 ; F, 4 M H C O O H + 0 . 3 0 M H C O O N H 4. O r d i n a t e : Absorbance at 2 5 4 n m (×), 3 6 0 n m (/,) a n d 578 n m C)), a n d 14C a c t i v i t y (o). a. S t a n d a r d p r o f i l e ; b, M e t a b o l i t e s f o r m e d a f t e r i n c u b a t i o n ( 4 5 m i n at 3 9 ° C ) o f salivary g l a n d e x t r a c t s w i t h [ 1 4 C ] F r u - 6 - P (2.3 p C • / 2 5 0 pl i n c u b a t i o n m i x t u r e ) ; Fru-6oP, 2 raM; Gln, 3 m M ; p H 6 . 8 5 ; c. S a m e as b e f o r e , w i t h 1 m M a c e t y l CoA a d d e d . N o t e d e c r e a s e o f N G I c - 6 - P a n d rise in N - a c e t y l a t e d s u g a r p h o s p h a t e s .

(d) Glucose 6-phosphate, fructose 6-phosphate (fractions 46--53). These were identified by appropriate enzymatic assays and identified furthermore by TLC in system A which also served to establish the proportion of radioactivity in each compound. In some experiments we noted a third labelled c o m p o u n d with a high RF value which, however, was not identified. (e) UDP-N-acetylhexosamines and UDP-hexoses (fractions 55--64). UDPderivatives of hexosamines and hexoses were not separated completely by column chromatography. Since in the present experiments significant radioactivity was never found in these fractions, further separation was not necessary. The yield of each c o m p o u n d after column and thin-layer chromatography was determined in a series of experiments with the Morgan-Elson reaction, or by measuring absorbance at 254 nm. NGlc-6-P was recovered at 90--95%, Glc6-P and Fru-6-P at 82%, AcNGlc-I-P and AcNGlc-6-P at 92 and 91%, respectively.

Pattern formed by incorporation of [14C]fructose 6-phosphate It was one of our objectives to study the capability of salivary glands to metabolize labelled precursors along the sequence leading from NGlc-6-P to UDPhexosamines, the building stones of mucoproteins. Fig. l b shows the pattern obtained after incubating salivary gland extracts from 140-h larvae with [14C]Fru-6-P. Most of the radioactivity resides in the hexose phosphate peak

251 but, in addition, radioactivity is found in the NGlc-6-P, AcNGlc-I-P and AcNGlc-6-P peaks. The small peak near fraction 30 is due to glycolytic endproducts. Thus, the activities of the aminotransferase and the acetyltransferase and of acetylglucosamine phosphomutase are readily demonstrable in the buffered extract from isolated salivary glands. The acetylated phosphates are formed, however, in minor amounts (22% of total activity incorporated; Table I, Expt. 1) which is to be expected in the absence of an acetyl CoA regenerating system. If the incubation mixture was supplemented by acetyl CoA (1 mM), the label in the NGlc-6-P was reduced by one half, while it increased 4-fold in the AcNGlc phosphates (Fig. lc; Table I, Expt. 2). It is notable that in both series of experiments radioactivity was totally absent from those fractions where the uridine derivatives were to be expected (Fig. lb,c; fractions 55--64). Addition of UTP (3 raM) and acetyl CoA (1 mM) to the medium did not result in labelling of the UDP aminosugars, although incorporation into AcNGlc-6-P and AcNGlc-I-P was enhanced 6-fold (Table I, Expt. 3). In a more recent study (Enghofer, E. and Kress, H., unpublished) UDPGlcNAc pyrophosphorylase activity could be demonstrated in gland extracts if EDTA was omitted and 3 mM Mg2÷ added. Despite this positive result, [14C]Fru-6-P incorporation into UDPGlcNAc could not be observed, since aminotransferase, i.e. the enzyme which channels Fru-6-P into the hexosamine pool, is only active in the presence of EDTA (unpublished data, see also ref. 18). Therefore it is not possible to establish concomitant activity of both enzymes in the same gland extract.

Comparison of the isotope experiments with the Morgan-Elson assay The chromatographic separation of enzymatic reaction products also allowed to assess the reliability of the Morgan and Elson reaction in the determination of NGlc-6-P. For this purpose, salivary gland extracts were prepared from (a) third instar larvae (140 h after oviposition), (b) prepupae (156 h after oviposition), and (c) third instar larvae (133 h after oviposition) injected with 10 ng of ~-ecdysone. Earlier observations had shown that aminotransferase activity decreases sharply prior to formation of the puparium [9] and that this drop is

TABLE I INCORPORATION OF [U-14C]FRUCTOSE 6-PHOSPHATE B Y S A L I V A R Y G L A N D E X T R A C T S F R O M D. V 1 R I L I S

INTO GLUCOSAMINE

METABOLITES

Incorporation of [U-14C]Fru-6-p (2.3 Ci/250 1 incubation mixture) into NGlc-6-P, AcNGlc-6-P and A c N G l c - I - P w a s d e t e r m i n e d a f t e r 4 5 - m i n i n c u b a t i o n s ( 3 9 ° C ) o f D. virilis s a l i v a r y g l a n d e x t r a c t s ( F r u - 6 - P , 2 r a M ; G i n , 3 r a M ; p H 6 . 8 5 ) . M e a n s o f t w o e x p e r i m e n t s . E x p t . 1: N o c o f a c t o r s ; E x p t . 2: In t h e p r e s e n c e o f 1 m M a c e t y l C o A ; E x p t . 3: In t h e p r e s e n c e o f 1 r a M a c e t y l C o A a n d 3 m M U T P . V a l u e s q u o t e d refer t o i n c o r p o r a t i o n o f 14C as c p m per gland p a i r .

Expt. 1 Expt. 2 Expt. 3

NGIc-6-/~

AcNGlc-6-P

AcNGlc-I-P

AcNGIc-I-P AcNGIc-6-P

NGlc-6-P A c N G l c - ( 1 + 6)-P

20 500 10 600 10 700

4 300 19 700 29 400

1600 4600 7000

0.37 0.23 0.24

3.5 0.44 0.29

252 T A B L E II I S O T O P E - A N D M O R G A N - E L S O N A S S A Y O F G L U C O S A M I N E 6 - P H O S P H A T E F O R M E D IN D. V I R I LIS SALIVARY GLAND EXTRACTS UNDER VARIOUS CONDITIONS D e t e r m i n a t i o n o f a m i n o t r a n s f e r a s e a c t i v i t y in salivary g l a n d e x t r a c t s ( F r u - 6 - P , 2 r a M ; G l n , 3 mM~ p H 6.82) by the Morgan-Elson assay and by incorporation of [U-14C] Fru-6-p (1.0 Ci]250 1 incubation mixt u r e ) i n t o N G l c - 6 - P , r e s p e c t i v e l y . M e a n s o f t w o o r m o r e e x p e r i m e n t s . V a l u e s in p a r e n t h e s e s are p e r c e n t ages. Morgan-Elson (nmol NGlc-6-P/gland pair per 45 min)

Isotope method (cpm/gland pair per 45 rain)

a. D i f f e r e n t d e v e l o p m e n t a l s t a g e Larvae Prepupae

3.51 (IO0) 0.13 (4)

8213 (IO0) 0 (0)

b. E f f e c t o f 1 0 n g e c d y s o n e Control Injected animals

3.44 (100) 0.26 (8)

5621 (100) 562 (10)

induced by administration of ecdysone [29]. The results (Table II) confirm the earlier observations but, in addition, they demonstrate that the Morgan-Elson test and the isotope experiment yield essentially identical results, thus strengthening our confidence in the validity of data obtained by means of the Morgan-Elson assay, to be described in the following section.

Glucosamine 6-phosphate and N-acetylglucosamine 6-phosphate synthesis in salivary gland ex tracts 1. The aminotransferase. NGlc-6-P can be synthesized by t w o different enzymes: the aminotransferase (EC 5.3.1.19) and glucosaminephosphate isomerase (EC 5.3.1.10). We have already studied the aminotransferase in crude extracts from D. virilis larvae, and shown that most of its activity resides in the salivary glands [9]. The kinetic data obtained with total extracts could be conformed in the present study: the pH optima (6.8) and Km values for Fru-6-P and Gln (Table III) were n o t significantly different in both types of extracts. For determination of the K m value with respect to Fru-6-P, the activity of phosphoglucose isomerase had to be considered. Due to the activity of this enzyme, a constant ratio of Fru-6-P and Glc-6-P (0.39 +_0.02) was attained in extracts from whole larvae during the first 5 min of incubation, even at the highest Fru-6-P concentrations tested [9]. In salivary glands, too, the initial concentration of Fru-6-P was significantly lowered during incubation, although the effect was less pronounced at higher concentrations (Table IVa). If the initial concentration of Fru-6-P exceeded 1 mM, no equilibrium between Fru-6-P and Glc-6-P was attained within the 45-min incubation interval, b u t Fru-6-P concentration was reduced at a constant rate. Plotting the initial Fru-6-P concentrations by the Lineweaver-Burk method would have been misleading with regard to the substrate affinity of aminotransferase, since phosphoglucose isomerase competes for Fru-6-P throughout the incubation time. Therefore the mean between the initial and the final concentrations of Fru-6-P, as determined

253

b y a separate measurement of phosphoglucose isomerase activity (Table IVa) was used in the Lineweaver-Burk plots. While in extracts from whole larvae saturation of aminotransferase never occurred, even at the highest F r u ~ - P concentration tested (400 mM), 40 mM were required for saturation of the enzyme in salivary gland extracts. An increase in the concentration of the second substrate, Gln, b e y o n d 1 mM resulted in a slight inhibition of enzyme activity in both types of extracts. The possibility that this effect was caused by interference of Gln with the MorganElson test could be excluded, since in control experiments a decrease in colour yield was only observed if Gln concentrations exceeded 20 mM (Enghofer, E., unpublished). Feedback inhibition by UDPGlcNAc was also observed, although complete inhibition of enzyme activity did not occur in gland extracts even at the highest inhibitor concentrations tested (Fig. 2). This has also been reported for the aminotransferase from other sources [30--33]. A concentration of 0.6 mM UDPGlcNAc was required for 50% inhibition of enzyme activity, while in extracts from total larvae only 0.025 mM were required (Fig. 2). This difference may be explained by the presence of factors which interfere with the binding of UDPGlcNAc to the enzyme. In rat liver the inhibitory effect of UDPGlcNAc on isolated aminotransferase is intensified b y AMP and Glc-6-P [32,34]. Extracts from isolated Drosophilia salivary glands behave in a similar way: in the presence of 0.5 mM UDPGlcNAc inhibition was 40%, while after addition of 10 mM AMP or Glc-6-P inhibition rose to 70% and 73%, respectively. The simultaneous presence of both effectors, however, cannot account for the 40-times greater sensitivity of the enzyme in extracts from whole lar-

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254 vae. Other factors like protein-protein interactions may be responsible in addition. 2. Glucosaminephosphate isomerase. Glucosaminephosphate isomerase, the second enzyme catalyzing the formation of NGlc-6-P had n o t been detected in crude extracts from total larvae [9]. In isolated salivary glands, however, NGlc-6-P isomerase activity could well be established. K m values for Fru-6-P and NH4C1 are shown in Table III. The actual concentrations of Fru-6-P were calculated in the same way as was done in the aminotransferase assay (see page 8 and Table IVa,b). In the presence of 20 mM Glc-6-P the enzyme exhibits a higher affinity for both substrates (Fig. 3; Table III). The kinetics of activation of the enzyme by Glc-6-P are revealed by plotting NGlc-6-P synthesis (V) as the reciprocal ( 1 / V - V0; where V0 is the activity in the absence of Glc-6-P) against 1/[Glc-6-P], according to the m e t h o d of Worcel et al. [35]. The plot is a straight line and yields an activation constant of 21 mM. In the presence of 20 mM Glc-6-P the pH optimum of NGlc-6-P isomerase is shifted from 6.6 to 7.4 and at the same time V increases by a factor of 2.5 (Fig. 4). Enzyme activity is also stimulated by AcNGlc-6-P, the maximal effect being observed at a concentration of 2 mM. Similar results have been reported for the isomerase from Musca domestica [28]. 3. Glucosamine acetyltransferase. Glucosamine acetyltransferase (EC 2.3.1.3) activity could easily be demonstrated. Fig. 5 shows that the pH optim u m for this enzyme is approx. 6.5. From the Lineweaver-Burk plots a Km of 2 mM for NGlc-6-P was calculated (Table III). The Km for acetyl CoA was n o t determined, b u t must be well below 0.5 mM, since at acetyl CoA concentrations of 0.75, 1.0 and 1.5 mM there was no significant difference in enzyme

T A B L E III K I N E T I C D A T A F O R E N Z Y M E S I N V O L V E D IN G L U C O S A M I N E M E T A B O L I S M IN D. V I R I L I S SALIVARY GLAND EXTRACTS K m (raM) a n d V m a x ( n m o l p r o d u c t / g l a n d p a i r p e r r a i n ) for three e n z y m e s o f t h e g l u c o s a r n i n e p a t h w a y In c r u d e e x t r a c t s f r o m i s o l a t e d salivary glands. I n c u b a t i o n t e m p e r a t u r e : 39°C. G l u c o s a m i n e p h o s p h a t e i s o m e r a s e a n d g l u c o s a m i n e a c e t y l t r a n s f e r a s e w e r e e i t h e r n o t d e t e c t e d or n o t a s s a y e d in w h o l e larval extracts.

Enzyme

Substrate

Extract: Salivary glands

W h o l e larvae Km

V

Km

Aminotransferase

Fru-6-P Gin

0.17 0.13

0.35 0.25

Isomerase

Fru-6-P Fru-6-P + 20 m M GIc-6-P NH4CI NH4C1 + 20 m M GIc-6-P

0.033 0.050 0.027 0.050

6.0 3.3 7.4 3.7

Acetyltransferase

N G l c - 6 -P Acetyl-CoA

0.45 --

* F r o m Kress and E n g h o f e r [ 9 ] .

2.0 <0.5?

0.30 0.16

T A B L E IV

255

E F F E C T O F P H O S H O G L U C O S E - I S O M E R A S E A C T I V I T Y ON C O N C E N T R A T I O N S O F F R U C T O S E 6 - P H O S H A T E A N D G L U C O S E 6 - P H O S P H A T E IN S A L I V A R Y G L A N D E X T R A C T S F R O M D. V I R I L I S Final c o n c e n t r a t i o n s (raM) o f F r u - 6 - P a n d GIc-6-P a f t e r 4 5 - r a i n i n c u b a t i o n s ( 3 9 ° C ) o f salivary g l a n d ext r a c t s in a b s e n c e (a) a n d p r e s e n c e (b) o f 20 m M GIc-6-P. Fru-6-P (mM):

1.0

a

Fru-6-P Glc-6-g

0.26 0.69

b

Fru-6-P GIc-6-P

1.92 16.60

2.5

5.0

10.0

0.87 1.03

---

---

4.71 17.66

20.0

6.7 3.2

---

8.10 19.40

16.31 22.00

80-

l

6(3

40

oT,

0

001

oT3

/

o's

lift0 0

o/e j e

02

0."-

06

0.8

10

1 / NH4C[ (mN1-1)

Fig. 3.

L i n e w e a v e r - B u r k p l o t s o f g l u e o s a m i n e p h o s p h a t e i s o m e r a s e a c t i v i t y in c r u d e e x t r a c t s o f i s o l a t e d salivary glands in the a b s e n c e (o) o r p r e s e n c e ( 2 0 raM) of GIc-6-P (e). Assays w i t h o u t GIc-6-P w e r e c a r r i e d o u t a t a p H o f 6 . 6 , t h e o t h e r s a t p H 7.4. 1 / V is in ( n m o l NGIc-6-P f o r m e d p e r g l a n d p a i r p e r r a i n ) -1 . E a c h p o i n t is t h e m e a n of t w o e x p e r i m e n t s . T o p : Fru-6-P variable, NH4C1 c o n s t a n t a t 2 0 raM. B o t t o m : N H 4 C I v a r i a b l e , Fru-6-P c o n s t a n t a t 10 raM.

256

V

ooB

007

-

0 06-

005 = 00z,003 002 001 0

6'2

6%

7'o

7'4

?%

812 pH

Fig. 4. I n f l u e n c e of p H o n the a c t i v i t y of g l u c o s a m i n e p h o s p h a t e i s o m e r a s e in c r u d e e x t r a c t s f r o m i s o l a t e d salivary glands. S t a n d a r d assay in t h e p r e s e n c e of 2 0 m M Glc-6-P ( $ ) o r w i t h o u t Glc-6-P (o). p H values w e r e m e a s u r e d in t h e r e a c t i o n m i x t u r e ( 1 5 0 #l) at t h e e n d o f i n c u b a t i o n . V is in n m o l NGlc-6-P f o r m e d p e r gland pair p e r rain. E a c h p o i n t is t h e m e a n o f t w o d e t e r m i n a t i o n s .

%

10o

80

60

1,0

20-

SiS

6'2

6'6

710

7'4

pH

Fig. 5. I n f l u e n c e of p H o n the a c t i v i t y of g l u c o s a m i n e a c e t y l t r a n s f e r a s e in c r u d e e x t r a c t s o f isolated saliv a r y glands. P H m e a s u r e d a t t h e e n d o f e a c h i n c u b a t i o n . M e a n s of t w o e x p e r i m e n t s , m a x i m a l activities set 100%.

257 activity, suggesting enzyme saturation at these concentrations. The V for AcNGlc-6-P formation was approx. 0.45 nmol/gland pair per min, and thus was considerably higher than that of the aminotransferase or the NGlc-6-P isomerase (Table III). Discussion In larval salivary glands NGlc-6-P may be formed by two different enzymes: glucosaminephosphate isomerase (glutamine forming) (EC 5.3.1.19) the aminotransferase and glucosaminephosphate isomerase (EC 5.3.1.10). The relatively low Km and high V values of the aminotransferase as compared to the isomerase (Table III) suggest that for the formation of NGlc-6-P the aminotransferase is predominantly responsible. While the aminotransferase catalyzes only NGlc-6-P formation (since the reversal of the reaction is practically not feasible, the isomerase catalyzes both formation of NGlc-6-P and its decomposition to Fru-6-P and NH~. Since the latter reaction is thermodynamically favoured, the possibility of a futile cycle exists. Most reports concerned with the isomerase, indeed, stress its catabolic function [21,36]. The presence of glucosamine acetyltransferase, however, which effectively removes NGlc-6-P from the equilibrium, should promote NGlc-6-P formation. This is supported by the finding [36] that in vitro AcNGlc-6-P is formed in a reaction mixture containing Fru-6-P, NH3, acetyl CoA, isomerase and N-acetyltransferase. In the presence of acetyltransferase, NGlc-6-P formation, rather than its decomposition, is apparently catalyzed by the isomerase. Obviously these conditions are met in salivary glands. The relatively high affinity of the acetyltransferase for NGlc6-P and acetyl CoA, in combination with its high V value (Table III), strongly suggests that NGlc-6-P formed by the aminotransferase is rapidly converted to AcNGlc-6-P. This is consistent with our finding that the ratio of NGlc-6-P to total AcNGlc phosphates formed during incubation of crude extracts decreased from 3.5 to 0.44 when acetyl CoA was added, and that the total radioactivity in hexosamines increased by approx. 30% (Table I, Expts. 1,2). Acetyltransferase activity is also high in rat liver [37] and bovine thyroid gland [38], where it is twice as active as the aminotransferase. The formation of NGlc-6-P is situated at a metabolic branch point and thus a most probable site for regulation. In larval salivary glands both the aminotransferase and the isomerase are subject to several control mechanisms. The most prominent feature of the aminotransferase is its sensitivity to feedback inhibition by UDPGlcNAc. We have already examined this effect using crude extracts from total larvae [9]. It is notable that the inhibition by UDPGlcNAc is more effective in homogenates from total larvae than in those from isolated salivary glands (Fig. 2). We suspect that this effect is due to the presence of factors which modify the enzyme-inhibitor interaction in both types of extract to different degrees. In gland extracts AMP and Glc-6-P significantly increase the affinity of the aminotransferase for UDPGlcNAc, thus intensifying its inhibitory effect. Similar effects were demonstrated for the isolated aminotransferase from rat liver [32,34]. Isomerase activity, too, is modified by Glc-6-P. In its presence the affinity of the enzyme for Fru-6-P and NH4C1 increases. Concentrations of 20 mM Glc-6-P

258 reduce the K~ values for both substrates to half the control values (Table III~. The Km values obtained in the presence of 20 mM Glc-6-P are almost identical to those stated for the enzyme from M. domestica at 5 mM Glc-6-P (Fru-6-P = 3 mM; NH4C1 = 3.3 mM; [28]), while the activation constant for Glc-6-P (21 mM) is approximately twice the value for the M. domestica enzyme (9.5 mM). For both enzymes Glc-6-P leads to a higher V in the turnover of both substrates (Table III). AcNGlc-6-P acts as a positive modulator for the isomerase in Drosophila as well as in Musca. In both species AcNGlc-6-P is a b o u t ten times as effective as Glc-6-P. Another c o m m o n feature is the shift of the pH optimum towards the alkaline region, when Glc-6-P is present. This a possibly important physiological quality. Whether the effects of Glc-6-P and/or AMP on glucosaminephosphate isomerase and aminotransferase activities are significant in vivo remains to be demonstrated. For Drosophila no data are available on the intracellular concentrations of these metabolites. In tissues and cells from other sources concentrations of Glc-6-P and AMP are far below the concentrations required for significant effects on isomerase and aminotransferase activity in vitro (Table V). Therefore we assume that these metabolites play a negligible role in the control of these enzymes. A similar reservation must be expressed for possible regulatory effects of AcNGlc-6-P on the isomerase, since its intracellular concentration might be very low (Table V). In contrast, a UDPGlcNAc concentration of 0.6 mM, which causes a 50% inhibition of aminotransferase activity in the salivary gland extract (Fig. 2), could well be within the physiological range, since values of 0.55 mM for rat liver and 0.37 mM for bovine thyroid gland have been reported (Table V). The isotope assay also indicated the presence of acetylglucosamine phosphomutase (EC 2.7.5.2) in the extracts which interconverts AcNGlc-6-P and AcNGlc-I-P. The ratio of the 1-P to the 6-P ester was found to be 0.33, if the medium was supplemented with acetyl CoA (Table I, Expt. 2), which is close to the equilibrium constant (0.16; [45]). Ratios of 0.18 in bovine thyroid gland [44] and of 0.33 in Zea rnays [16] were found. In rat liver, where most enzymes involved in glucosamine metabolism were examined [37], acetylgiucosamine phosphomutase showed by far the highest specific activity. The absence of UDPNAc hexosamines among the products was rather puzzling b u t has n o w been traced to the lack of Mg 2÷ in the medium. Consequently, UDPGlcNAc could be synthesized from AcNGlc-I-P only in the absence of EDTA (Enghofer, E. and Kress, H., unpublished). [14C]Fru-6-P incorporation into UDPGlcNAc, however, could n o t be established, since aminotransferase (see Results) as well as isomerase (unpublished data, see also ref. 28), both channelling Fru-6-P into the glucosamine pathway, require the presence of EDTA. The chromatographic isolation of all metabolites from each single experiment permitted estimation of the proportion of the glucosamine vs. the glycolytic pathway. Since after a 45 min incubation the pools are probably saturated with radioactive lable, the relative metabolite concentrations are indicated by their ratios of total radioactivity. If acetyl CoA and UTP were added to the medium, the ratio of NGlc-6-P to AcNGlc-(1 + 6)-P radioactivity was

FOR SIGNIFICANT

EFFECTS

ON THE GLUCO-

0.16---0.6 [ 3 9 , 4 0 ]

0.25--0.6 [40,43]

0.55 [39]

GIc-6-P

AMP AcNGlc-6-P

UDPGIcNAc

R a t Hver

[44]

0.37 [44]

0.10

--

--

--

--

Bovine thyroid gland

0.15--0.45 [42]

--

Rat kidney cortex

--

--

--

~0.24

[41]

Myxamoebae

1--2 0.6

5--10 5---10

Concentrations required for significant effects in salivary gland extracts (mM)

N G I c - 6 - P i s o m e r a s e a n d a m i n o t r a n s f e r a s e are s u b j e c t t o c o n t r o l b y v a r i o u s e f f e c t o r s (see t e x t ) w h o s e c o n c e n t r a t i o n s are n o t k n o w n i n s a l i v a r y g l a n d s o f D . virilis. In o r d e r to e s t i m a t e t h e p h y s i o l o g i c a l s i g n i f i c a n c e o f t h e s e e f f e c t o r s , t h e i r levels ( r a M ) in t i s s u e s a n d cells f r o m o t h e r s o u r c e s a r e l i s t e d t o g e t h e r w i t h t h e r e f e r e n c e s .

SUBSTRATE LEVELS IN VARIOUS TISSUES AND CELLS IN COMPARISON WITH THOSE REQUIRED S A M I N E 6 - P H O S P H A T E S Y N T H E S I Z I N G E N Z Y M E S I N D. V I R I L 1 S S A L I V A R Y G L A N D E X T R A C T S

TABLE V

t~ O1 ¢.O

260 0.44 and 0.29, respectively (Table I, Expts. 2 and 3), i.e. in favour of the acetylated products. In the assay a total of 5 • 106 cpm of [14C]Fru-6-P were added to the reaction mixture. At the end of the experiment 2 • 106 cpm were recovered in the Fru-6-P and Glc-6-P (hexose 6-phosphate) fraction. If Acetyl CoA was present, 0.7 • 106 cpm were recovered in NGlc-6-P and in the N-acetylated derivatives. Therefore, roughly 2.3 • 106 cpm could have been metabolized via the glycolytic and other pathways. On this basis the rate of NGlc-6-P synthesis represents approximately 30% of the glycolytic rate. Although other explanations cannot be ruled out for these figures, a comparison with corresponding data for rat liver (0.5-2.0%; [32]) and rat skin (15--20%; [46]) indicates a relatively high flux of Fru-6-P through the glucosamine pathway in larval salivary glands. This is in line with the high specific activity of aminotransferase in the glands [9], which is about 70 times the specific activity in rat liver [18], and 150 times the specific activity in bovine thyroid glands [38]. The exceptionally high capacity for aminosugar synthesis in 140-h larvae demonstrates clearly that the salivary glands of D. virilis are well equipped for their predominant cellular activity at this developmental stage, the biosynthesis of secretory mucoprotein. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (SFB

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