Characterization of two glyceraldehyde-3-phosphate dehydrogenase

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enzymes had the same substrate specificity but different catalytic and molecular ... Downloaded from .... all experiments according to the method of Furfine and Velick (7). The ... NADH as substrates, as described by Bergmeyer (3). ... PL sensitivity ofGAPDH activity in cell-free extracts of S. arenae TU469 grown in synthetic ...
Vol. 153, No. 2

JOURNAL OF BACTERIOLOGY, Feb. 1983, p. 930-936 0021-9193/83/020930-07$02.00/O Copyright 0 1983, American Society for Microbiology

Characterization of Two Glyceraldehyde-3-Phosphate Dehydrogenase Isoenzymes from the Pentalenolactone Producer Streptomyces arenae KARL-HEINZ MAURER, FRIEDHELM PFEIFFER,t HARTMUT ZEHENDERJ AND DIETER -' MECKE* Physiologisch-chemisches Institut der Universitat Tubingen, D-7400 Tubingen, Federal Republic of Germany

Received 13 September 1982/Accepted 29 November 1982

Pentalenolactone (PL) irreversibly inactivates the enzyme glyceraldehyde-3phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD' oxidoreductase (phosphorylating)] (EC 1.2.1.12) and thus is a potent inhibitor of glycolysis in both procaryotic and eucaryotic cells. We showed that PL-producing strain Streptomyces arenae TU469 contains a PL-insensitive glyceraldehyde-3-phosphate dehydrogenase under conditions of PL production. In complex media no PL production was observed, and a PL-sensitive glyceraldehyde-3-phosphate dehydrogenase, rather than the insensitive enzyme, could be detected. The enzymes had the same substrate specificity but different catalytic and molecular properties. The apparent Km values of the PL-insensitive and PL-sensitive enzymes for glyceraldehyde-3-phosphate were 100 and 250 ,uM, respectively, and the PL-sensitive enzyme was strongly inhibited by PL under conditions in which the PL-insensitive enzyme was not inhibited. The physical properties of the PLinsensitive enzyme suggest that the protein is an octamer, whereas the PLsensitive enzyme, like other glyceraldehyde-3-phosphate dehydrogenases, appears to be a tetramer. enzymes are strictly homologous and show sequence identities of 50 to 60%o. Furthermore, each of these enzymes that has so far been characterized has a molecular weight of 146,000 and consists of four identical subunits (8). Streptomyces arenae TU469 produces PL in a minimal medium containing glucose or mannitol as the sole carbon source (10). Despite the presence of PL, growth or glucose utilization is not inhibited. In a preliminary report we have described a PL-insensitive GAPDH which is responsible for this resistance of S. arenae

The antibiotic pentalenolactone (PL) (Fig. 1) is one of the few sesquiterpene lactones produced by procaryotes (4). In the fermented broth of several strains of actinomycetes (11, 16, 18), PL occurs in three different forms: chlorohydrin, diol, and epoxide (1, 10). Several intermediates and metabolites of PL have also been described (4). PL is a potent inhibitor of the glycolytic and gluconeogenetic pathways in both procaryotic and eucaryotic organisms (6, 9). In vitro, an irreversible inactivation of the enzyme glyceraldehyde-3-phosphate dehydrogenase [Dglyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating); EC 1.2.1.12] (GAPDH), of microbial, plant, and mammalian origin, by 10-6 M PL has been shown (6, 9, 15). The action of the antibiotic seems to be restricted to this enzyme, as the addition of noncarbohydrate substrates, such as pyruvate, to the culture medium restores normal growth activity to microorganisms [10]. The NAD+-dependent GAPDH has been isolated and characterized from a number of procaryotic and eucaryotic sources. The different

TU469 (20). We now report that during cultivation in complex media containing peptone and yeast extract, no PL was produced. In these media an isoenzyme of GAPDH was formed, exhibiting almost the same sensitivity to inactivation by the antibiotic as the enzymes from other sources. The two isoenzymes from S. arenae TU469 were isolated, and their molecular structures were characterized. They showed similar catalytic properties compared with the enzymes known so far, but they were different in respect to structural composition.

t Present address: Max-Planck Institut futr Psychiatrie, Abt. Neurochemie, D-8033 Martinsried, Federal Republic of Germany. t Present address: Biochemisches Institut der Universitat Freiburg, D-7800 Freiburg, Federal Republic of Germany.

MATERIALS AND METHODS Chemicals and reagents. All reagents were of analytical grade. Glyceraldehyde-3-phosphate-diethylacetal 930

GAPDH ISOENZYMES

VOL. 153, 1983

CH3

COOH

o 0P FIG. 1. PL

n0

(chlorohydrin

form).

(cyclohexylammonium salt), NAD+, rabbit muscle GAPDH, and the reference proteins for molecular weight determination were obtained fr rom Boehringer Mannheim Corp., Mannheim, Federal Republic of Germany. DEAE-Sephacel and Blue Sepharose CL6B were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Celite 535 was from Serva, Heidelberg, Federal Republic of Germany. 13 io-Gel A 0.5 m and Bio-Gel HTP were from Bio-Rac I Laboratories, Richmond, Calif. PL was isolated as chlorohydrin froim the acidified fermentation broth of a 4,000-liter fern mentation of S. arenae TU469, carried out at the CJesellschaft fiOr Biotechnologische Forschung, Braun kschweig-Stockheim, Federal Republic of Germany. IPL was purified by chromatography on Amberlite-XAID 2 and Sephadex LH-20 and by high-pressure liqui4d chromatography on Merck LiChrosorb RP 18. In all experiments the chlorohydrin form of PL was used I. Organisn. S. arenae TU469 (DSIN4 40 734) was kindly provided by H. Zahner, Institu It fur Mikrobiologie, Tubingen, Federal Republic of ( Jermany. Media and culture conditions. S. arenae TU469 produced about 3,ug of PL per ml in sy nthetic medium containing 4% mannitol, 0.25% asrparagine, 0.2% (NH4)2SO4, 0.1% NaCI, 0.3% K2HPO )4 3H20, 0.1% MgSO4* 7H20, 0.04% CaC12* 2H20, 0.002% FeSO4 * 7H20, and 0.001% ZnSO4* 7H20 at p tH 6.2 and 30°C. Wickerham medium (23) was used as ccDmplex medium and contained 0.5% peptone, 0.3% yea st extract, 0.3% malt extract, and 3% glucose at pH 7.( D and 30°C. -

For enzyme production, cells were grown in a 20liter fermentor (Giovanola Intensor I b20; Monthey, Switzerland) at 30°C for 20 h at pH 7.0. .The mycelium harvested by centrifugation and vvashed with 50 mM Tris-chloride buffer (pH 8.0) cointaining 5 mM mercaptoethanol, 5 mM EDTA, and 1 ,uM NAD+ (buffer A). The cells were suspended in 1 volume of buffer A and 1 volume of glass beads (diameter, 0.5 mm; Braun-Melsungen, Federal Repu blic of Germany). Homogenization was carried out in a laboratory blender at high speed for 5 min at 4°C. TFhe supernatant was decanted, and the glass beads weire washed with buffer A. The pooled supernatants werce centrifuged at 6,000 x g for 20 min to remove cell de -bris. For some experiments S. arenae TU '469 was grown in 100 ml of medium in 1-liter Erlenr neyer flasks at with shaking. Washed cells wtere broken by ultrasonic disruption in a Branson Soni fier 12b for 30 s was

30°C

in two 15-s pulses at

0°C.

931

Purification of PL-sensitive and PL-insensitive GADPH from S. arenae TU469. Both isoenzymes were prepared by the following procedure, in which all operations were carried out at 4°C. To the crude extract 1 g of Celite per 100 mg of protein was added, and after 30 min of constant stirring, the suspension was brought to 85% saturation by the addition of solid ammonium sulfate (559 g/liter) over the next 30 min. The suspension was allowed to stand for 1 h and was then centrifuged for 30 min at 15,000 x g. The pellet was resuspended in buffer A saturated to 85% with ammonium sulfate and poured into a column (6-cm diameter). After settling, the enzyme was eluted with a 600-ml linear gradient of 85 to 40% saturated ammonium sulfate. Fractions containing the enzyme were dialyzed for 24 h against three changes of 5 liters of buffer A. The extract was applied to a column (1 by 12 cm) of DEAE-Sephacel equilibrated in buffer A. The enzyme was eluted with a 300-mI linear gradient of 0 to 0.5 M NaCl in buffer A and then applied to a column (2 by 150 cm) of Bio-Gel A 0.5m equilibrated in buffer A. Fractions with high activity were pooled and dialyzed overnight against two changes of 2 liters of 10 mM potassium phosphate buffer (pH 6.9) containing 5 mM mercaptoethanol. The dialyzed preparation was applied to a column (1 by 15 cm) of Bio-Gel HTP equilibrated with 10 mM potassium phosphate buffer. GAPDH was eluted with a 200-ml linear gradient of 10 to 200 mM potassium phosphate at pH 6.9. Active fractions were pooled, chromatographed on a column (2 by 50 cm) of Bio-Gel P6 equilibrated with buffer A, and stored at 40C. GAPDH assay. GAPDH activity was measured spectrophotometrically with NAD+ as the substrate, according to the method of Furfine and Velick (7). The standard assay mixture (2.5 ml) contained 50 mM Trischloride buffer (pH 8.0), 4 mM mercaptoethanol, 4 mM EDTA, 2 mM NAD+, and 4 mM Na2HAsO4. The reaction was started by the addition of glyceraldehyde3-phosphate to a final concentration of 0.4 mM. Enzyme inactivation was measured by adding 10 nmol of PL (4 ,uM, unless otherwise stated) to the assay mixture. After 1 h of incubation at 26°C, the reaction was started by addition of glyceraldehyde-3phosphate. (PL was dissolved in 50%o methanol. An equal amount was added to the reference samples.) The increase in absorbance at 340 nm was measured, and the activity was calculated from the initial reaction velocity at 260C. One unit of GAPDH was defined as the amount of the enzyme which catalyzes the reduction of 1 ±mol of NAD+ per min. D-Glyceraldehyde-3-phosphate was used for the determination of the substrate specificity, whereas DLglyceraldehyde-3-phosphate was used for the routine assays. To test the reversibility of the reaction and the inhibition of the enzyme with iodoacetic acid, the assay was performed with 1,3-diphosphoglycerate and NADH as substrates, as described by Bergmeyer (3). PL determination. The concentration of PL was determined by inhibition of 13 mU of rabbit muscle GAPDH after incubation for 1 h at 26°C in the assay mixture. The inhibition of the enzyme proved to be a function of the PL concentration. PL concentrations could be estimated by comparison with a standard curve made with pure PL. Protein determination was by the procedure of Lowry et al. (13).

932

MAURER ET AL.

J. BACTERIOL.

RESULTS

medium to Wickerham medium (Fig. 2). The PLsensitive GAPDH activity began to appear under these conditions after 3 h. Only the PLsensitive activity was found 20 h after transfer. The possibility of an infection with another strain could be excluded. In another set of experiments, cells were transferred from Wickerham medium to synthetic medium (Fig. 3). PL-insensitive activity first appeared after 12 h, and 8 h later PL could be detected. At the same time, the PL-sensitive enzyme activity decreased rapidly. After 48 h only the PL-insensitive GAPDH activity was found. In a control experiment in which cells were transferred from Wickerham medium back to Wickerham medium, the specific activity of the sensitive enzyme reached a maximum of 0.9 U/mg after 24 h and decreased to 0.45 U/mg after 48 h. Nearly the same specific activity of PLinsensitive enzyme activity was found 48 h after cells were transferred from Wickerham medium to synthetic medium. Cells grown in Wickerham medium reached twice the biomass and, accordingly, twice the amount of GAPDH activity compared with cells grown in synthetic medium. After the addition of PL (0.2 ,ug/ml) to Wickerham medium, there was a slow appearance of PL-insensitive GAPDH activity which followed the total inhibition of the PL-sensitive GAPDH activity (Fig. 4). The cells were not strongly

PL sensitvity of GAPDH activity in S. arenae TU469 under various culture conditions. When grown in the synthetic medium described above, S. arenae TU469 produced PL to a concentration of 10 ,uM after 50 h in submersed culture, whereas no PL was produced by this microorganism during 90 h of submersed culture in Wickerham medium. During the production phase of PL, no inhibition of growth or of glucose utilization was observed. Cell-free extracts of S. arenae TU469 grown under both culture conditions were compared with respect to PL sensitivity of GAPDH activity. Although the activity was inactivated by more than 90%o at 0.4 FM PL after growth in Wickerham medium, it was inactivated by less than 5% at 0.4 ,M PL after growth in the synthetic medium. Thus, the PL sensitivity of the enzyme activity was completely different under the two culture conditions. In further experiments a PL concentration of 4 ,uM was used to differentiate between PLsensitive and PL-insensitive GAPDH activity. A time course of PL sensitivity and PL insensitivity was followed by first growing cells for 24 h in either synthetic or Wickerham medium. Cells were harvested by centrifugation, washed twice with sterile 0.9%o NaCl, and then transferred to the other medium. In one set of experiments, cells were transferred from synthetic

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FIG. 2. PL sensitivity of GAPDH activity in cell-free extracts of S. arenae TU469 grown in synthetic medium and transferred to Wickerham medium. PL sensitivity was measured as described in the text. The increasing inhibition corresponds to the appearance of PL-sensitive GAPDH. Inhibition at 100%0 indicates the PL-sensitive form of the enzyme, inhibition at 0o indicates the PL-insensitive form. The specific activity was 0.45 U/mg throughout the experiment. Symbols: A, mycelium dry weight; 0, % inhibition.

GAPDH ISOENZYMES

VOL. 153, 1983

7i 2000.6-

300 E

1A E 100 ,,00

50

60

1200

80

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FIG. 3.. Enzyme activity in cell-free extracts of S. arenae TU469 grown in Wickerham medium and transferred to synthetic medium. Enzyme activity and PL sensitivity were tested as described in the text. According to the PL sensitivity, the enzyme activity was differentiated between the PL-insensitive and PLsensitive forms of GAPDH. Symbols: 0, PL-insensitive GAPDH; 0, PL-sensitive GAPDH; 0, PL; A, mycelium dry weight.

933

materials therefore had to be avoided. Furthermore, in contrast to GAPDH from other sources, both isoenzymes did not bind to Blue Sepharose CL-6B, and therefore affinity chromatography could not be used on this material. After purification, the PL-insensitive enzyme was stable at 4°C for up to 6 months, whereas the PL-sensitive enzyme lost activity with a halflife of 7 days in buffer A. PL sensitivity of the GAPDH isoenzymes. Table 4 lists the different PL sensitivities of the two isoenzymes of S. arenae TU469. The purified isoenzymes showed the same PL sensitivities as described for cell-free extracts, thus excluding a PL-inactivating mechanism. The inhibition of several GAPDHs by PL is irreversible and follows first-order kinetics with different rates (9, 15). To measure PL sensitivity, it was therefore necessary to standardize the percent inhibition and the incubation time. Relative PL sensitivity is expressed as the concentration of antibiotic necessary for 90% inhibition after 1 h of incubation at 26°C divided by the concentration of PL necessary for 90% inhibition of the same activity of rabbit muscle GAPDH. The relative sensitivities of GAPDHs from various sources and from

affected by the inhibition of the PL-sensitive GAPDH, as they were able to grow in complex medium without catabolizing glucose. Purification of two GAPDH isoenzymes. To investigate the molecular mechanism of the development of PL resistance, GAPDH was isolated from cells grown in Wickerham medium and in synthetic medium. Although the same purification scheme was followed, differences in the elution profiles and the final specific activities of the enzymes purified from the two sources indicated the presence of two isoenzymes; i.e., the PL-sensitive enzyme eluted at 0.07 M potassium phosphate from the Bio-Gel HTP column, whereas the PL-insensitive enzyme eluted at 0.04 M. PL-insensitive GAPDH from S. arenae TU469 was purified 50-fold, with a recovery of 25% compared with the crude extract (Table 1). PL-sensitive GAPDH was purified 93-fold, with a recovery of 10% (Table 2). After purification, both forms of the enzyme showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 5). Also, the final specific activities were in the range known for GAPDH from other sources (Table 3). During initial purification experiments, we realized that Dextran-containing chromatography materials inactivated both enzymes. These

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FIG. 4. Enzyme activity in cell-free extracts of S. arenae TU469 grown in Wickerham medium, after inactivation in vivo with 0.3 p.g of PL/ml. Enzyme activity and PL sensitivity were tested as described in the text. According to PL sensitivity, the enzyme activity was differentiated between PL-insensitive and PL-sensitive forms of GAPDH activity. Symbols: 0, PL-insensitive GAPDH; 0, PL-sensitive GAPDH; A, mycelium dry weight in PL culture; A, mycelium dry weight in control.

934

J. BACTERIOL.

MAURER ET AL.

TABLE 1. Purification of PL-insensitive GAPDH from S. arenae TU469 Total Total Vol Recovery Sp act (m) protein (% Purification step activity (U/Mg) (U) (mg) 280 480 686 0.65 100 Cell extract 212 47 4.5 46 71 Ammonium sulfate eluate 12 314 27 65 11 DEAE-Sephacel eluate 13 13 37 170 36 Bio-Gel A 0.5m eluate 3.6 33 25 120 30 Bio-Gel HTP eluate

Purification (fold)

1 6.8 18 19 50

TABLE 2. Purification of PL-sensitive GAPDH from S. arenae TU469

Total

Total protein (mg)

Sp (U/mg)

Recovery (%

Purification (fold)

3,340 717 873 586 362

2,780 109 25 10 3.3

1.2 6.5 32 58 112

100 22 26 17.5 11

1 5.4 27 49 93

Vol Purification step (ml) Purification stepactivity

~(U)

Cell extract Ammonium sulfate eluate DEAE-Sephacel eluate Bio-Gel A 0.5m eluate Bio-Gel HTP eluate

1,000 640 48 77 33

S. arenae TU469 are listed in Table 3. Compared with PL-sensitive GAPDHs from various sources, the PL-sensitive isoenzyme showed relatively low sensitivity, being 16 to 100 times less sensitive than other enzymes. The PLinsensitive isoenzyme, however, was more than O0s times less sensitive than GAPDH from other sources (Table 3). Substrate kinetics. Both isoenzymes of GAPDH of S. arenae TU469 require as substrates NAD+, arsenate or phosphate, and D-

glyceraldehyde-3-phosphate for their activity. These isoenzymes are D-glyceraldehyde-3-phosphate:NAD+ oxidoreductases (phosphorylating), as NADP+ is not accepted as a substrate and both enzymes reduce 1,3-diphosphoglycerate with NADH. The preparations of the two enzymes showed similar substrate kinetics for the substrates NAD+ and glyceraldehyde-3phosphate. The apparent Km for NAD+ was 120 ,uM for the PL-insensitive form and 110 ,uM for the PL-sensitive form. For glyceraldehyde-3phosphate an apparent Km value of 100 FM was obtained for the PL-insensitive form, and an apparent Km value of 250 ,uM was obtained for the PL-sensitive form. A comparison with enzymes from other sources is given in Table 3. Iodoacetic acid was incubated with 40 mU of each enzyme preparation for 5 min in the assay which used 1,3-diphosphoglycerate and NADH as substrates. The PL-sensitive isoenzyme showed 50%o inhibition in the presence of 200 ILM iodoacetic acid, whereas 1,200 ,uM iodoacetic acid was necessary for 50%o inhibition of the PL-insensitive form. Molecular weight. The molecular weights of the native enzymes were estimated by chromatography of 0.5 mg of enzyme on a Bio-Gel A

act

0.5m column (2 by 150 cm). The reference proteins were catalase, aldolase, fumarase, and rabbit muscle GAPDH. PL-sensitive GAPDH from S. arenae TU469 eluted before rabbit mus-

A BC D

FIG. 5. Sodium dodecyl sulfate-polyacrylamide gel

electrpphoresis of the purified enzymes. An 11 to 15% gradient gel was run according to the method of Laemmli (12). Samples were prepared by incubation at 100°C for 3 min in the presence of 1% sodium dodecyl sulfate and 1% mercaptoethanol. Lanes: A, reference proteins bovine serum albumin, ovalbumin, trypsinogen, lactoglobulin, and lysozyme; B, PL-insensitive GAPDH from S. arenae TU469; C, PL-sensitive GAPDH from S. arenae TU469; D, GAPDH from rabbit muscle.

VOL. 153, 1983

935

GAPDH ISOENZYMES TABLE 3. Comparison of NAD+-dependent GAPDHs from different sourcesa Apparent Km (RM)

Source

Mol wt

Rabbit (7) Spinach (21) Yeast (22) E. coli (2) S. arenae TU469 PL sensitive PL insensitive

Mol wt subunit

No. of

Sp act"

for:

(U/mg)

subunits

NAD+

GAPd

13 NDe 44 166

90 ND 160 142

164 80 100 40

110 120

250 100

112 33

36,500 37,000

146,000 145,000 146,000 144,000

36,500 35,000

4 4 4 4

180,000 290,000

43,000 37,000

4 8

Relative sensitivity to inactivation

by

PLC

1 5.7 (15) 3.2 (9) 1.4 (9) 100 105

Numbers within parentheses are reference numbers. Specific activity of purified enzyme. c Expressed as PL concentration required for 90% inhibition divided by that required for 90% inhibition of rabbit muscle enzyme. d GAP, Glyceraldehyde-3-phosphate. ' ND, Not determined. a

b

cle GAPDH but after fumarase. The PL-insensitive enzyme eluted before all reference proteins used. By comparison with the elution volumes of the reference proteins, the molecular weights of the isoenzymes were calculated to be 280,000 for the PL-insensitive form and 180,000 for the PL-sensitive form. Upon gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol, the PL-insensitive enzyme showed one subunit with an apparent molecular weight of 37,000, whereas the PL-sensitive enzyme gave one subunit with an apparent molecular weight of 43,000 (Fig. 5). Thus, the PL-sensitive GAPDH probably consists offour subunits, in contrast to eight subunits in the PL-insensitive form. DISCUSSION S. arenae TU469 produces PL, an antibiotic shown to inactivate GADPH from various sources (9, 15). The production of PL is strongly dependent on culture conditions. Under conditions of PL production, the organism strictly TABLE 4. PL sensitivity of purified GAPDH from rabbit muscle and from S. arenae TU469 Inhibition (%) of 50 mU of GAPDH activitya from:

S. arenae TU469

PL concn

(M)

Rabbit (PL sensitive)

4 x 10-6

100

4 x 10-7 4 x 10-8

100

4 x 10-9

100 90

PL insensitive