Adenylate kinase isoenzymes in Aspergillus nidulans

FEMS Microbiology Letters 78 (1991) 133-138 © 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50 Published by Elsevier

ADONIS 037810979100116S

133

FEMSLE 04313

Adenylate kinase isoenzymes in Aspergillus nidulans

Van-Quyen Hoang, Walter Jaklitsch and Michael C. Scrutton

Division of Biomolecular Sciences, King's College, London, U.K.

Received 7 August 1990 Revision received 16 October 1990

Accepted 17 October 1990

Key words: Adenylate kinase; Isoenzyme; Aspergillus nidulans

1. SUMMARY

Adenylate kinase isoenzymes localised in the mitochondria and in the cytosol have been detected in extracts of glucose-grown Aspergillus nidulans using specific staining after electrophoresis on cellulose acetate. The isoenzymes have similar g m values for AMP, ADP and MgATP 2- but may differ in the mechanism used for inter- nucleotide phosphate transfer.

2. INTRODUCTION

Adenylate kinase has an important role in the regulation of cellular adenylate concentrations [1]. Organelle-specific isoenzymes of adenylate kinase have been described in mammalian species [2-4]. In contrast, such isoenzymes have not been reported in fungi but in glucose-grown Sac- charomyces cerevisiae the single adenylate kinase species is apparently localised in the cytosol [5]. In Aspergillus nidulans adenylate kinase, when used as a putative marker for the mitochondrial inter-

Correspondence to: C. Scrutton, Division of Biomolecular Sci- ences, King's College, Campden Hill Road, London W8 7AH, U.K.

membrane space [6], was present in both the mitochondria and the cytosol [7]. We have now shown that organdie-specific adenylate kinase isoenzymes occur in A. nidulans and have char- acterised the kinetic properties of the separated isoenzymes.

3. MATERIALS AND METHODS

Aspergillus nidulans R21y paba- was grown in a glucose/salts medium containing p-aminoben- zoic acid [8]. Cultures were harvested in the growth phase 18-24 h after inoculation with conidia.The mycelium was incubated with Novozyme 234 (25 mg per g mycelium) in 0.2 M potassium phosphate, pH 6.4, containing 0.6 M sucrose for 35 rnin at 30°C and then washed first with phosphate/sucrose and subsequently with 0.01 M Tris-C1, pH 7.5, containing 2 mM MgC12, 1 mM EDTA, 0.25 M sucrose and 4/~M phenylmethyl- sulfonylfluoride (the Tris-sucrose buffer). The washed mycelium was suspended in this medium and disrupted using low pressure N 2 cavitation (5.2 MPa) [7]. Mitochondrial and cytosolic fractions were isolated from this extract by differential centrifugation [7]. The identity of such fractions has been established by marker enzyme analysis [7]. It was verified here by determination of the

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Fig. 1. Cellulose acetate strip electrophoretic analysis of adenylate kinase i soenzymes in fractions derived by (NH 4)2504 fractionation of the cytosolic fraction. The cytosolic fraction was prepared by the Novozyme/N 2 cavitation procedure and was fractionated by (NH4)2SO 4 precipitation and extraction. Fractions which contained significant adenylate kinase activity were desalted using PD-10 columns (Pharmacia), concentrated and applied to cellulose acetate strips. Adenylate kinase was detected by measurement of ATP formation from ADP. The lanes were loaded with unfractionated cytosolic fraction (1); 55% (NH4)2SO 4 extract (2); 50% (NH4)2SO 4 extract (3); 45% (NH4)2SO 4 extract (4); 40% (NH4)2SO 4 extract (5). The letters indicate the position of migration of the mitochondrial (M)

and cytosolic (C) isoenzymes respectively.

d is t r ibut ion of citrate synthase, NAD-isoci t ra te dehydrogenase and glucose-6-phosphate dehydrogenase. Mitochondria l enzyme activities were ex- pressed by sonicat ion for 3 × 15 s at 2°C. Where necessary fractions were concentra ted 10- to 20- fold using Centr icon C30 microconcentrators .

Adenyla te kinase was measured by conversion of A D P to ATP + A M P in the presence of hexokinase and glucose-6-phosphate dehydrogenase as described by Chiu et al. [5]. N A D P H format ion was entirely dependent on the addi t ion

of ADP. In some cases adenylate kinase was mea-

sured by product ion of A D P from A TP + A M P in the presence of pyruvate kinase and lactate dehydrogenase [5]. N A D H oxidat ion unrelated to adenylate kinase was estimated by omission of

AMP. Cellulose acetate strip electrophoresis was per-

formed for 35-40 min at 2 0 - 2 5 ° C in 0.022 M

potass ium phosphate, pH 7.4, and at a current of 3 m A per strip [9]. Adenyla te kinase was located

by the agar overlay method [9] using assay condi-

t ions adapted from those for detect ion on starch gel [10].

The cytosolic isoenzyme was part ial ly purified from the 35000 x g superna tan t fraction by (NH4)2SO 4 extraction. Adenyla te kinase was precipitated by addi t ion of solid (NH4)2SO 4 to a final concent ra t ion of 60% (w/v) . After stirring for 15 min at 2°C precipitated protein was col- lected by centr i fugat ion at 35 000 x g for 15 min. The precipitate was extracted with 4 ml of the Tris-sucrose buffer conta in ing 55% ( w / v ) (NH4) 2

SO 4. After suspension in the (NH4)2SO 4 solut ion and stirring for 10 min at 2°C, undissolved material was removed by centr i fugat ion at 35 000 x g for 5 min. This procedure was repeated in succession using Tris-sucrose buffer conta in ing

50% (w/v) , 45% ( w / v ) and 40% ( w / v ) (NH4)2SO 4. Adenyla te kinase was detected in all the fractions

bu t the cytosolic isoenzyme was markedly enriched in the 55% (NH4)2SO 4 extract (Fig. 1) which was used for the initial rate studies.

The mitochondria l isoenzyme was obta ined by

Fig. 2. Cellulose acetate strip electrophoretic analysis Of adenylate kinase activity in cell-free extract and in mitochondrial and cytosofic fractions derived from that extract by differential centrifugation. Cell-free extracts containing intact mitochondria were prepared and fractionated to yield mitochondrial and cytosolic fractions. Mitochondria were disrupted by sonication for 3 x 15 s. The cytosolic fraction was concentrated 10-fold before application to cellulose acetate strips. Adenylate kinase was detected either by measurement of ATP formation from ADP (A) or by measurement of ADP formation from ATP (B). The lanes were loaded in (A) with mitochondrial fraction (1), cytosolic fraction (2), cell-free extract (3) and rabbit skeletal muscle adenylate kinase (4); and in (B)

with rabbit skeletal muscle adenylate kinase (1), mitochondrial fraction (2), cell-free extract (3), and cytosolic fraction (4).

sonication of isolated mitochondrial fraction fol- lowed by removal of insoluble protein by centrifugation at 35 000 x g for 10 min. Electrophoretic analysis confirmed that the resulting preparation contained a single species of adenylate kinase with a mobility corresponding to that shown for the mitochondrial isoenzyme in Fig. 2.

ATP, ADP, AMP, NADP, NADH, rabbit skeletal muscle adenylate kinase, pyruvate kinase, hexokinase, lactate dehydrogenase and glucose-6- phosphate dehydrogenase were obtained from Sigma Chemical Co.; Novozyme 234 from Novo Industries; and cellulose acetate strips from British Drug Houses.

4. RESULTS

4.1. Isoenzyme analysis Cell-free extracts of glucose-grown A. nidulans

prepared by sonication or by Novozyme treat- ment / low-pressure N 2 cavitation showed two bands of adenylate kinase as detected by ATP formation from ADP (positive stain) (Fig. 2A) or by ADP formation from ATP (negative stain) (Fig.2B). Fig. 2, track (3) shows these results for an extract prepared by N o v o z y m e / N 2 cavitation. This staining is specific for adenylate kinase since no bands were detected in the absence of ADP (positive stain) or AMP (negative stain) (data not shown). When the extract prepared by Novo- z y m e / N 2 cavitation was fractionated by differential centrifugation the mitochondrial fraction contained only the slower-moving isoenzyme (track (1) in A; track (2) in B of Fig. 2). This isoenzyme migrated to a position similar to that found for muscle adenylate kinase which was run as a marker (track (4) in A; track (1) in B of Fig. 2). The cytosolic fraction (35000 X g supernatant fraction) was enriched in the faster-moving isoenzyme which was entirely absent from the mitochondrial fraction but also contained some of the slower- moving isoenzyme (track (2) in A; track (4) in B of Fig. 2). This contamination would be expected since the cytosolic fraction contained significant amounts of t h e mitochondrial marker enzyme, citrate synthase, as a consequence of damage to this organelle during the preparation.

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4.2. Initial rate studies Initial rate studies were performed on the sep-

arated mitochondrial and cytosolic isoenzymes. The apparent K m values measured for ADP in the direction of ATP synthesis, and for MgATP 2- and AMP in the direction of ADP synthesis are summarised in Table 1. Linear relationships between reciprocal initial velocity and reciprocal nucleotide concentration were observed for both isoenzymes when either MgATP 2- or AMP was the varied substrate except at high nucleotide concentration where there was some tendency to substrate inhibition. Similar values were obtained for the apparent K m for MgATP 2- for both isoenzymes but the cytosolic isoenzyme exhibited a significantly more favourable g m for AMP (Table 1).

For ADP the situation is more complex since the enzyme must interact with two molecules of this substrate binding at two distinct sites [12]. The substrate is postulated to be ADP 3- at the phosphate donor site and either ADP 3- or M g A D P - at the phosphate acceptor site [13]. Under the conditions used both ADP 3- and M g A D P - were present. For the cytosolic isoenzyme the relationship between reciprocal initial rate and reciprocal [ADP] total deviated markedly from linearity at low concentrations of ADP. A linear relationship was, however, observed over the entire range of ADP concentration tested if reciprocal initial rate was plotted against reciprocal total [ADP] 2 (Fig. 3A). This was used to estimate the apparent K m for ADP (Table 1).

Table 1

Kinetic constants for the mitochondrial and cytosolic isoenzymes of A. nidulans adenylate kinase

Apparent K m values were determined in studies similar to those described for ADP in Fig. 3.

Substrate Apparent K m (mM) for:

Mitochondrial Cytosolic isoenzyme isoenzyme

MgATP 2- 0.10 +0.04 0.11 +0.03 ADP 0.23 +0.05 0.31 +0.05 AMP 0.045 + 0.01 0.024 + 0.005 *

• P < 0 .01 .

136

T

E

E

>-

I/[ADP] 2 , mM -2

20 4-0 60 80 1 O0 i , i i

moo A 1 f

750 •

500

250.

0 o ~ 6

I/[ADP], mM -I 12

T

E

E

600 -

500 -

400 -

,300 -

200 -

1 O0 -

0

r

i'0 2'0 3'o 4'0 5'0 1/[ADP], mM -I

60

Fig. 3. Reciprocal initial rate of ATP formation by the cytosolic (A) and mitochondrial (B) isoenzymes of adenylate kinase measured as a function of reciprocal total ADP concentration. The mitochondrial and cytosolic isoenzymes were prepared and adenylate kinase assayed as ATP formation from ADP. The assay system contained, in 1.0 ml, 100 mM Tris-Cl, pH 8.0, 10 mM MgC12, 5 mM glucose, 0.4 mM NADP, 5 IU hexokinase, 5 IU glucose-6-phosphate dehydrogenase and the concentrations of ADP as indicated. The data points shown are means + S.E.M. from 3 experiments with determinations performed in triplicate. In (A) the open circles indicate

reciprocal initial rate plotted as a function of reciprocal [ADP] and the closed circles as a function of reciprocal [ADP] 2.

For the mitochondrial isoenzyme the relationship between reciprocal initial rate and reciprocal total [ADP] was linear over the range of concentration tested (Fig. 3B). No deviation from linearity was observed at low [ADP] even though the range used (0.02-1.5 mM) extended well be- yond that employed in the studies on the cytosolic isoenzyme (0.1-3.0 mM). Since the two isoenzymes exhibited similar apparent K m values for ADP (Table 1) the use of lower concentrations of this substrate should have allowed detection of any deviation from linearity.

5. DISCUSSION

These studies provide the first definitive evi- dence for the presence of organdie-specific isoenzymes of adenylate kinase in fungi (Fig. 2) and also, when taken together with our earlier report [7], for mitochondrial adenylate kinase activity in these organisms. The findings therefore resemble those previously reported for mammalian liver [2,3]. The data of Fig. 2, however, only demon- strate that two forms of adenylate kinase are present in A. nidulans which differ in net charge, and do not establish that these isoenzymes are unique gene products.

The initial rate studies (Fig. 3) suggest, however, that these adenylate kinase isoenzymes may catalyse interconversion of ATP, ADP and AMP using different mechanisms. The cytosolic isoenzyme of A. nidulans adenylate kinase appears to use a random order mechanism with isomerisation of the ternary complex as the rate-determining step. This mechanism has previously been postulated to describe catalysis by yeast adenylate kinase [13] and predicts the observed linear relationship between reciprocal initial rate and reciprocal total [ADP] 2 (Fig. 3A) [14]. A different mechanism is apparently used by the mitochondrial isoenzyme since here a linear relationship is obtained between reciprocal initial rate and reciprocal [ADP] even when examined over a wider range of total ADP concentration (Fig. 3B). These latter data indicate a mechanism in which an irreversible step, e.g. release of a product at low concentration, separates addition of the two mole-

ADP AMP ADP ATP

, t ; T E E-ADP E-P E-P E

I ADP

Fig. 4. Proposed reaction sequence for the mitochondrial isoenzyme.

cules of ADP [14] as is shown in the reaction sequence of Fig. 4 in which AMP dissociates from the enzyme prior to addition of the second mole- cule of ADP. Support for this postulate has been obtained in preliminary studies in which addition of non-saturating [AMP] (0.1 mM) induced significant non-linearity in the relationship between reciprocal initial rate and reciprocal [ADP] (data not shown).

REFERENCES

[1] Atkinson, D.E. (1977) Cellular Energy Metabolism and its Regulation, pp. 85-110, Academic Press Inc.,New York.

[2] Markland, F.S. and Wadkins, C.L. (1966) J. Biol. Chem., 241, 4124-4135.

[3] Criss, W.E. (1970) J. Biol. Chem., 245, 6352-6356.

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[4] Khoo, J.C. and Russell, P.J. (1972) Biochem. Biophys. Acta 268, 98-101.

[5] Chiu, C.S., Su, S. and Russell, P.J. (1967) Biochem. Bio- phys. Acta 132, 361-369.

[6] Brdiczka, D., Pette, D., Brunner, G. and Miller, F. (1968) Eur. J. Biochem. 5, 294-304.

[7] Singh, M., Scrutton, N.S. and Scrutton, M.C. (1988) J. Gen. Microbiol. 134, 643-654.

[8] Osmani, S.A., Marston, F.A.O., Selmes, I.P. and Scrutton, M.C. (1981) Eur. J. Biochem., 118, 271-278.

[9] Osmani, S.A. and Scrutton, M.C. (1983) Eur. J. Biochem. 133, 551-560.

[10] Fildes, R.A. and Harris, H. (1966) Nature (Lond.) 209, 261-263.

[12] Rao, B.D.N., Cohn, M. and Noda, L. (1978) J. Biol. Chem. 253, 1149-1158.

[13] Su, S. and Russell, P.J. (1968) J. Biol. Chem. 243, 3826- 3833.

[14] Cleland, W.W. (1963) Biochem. Biophys. Acta. 67, 188- 196.

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Adenylate kinase isoenzymes in Aspergillus nidulans