ARCHIVES
Vol.
OF BIOCHEMISTRY
291, No. 2, December,
ANI)
BIOPHYSICS
pp. 225-230,
1991
The NAD-Dependent Glutamate Dehydrogenase from Dictyostelium discoideum: Purification and Properties F. Pamula and J. F. Wheldrake’ School of Biological Sciences, The Flinders University of South Australia, GPO Box 2100, Adelaide 5001, Australia
Received
April
8, 1991, and in revised
form
August
1, 1991
The NAD-dependent glutamate dehydrogenase (GDH) from Dictyoetelium discoideum was purified 1 lOl-fold with a yield of 23.4%. The enzyme has an apparent AZ, of 356 kDa, determined using Sephacryl 5400, and a subunit molecular weight of 54 kDa on SDS-polyacrylamide gel electrophoresis. The K,,,s for a-ketoglutarate, NADH,andNHaare0.36+0.03mM, 16.0*0.1p~,and 34.5 + 2.7 mM, respectively. The purified enzyme has a pH optimum of pH 7.25-7.5. At 0.1 mM, ADP and AMP stimulate GDH activity 26 and 102%. respectively. Halfmaximal activity in the presence of 0.1 mM AMP for cu-ketoglutarate, NADH, and NHf is reached at 2.3 + 0.1 mM, 71.4 +- 6.5 pM, and 27.9 + 3.6 mM, respectively. 0 1991
Academic
Press,
Inc.
The cellular slime mold Dictyostelium dkcoideum is a free living soil microorganism (1). Amoebae, on depletion of their bacterial food source, aggregate in response to pulses of CAMP and then undergo a series of morphological changes resulting in the formation of a multicellular fruiting body. The fruiting body consists of about lo5 cells and is composed of two main cell types, spore and nonviable stalk cells in a 4:l ratio (2). This organism therefore has many of the properties of advanced developmental systems without their inherent complexity. In D. discoideum, amino acid starvation (3-7) is the cue to proceed through morphogenesis. Once the process commences amoebae stop feeding and rely on their endogenous reserves to provide the energy for development. Protein and RNA appear to provide the main source of this energy because total protein decreases 50% (8) and RNA 40% (9), whereas total lipid (10) and carbohydrate content (11) decrease little during morphogenesis. Although total carbohydrate decreases only slightly, many new carbohydrates are synthesised during morphogenesis (12); yet no more than 70% of these new carbohydrates i To whom
correspondence
should
0003-9861/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
be addressed.
are thought to be derived from glycogen breakdown (13). The remainder presumably are derived from the breakdown of endogenous protein and RNA. The transition from growth to morphogenesis in D. discoideum is regulated by CAMP, differentiation inducing factor (DIF),2 adenosine, and ammonia (14). Early in development CAMP serves as a chemotactic signal and induces cell differentiation while later in morphogenesis, CAMP stimulates prespore differentiation. In the early stages of development ammonia is a CAMP antagonist, inhibiting both CAMP accumulation (1516) and CAMPinduced gene expression (17). Later in morphogenesis ammonia also promotes prespore differentiation and represses stalk cell differentiation (U-20). It appears to do this by stimulating CAMP accumulation in prespore cells and inhibiting the accumulation of CAMP in prestalk cells (21). DIF directs prestalk differentiation while adenosine repressesprespore differentiation (14). These morphogens therefore are important regulatory molecules for the construction of the fruiting body. Glutamate dehydrogenase (GDH) catalyses the interconversion of L-glutamate and a-ketoglutaratez a-ketoglutarate
+ NAD(P)H
+ NH: $
NAD(P)+
+ L-glutamate + H20.
GDH lies at an important branchpoint in metabolism as it provides a link between amino acid metabolism and the tricarboxylic acid cycle. In many prokaryotes and some lower eukaryotes distinct NAD- and a NADP-dependent GDH enzymes are present. When both enzymes are present NAD-GDH functions in the deamination of L-glu* Abbreviations used: EDTA, ethylenediaminetetraacetate; Tris, tris(hydroxymethyl)aminomethane; bii-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; Triton X-100, isooctylphenoxypolyethoxyethanol; DIF, differentiation inducing factor; GDH, glutamate dehydrogenase; SDS, sodium dodecyl sulfate; FPLC, fast protein liquid chromatography. 225
Inc. reserved.
226
PAMULA
AND
WHELDRAKE
TABLE
Steps in the Purification Total Purification
step
volume
Total
(ml)
Crude extract Activated extract DE-52 Matrex Blue A TSK Phenyl-5PW Mono Q Note.
of the NAD-Dependent
200 200 235 42.5 16 6
See Experimental
Procedures
protein (mg) 1933.3 1733.3 275.0 4.0 1.6 0.4
for a detailed
description
r----lo”‘” ---------_
Specific activity W/md 0.02 0.06 0.18 10.53 18.07 25.57
Purification (-fold) 1.0 2.7 7.5 453.8 778.8 1101.9
Yield (%) 100.0 238.3 107.1 94.1 64.3 23.4
of procedures.
EXPERIMENTAL
PROCEDURES
Materials. DE-52 was purchased from Whatman, Matrex Blue A was purchased from Amicon, SephacrylS400 and Mono Q HR 5/5 were purchased from Pharmacia, and TSK Phenyl-5PW (75 X 8 mm) was purchased from LKB Bromma. AMP, ADP, and AMP were purchased from the Sigma Chemical Co. NAD+, NADH, NADPH, a-ketoglutarate, and bovine serum albumin were obtained from Boehringer. All other
reagents were of the highest purity available. Buffers. Buffer A consisted of 25 mM Tris-HCl (pH 8.0) and buffer B 0.1% (w/v) Triton X-100 in 25 mM Tris-HCl (pH 8.0). Cell culture. For enzyme purification, D. discoideum (strain V12) was grown as described by Marshall and Wheldrake (24), washed free of bacterial contaminants with KK2 as previously described (25), and then frozen at -20°C until required. Enzyme purification. Washed cells (50 ml packed cells) were thawed, resuspended to 150 ml with buffer B, and then sonicated (3X 20-s bursts) with a Sony sonicator probe at the maximum setting. The extract was
0.10
- 0.05
1 -
0
activity W)
from D. discoideum
0.15
.E E B E
Dehydrogenase
44.9 106.9 48.1 42.2 28.8 10.5
and NADP-GDH in the amination of cy-ketoglutarate. The change in total protein, RNA, and carbohydrate that occur during development must require metabolic reorientation. Since GDH occupies a pivotal position in amino acid metabolism and since one of the substrates of GDH is the ammonium ion, the enzyme may play a regulatory role in the transition from growth to morphogenesisand terminal differentiation in slime moulds. The NADP-GDH from D. discoideum has been purified to homogeneity (22) and because of its low levels it is probably not a developmental control point. By contrast, the NADGDH enzyme has some interesting properties. It is activated up to lo-fold in crude extracts upon standing at 4°C. The degree of latency varies during morphogenesis with the highest level found in vegetative stage cells and the lowest in aggregation stage cells. Finally, activation does not appear due to proteolysis but the activation of NAD-GDH can be partially inhibited by EDTA and restored by the addition of equal molar concentration Mg2+ ions (23). As a first step in resolving the nature of this activation we here present the purification and in vitro properties of the NAD-dependent GDH from D. discoideum.
7
Glutamate Total
tamate
rE
I
20
40 Fraction
60
SO
0.00 100
number
FIG. 1. The elution profile of NAD-GDH from Matrex Blue A. Enzyme activity, (0) 100 mM NaCl and 1 mM NAD+ (---). See Experimental Procedures for details.
FIG. 2.
SDS-polyacrylamide gel electrophoresis of samples taken after the various stages of the purification of glutamate dehydrogenase. Lane 1, markers (phosphorylase b, 94 kDa; albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 20.1 kDa; and (Yla&albumin, 14.4 kDa); lane 2, crude extract; lane 3, crude extract (activated); lane 4, DE-52 eluate; lane 5, Matrex Blue A eluate; lane 6, TSK Phenyl-5PW eluate; and lane 7, Mono Q eluate.
NAD-DEPENDENT
GLUTAMATE
DEHYDROGENASE 50
r-----l25
-----
12
I
-
20
I
.
1
.
227
discoideum I
’
I
.
I
’
1
’
40 -
-15
1 -x m
-10
g 5l it h
/
/ /
Dictyostelium
E
0-
/ 4-
FROM
/ /
-5
10
-
/ 0 0
8
16
24
Fraction
32
40
0
5.0
number
FIG. 3. The elution profile of NAD-GDH Enzyme activity (a) and propylene glycol mental Procedures for details.
from TSK Phenyl-5PW. (---) gradient. See Experi-
centrifuged (100,OOOg at 4°C for 35 min), and the high speed supematant was collected, made up to 200 ml with buffer B, adjusted to pH 8.0 with NaOH, and then left overnight at 4°C. The high speed supernatant was then applied to a DE-52 column (44 X 3.2 cm) preequilibrated with buffer A and washed for 24 h with 25 mM NaCl in buffer A at 40 ml * h-l. Enzyme elution was with 175 mM NaCl in buffer A at 60 ml * h-i. The active fractions were pooled and then desalted by repeatedly concentrating using an Amicon ultrafiltration unit with a YMlOO filter and then diluting the sample by the addition of buffer A. The concentrate was applied to a column, preequilibrated with buffer A, of Matrex Blue A (48 X 1.6 cm) at 30 ml * h-l. To remove loosely bound material the column was washed overnight with 25 mM NaCl in buffer A and the enzyme eluted with 1 mM NAD+ and 100 mM NaCl in buffer A at 12 ml. h-‘. Active fractions were pooled, concentrated, brought up to 1 M (NH&SO,, and then applied to the TSK Phenyl-5PW (75 X 8 mm) column. The enzyme was eluted with a linear gradient starting from 1 M (NH&SO, and 0% propylene glycol and changing to 0.25 M (NH,)sSOI and 15% (w/v) propylene glycol (both in buffer A) in 20 ml, followed with 10 ml 20% propylene glycol at a flow rate of 60 ml * h-i. Active fractions were pooled, filtered as before, and then applied to a Mono Q HR 5/5 column, The column was washed with 2 ml (O-O.25 M NaCl) and the enzyme eluted with a linear salt gradient (0.25-0.65 M NaCl) in 23 ml at 60 ml. h-i, all in buffer A. Pure enzyme was found in fractions
5.2
5.4
I
I
5.6
5.8
6.0
LdJw FIG. 6. The M, of the native enzyme was determined using Sephacryl S400 in 0.2 M NaCl in buffer A. M, markers are thyroglobulin, 669 kDa; ferritin, 450 kDa; beef liver GDH, 350 kDa; and catalase, 240 kDa. Purified D. discoideum GDH (7).
Glutamate dehydrogenase assay. NAD-dependent GDH was assayed by monitoring the oxidation of NADH at 30°C with a Hitachi recording spectrophotometer as described by Mazo (26). The assay was commenced with the addition of enzyme to the reaction mixture containing 0.2 mM NADH, 2.5 mM cu-ketoglutarate, and 50 mM NH&l in 100 mM TrisHCl (pH 7.5). One unit (U) of enzyme activity is defined as the amount of enzyme required to oxidize 1 pmol of substrate * min-‘. For the pH activity profile, a mixed buffer containing 50 mM bis-Tris-HCl and 50 mM Tris-HCl was used from pH 6.5-8.5. Protein determinations. Protein assays were performed using the Bradford method (27) and bovine serum albumin was used to construct the standard curve. Gel electrophoresis. SDS-polyacrylamide gel electrophoresis was performed essentially as described by Laemmli (28) using the Bio-Rad Mini-Protean gel electrophoresis apparatus as described by the manufacturer. Determination of native M,. The M, of the native enzyme was estimated using Sephacryl S400 (50 X 1 cm). Purified enzyme and standards were made up in buffer A containing 0.2 M NaCl. Samples were
r
5
0
20
10
Fraction
FIG. 4. The elution profile activity (0) and salt gradient details.
30
uee-e-ao.o 40
50 Velocity
number
of NAD-GDH from (---). See Experimental
Mono Q. Enzyme Procedures for
FIG. 6. Eadie-Hofstee plot for cY-ketoglutarate: enzyme activity plotted against enzyme activity over a-ketoglutarate concentration with 0.1 mM AMP (0), r = 0.98, or without AMP (O), r = 0.96.
228
PAMULA
AND
WHELDRAKE
r
1.5 r E ‘i
.r E a
1.2
0.9
5 ; .$ ‘G
0.6
g
0.3
I
0.0 6.0
6.5
7.0
Velocity
FIG. ‘7. Eadie-Hofstee plot for NADH: enzyme activity enzyme activity over NADH concentration with 0.1 = 0.98, or without AMP (O), r = 0.99.
RESULTS
of Glutamate Dehydrogenase
After overnight activation of the crude supernatant at 4°C the first step in the purification involved DE-52 (Table I). After eluting from DE-52 the desalted eluate was applied to a Matrex Blue A column. The column was washed with 25 mM NaCl to remove loosely bound material and the enzyme was eluted with 1 mM NAD+ and 100 mM NaCl (Fig. 1). This step resulted in a 60-fold purification. Examination of a sample by polyacrylamide gel electrophoresis from this step showsthree major bands (Fig. 2). The active fractions were then applied, in the presence of (NHJ2S04, to a hydrophobic interaction col-
FIG. 9. pH activity profile of glutamate mental Procedures for details.
6.5
9.0
mM
plotted against AMP (O), r
dehydrogenase.
See Experi-
umn (Fig. 3) attached to the Pharmacia FPLC system. Elution of the enzyme from this resin resulted in the removal of at least one major contaminant (Fig. 2) and the enzyme was purified approximately 2-fold. The active fractions were desalted before being applied to and eluted from the Mono Q column (Fig. 4), again using the FPLC. This final step resulted in a single band on SDS-polyacrylamide gel electrophoresis (Fig. 2). The enzyme was purified llOl-fold with a yield of 23.4% (Table I). The subunit molecular weight of the purified enzyme was 54 kDa, as determined by SDS-polyacrylamide gel electrophoresis (Fig. 2), while the native M, was 356 kDa by Sephacryl S400 (Fig. 5). On this basis the structure of the native enzyme is probably hexameric, which is similar to that found in most organisms (29). Eadie-Hofstee plots for a-ketoglutarate, NADH, and NH: are shown in Figs. 6-8. The K,,, values are 0.36 + 0.03 mM for a-ketoglutarate (Fig. 6), 16.0 + 0.1 I.LM for NADH (Fig. 7), and 34.5 f 2.7 mM for NH: (Fig. 8). The pH
0.3
Velocity
FIG. 8. Eadie-Hofstee plot for NH&l: enzyme activity enzyme activity over NH&l concentration with 0.1 = 0.93, or without AMP (O), r = 0.96.
6.0
PH
plotted against mM AMP (O), r
applied directly to the column in 0.5 ml and developed at a 10 ml * h-’ using the same buffer. The elution peak was found by measuring activity (GDH) or absorbance at 280 nm.
Purification
1
7.5
0.6
0.9
Concentration
FIG. 10. The effect of various (0) on NAD-GDH activity.
concentrations
1.2
1.5
(mM)
of AMP
(0) and ADP
NAD-DEPENDENT
GLUTAMATE
DEHYDROGENASE
activity profile shows the purified enzyme has an optimum of pH 7.25-7.5 (Fig. 9). Effect of AMP
and ADP on Glutamate
Dehydrogenase
The purified enzyme was tested with a number of nucleotides known to be allosteric modifiers of mammalian GDHs to determine their effect on enzymatic activity. ADP had a slight stimulatory effect, 25% at 0.1 mM, whereas AMP was highly stimulatory, 102% at 0.1 mM, and dAMP stimulated GDH activity 67% at 0.1 mM. Other nucleotides tested had no effect (data not shown). Since both AMP and ADP had a stimulatory effect on the purified enzyme, these nucleotides were tested over a range of concentrations. The enzyme is stimulated by low concentrations of AMP but at concentrations above 0.3 mM, AMP slightly inhibits the reaction (Fig. 10). By contrast ADP is not nearly as stimulatory at the same concentrations as AMP (Fig. 10). In view of the possible regulatory role of AMP the K,s were reexamined in the presence of 0.1 mM AMP. ‘The K, for cY-ketoglutarate was 2.3 + 0.1 mM (Fig. 6), 71.4 f 5.5 pM for NADH (Fig. 7), and 27.9 + 3.6 mM for NH: (Fig. 8). Coenzyme Specificity
of NAD-GDH
from D. discoideum
Previous workers (30) have partially purified two forms of GDH from D. dkcoideum, a mitochondrial enzyme that can utilize both NAD+ and NADP+, and an extramitochondrial enzyme that is NAD+ specific. Our purified NAD-dependent GDH showed some activity when NADPH was substituted for NADH in the assay mixture (data not shown). The level of activity was just above background and some doubt exists as to whether NADPH can substitute for NADH in the amination of cY-ketoglutarate. DISCUSSION After overnight incubation the crude extract was fractionated using a DE-52 column which resulted in a 3-fold purification and a considerable reduction of protein (Table I). The next step in the purification, the use of the Matrex Blue A resin, was the most effective, resulting in a 60fold purification, and examination of the eluate by polyacrylamide gel electrophoresis (Fig. 2) shows three major bands. The use of the hydrophobic column caused a 32% loss of enzyme activity (Table I) but was effective in the removal of one major contaminant. The last step, a Mono Q column, also caused an appreciable loss (64%) of enzyme activity, but was required to remove the remaining contaminants (Fig. 2). These losses are probably due to a combination of the instability of the enzyme and a combination of high dilution and/or absorptive losses. Despite this the final yield was 0.4 mg of pure NAD-GDH (23.4%) and a llOl-fold purification was achieved in approximately 5 days.
FROM
Dictyostelium
229
dixoideum
AMP has two interesting effects, it increases both V,, and the K,,, for a-ketoglutarate and NADH (six- and fourfold, respectively) when assayed in the direction of L-glutamate formation. Hence, the affinity of the NADdependent GDH for two of its substrates decreases. During the vegetative stage, slime molds are actively feeding and macromolecules are freely available for metabolism so that it is expected that the AMP concentration is low. Since starvation is the cue for differentiation it is expected that the level of AMP would increase. The increase in AMP concentration and hence V,, would have the effect of increasing the rate of formation of both a-ketoglutarate and NADH, thereby increasing the cell’s energy balance. Late in morphogenesis, the total level of GDH decreases. This presumably reflects a decrease in protein catabolism during late morphogenesis (31, 12) and hence a general decrease in the availability of amino acids. It has been shown that the concentrations of glutamate (32) and glutamine (33) increase substantially during morphogenesis. In addition, there is a general slow down in the TCA cycle late in morphogenesis (34, 35). The reasons for the accumulation of glutamate and glutamine are not known but a decrease in GDH would be expected to be a precondition for such an event. The regulation exerted by AMP over NAD-GDH makes good metabolic sense and is in agreement with what is known about slime mold metabolism. It would be of interest to see if the NAD-dependent GDH enzyme is sensitive to AMP throughout morphogenesis and what effect, if any, activation of this enzyme has on its ability to respond to AMP. We are in the process of pursuing these points. REFERENCES 1. Sussman, 61,426-435. 2. Loomis,
M.,
and Bradley,
W. F. (1988)
ZSZAths
S. G. (1954)
Arch.
Sci. Zmmunol.
3. Darmon, M., and Klein, C. (1978) 4. Lee, K. C. (1972) J. Gen. Microbial.
S., Skrinska,
6. Marin, 7. Marin,
48,110-117. 60, 389-395.
8. Mizukami, 9. Walsh, 10. Long,
Deu. Biol. Dev. Biol.
Y., and Iwabuchi, J., and Wright,
M. (1970)
B. E. (1978)
B. H., and Coe, E. L. (1974)
11. Rosness, P. A., and Wright, 164,60-72. 12. Hames, B. D., and Ashworth, 325. 13. Hames, 315. 14. Williams,
B:D.,
and Ashworth,
J. G. (1988)
Biophys.
1,25-30.
Deu. Biol. 63,377-389. 72,457-471.
J. P., Froshauer, 5. Margolskee, (1980) Deu. Biol. 74,409-421. F. T. (1976) F. T. (1977)
B&hem.
R., and Lodish,
Ezp.
Cell Res. 63,317-324.
J. Gen. Microbial. J. Biol.
B. E. (1974)
H. F.
C&m. Arch.
108,
57-62.
249,521~529. Biochem.
Biophys.
J. M. (1974)
Biochem.
J. 142,
J. M. (1974)
B&hem.
J. 142,301-
Development
317-
103,1-16.
15. Schindler, J., and Sussman, M. (1979) Deu. Genet. 1, 13-20. G. B., Elder, E. M., and Sussman, M. (1984) Deu. Biol. 16. Williams, 105,377-388.
230
PAMULA
AND
WHELDRAKE
17. Kay, R. R. (1979) J. Embryol. Ezp. Morphol. 52,171-182. 18. Schindler, J., and Sussman, M. (1977) J. Mol. Biol. 116, 161-169. 19. Gross, J. D., Bradbury, J., Kay, R. R., and Peacey, M. J. (1983) Nature 303,244-245.
28. Laemmli,
20. Wang, M., and Schaap, P. (1989) Development 105,569-574. 21. Riley, B. B., and Barclay, S. L. (1990) Development 109, 715-722. 22. Pamula, F., and Wheldrake, J. F. (1991) Mol. Cell. Biochem., in press. 23. Pamula, F., and Wheldrake, J. F. (1990) Biochem. Znt. 20,623-631. 24. Marshall, J., and Wheldrake, J. F. (1990) Biochem. Znt. 21, 615622. 25. Pamula, F., and Wheldrake, J. F. (1988) Biochem. Znt. 1'7,535-543.
31. White, G. J., and Sussman, 285-293.
26. Maze, M. J. (1978) J. Bacterial. 133, 27. Bradford, M. M. (1976) And. B&hem.
780-785. 72,248-254.
U. K. (1970)
Nature
227,
660-665.
13,879-886. 30. Langridge, W. H. R., Komuniecki, P., and DeToma, Arch. Biochem. Biophys. 178,581-587.
29. Gore,
32. Wright,
M. G. (1981)
Znt. J. Biochem.
B. E., and Bard,
M. (1961) S. (1963)
B&him.
B&him.
F. J. (1977)
Biophys. Biophys.
Acta
Acta
53,
71,45-
49. 33. Klein, G., Cotter, D. A., Martin, Biochem. 93, 135-142. 34. Kelly,
P. J., Kelleher,
J.-B.,
and Satre,
M. (1990)
Eur. J.
J. K., and Wright,
B. E. (1979)
Biochem.
J.
J. K., and Wright,
B. E. (1979)
B&hem.
J.
184,581-588. 35. Kelly,
P. J., Kelleher,
184,589-597.
Vol.
OF BIOCHEMISTRY
291, No. 2, December,
ANI)
BIOPHYSICS
pp. 225-230,
1991
The NAD-Dependent Glutamate Dehydrogenase from Dictyostelium discoideum: Purification and Properties F. Pamula and J. F. Wheldrake’ School of Biological Sciences, The Flinders University of South Australia, GPO Box 2100, Adelaide 5001, Australia
Received
April
8, 1991, and in revised
form
August
1, 1991
The NAD-dependent glutamate dehydrogenase (GDH) from Dictyoetelium discoideum was purified 1 lOl-fold with a yield of 23.4%. The enzyme has an apparent AZ, of 356 kDa, determined using Sephacryl 5400, and a subunit molecular weight of 54 kDa on SDS-polyacrylamide gel electrophoresis. The K,,,s for a-ketoglutarate, NADH,andNHaare0.36+0.03mM, 16.0*0.1p~,and 34.5 + 2.7 mM, respectively. The purified enzyme has a pH optimum of pH 7.25-7.5. At 0.1 mM, ADP and AMP stimulate GDH activity 26 and 102%. respectively. Halfmaximal activity in the presence of 0.1 mM AMP for cu-ketoglutarate, NADH, and NHf is reached at 2.3 + 0.1 mM, 71.4 +- 6.5 pM, and 27.9 + 3.6 mM, respectively. 0 1991
Academic
Press,
Inc.
The cellular slime mold Dictyostelium dkcoideum is a free living soil microorganism (1). Amoebae, on depletion of their bacterial food source, aggregate in response to pulses of CAMP and then undergo a series of morphological changes resulting in the formation of a multicellular fruiting body. The fruiting body consists of about lo5 cells and is composed of two main cell types, spore and nonviable stalk cells in a 4:l ratio (2). This organism therefore has many of the properties of advanced developmental systems without their inherent complexity. In D. discoideum, amino acid starvation (3-7) is the cue to proceed through morphogenesis. Once the process commences amoebae stop feeding and rely on their endogenous reserves to provide the energy for development. Protein and RNA appear to provide the main source of this energy because total protein decreases 50% (8) and RNA 40% (9), whereas total lipid (10) and carbohydrate content (11) decrease little during morphogenesis. Although total carbohydrate decreases only slightly, many new carbohydrates are synthesised during morphogenesis (12); yet no more than 70% of these new carbohydrates i To whom
correspondence
should
0003-9861/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
be addressed.
are thought to be derived from glycogen breakdown (13). The remainder presumably are derived from the breakdown of endogenous protein and RNA. The transition from growth to morphogenesis in D. discoideum is regulated by CAMP, differentiation inducing factor (DIF),2 adenosine, and ammonia (14). Early in development CAMP serves as a chemotactic signal and induces cell differentiation while later in morphogenesis, CAMP stimulates prespore differentiation. In the early stages of development ammonia is a CAMP antagonist, inhibiting both CAMP accumulation (1516) and CAMPinduced gene expression (17). Later in morphogenesis ammonia also promotes prespore differentiation and represses stalk cell differentiation (U-20). It appears to do this by stimulating CAMP accumulation in prespore cells and inhibiting the accumulation of CAMP in prestalk cells (21). DIF directs prestalk differentiation while adenosine repressesprespore differentiation (14). These morphogens therefore are important regulatory molecules for the construction of the fruiting body. Glutamate dehydrogenase (GDH) catalyses the interconversion of L-glutamate and a-ketoglutaratez a-ketoglutarate
+ NAD(P)H
+ NH: $
NAD(P)+
+ L-glutamate + H20.
GDH lies at an important branchpoint in metabolism as it provides a link between amino acid metabolism and the tricarboxylic acid cycle. In many prokaryotes and some lower eukaryotes distinct NAD- and a NADP-dependent GDH enzymes are present. When both enzymes are present NAD-GDH functions in the deamination of L-glu* Abbreviations used: EDTA, ethylenediaminetetraacetate; Tris, tris(hydroxymethyl)aminomethane; bii-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; Triton X-100, isooctylphenoxypolyethoxyethanol; DIF, differentiation inducing factor; GDH, glutamate dehydrogenase; SDS, sodium dodecyl sulfate; FPLC, fast protein liquid chromatography. 225
Inc. reserved.
226
PAMULA
AND
WHELDRAKE
TABLE
Steps in the Purification Total Purification
step
volume
Total
(ml)
Crude extract Activated extract DE-52 Matrex Blue A TSK Phenyl-5PW Mono Q Note.
of the NAD-Dependent
200 200 235 42.5 16 6
See Experimental
Procedures
protein (mg) 1933.3 1733.3 275.0 4.0 1.6 0.4
for a detailed
description
r----lo”‘” ---------_
Specific activity W/md 0.02 0.06 0.18 10.53 18.07 25.57
Purification (-fold) 1.0 2.7 7.5 453.8 778.8 1101.9
Yield (%) 100.0 238.3 107.1 94.1 64.3 23.4
of procedures.
EXPERIMENTAL
PROCEDURES
Materials. DE-52 was purchased from Whatman, Matrex Blue A was purchased from Amicon, SephacrylS400 and Mono Q HR 5/5 were purchased from Pharmacia, and TSK Phenyl-5PW (75 X 8 mm) was purchased from LKB Bromma. AMP, ADP, and AMP were purchased from the Sigma Chemical Co. NAD+, NADH, NADPH, a-ketoglutarate, and bovine serum albumin were obtained from Boehringer. All other
reagents were of the highest purity available. Buffers. Buffer A consisted of 25 mM Tris-HCl (pH 8.0) and buffer B 0.1% (w/v) Triton X-100 in 25 mM Tris-HCl (pH 8.0). Cell culture. For enzyme purification, D. discoideum (strain V12) was grown as described by Marshall and Wheldrake (24), washed free of bacterial contaminants with KK2 as previously described (25), and then frozen at -20°C until required. Enzyme purification. Washed cells (50 ml packed cells) were thawed, resuspended to 150 ml with buffer B, and then sonicated (3X 20-s bursts) with a Sony sonicator probe at the maximum setting. The extract was
0.10
- 0.05
1 -
0
activity W)
from D. discoideum
0.15
.E E B E
Dehydrogenase
44.9 106.9 48.1 42.2 28.8 10.5
and NADP-GDH in the amination of cy-ketoglutarate. The change in total protein, RNA, and carbohydrate that occur during development must require metabolic reorientation. Since GDH occupies a pivotal position in amino acid metabolism and since one of the substrates of GDH is the ammonium ion, the enzyme may play a regulatory role in the transition from growth to morphogenesisand terminal differentiation in slime moulds. The NADP-GDH from D. discoideum has been purified to homogeneity (22) and because of its low levels it is probably not a developmental control point. By contrast, the NADGDH enzyme has some interesting properties. It is activated up to lo-fold in crude extracts upon standing at 4°C. The degree of latency varies during morphogenesis with the highest level found in vegetative stage cells and the lowest in aggregation stage cells. Finally, activation does not appear due to proteolysis but the activation of NAD-GDH can be partially inhibited by EDTA and restored by the addition of equal molar concentration Mg2+ ions (23). As a first step in resolving the nature of this activation we here present the purification and in vitro properties of the NAD-dependent GDH from D. discoideum.
7
Glutamate Total
tamate
rE
I
20
40 Fraction
60
SO
0.00 100
number
FIG. 1. The elution profile of NAD-GDH from Matrex Blue A. Enzyme activity, (0) 100 mM NaCl and 1 mM NAD+ (---). See Experimental Procedures for details.
FIG. 2.
SDS-polyacrylamide gel electrophoresis of samples taken after the various stages of the purification of glutamate dehydrogenase. Lane 1, markers (phosphorylase b, 94 kDa; albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 20.1 kDa; and (Yla&albumin, 14.4 kDa); lane 2, crude extract; lane 3, crude extract (activated); lane 4, DE-52 eluate; lane 5, Matrex Blue A eluate; lane 6, TSK Phenyl-5PW eluate; and lane 7, Mono Q eluate.
NAD-DEPENDENT
GLUTAMATE
DEHYDROGENASE 50
r-----l25
-----
12
I
-
20
I
.
1
.
227
discoideum I
’
I
.
I
’
1
’
40 -
-15
1 -x m
-10
g 5l it h
/
/ /
Dictyostelium
E
0-
/ 4-
FROM
/ /
-5
10
-
/ 0 0
8
16
24
Fraction
32
40
0
5.0
number
FIG. 3. The elution profile of NAD-GDH Enzyme activity (a) and propylene glycol mental Procedures for details.
from TSK Phenyl-5PW. (---) gradient. See Experi-
centrifuged (100,OOOg at 4°C for 35 min), and the high speed supematant was collected, made up to 200 ml with buffer B, adjusted to pH 8.0 with NaOH, and then left overnight at 4°C. The high speed supernatant was then applied to a DE-52 column (44 X 3.2 cm) preequilibrated with buffer A and washed for 24 h with 25 mM NaCl in buffer A at 40 ml * h-l. Enzyme elution was with 175 mM NaCl in buffer A at 60 ml * h-i. The active fractions were pooled and then desalted by repeatedly concentrating using an Amicon ultrafiltration unit with a YMlOO filter and then diluting the sample by the addition of buffer A. The concentrate was applied to a column, preequilibrated with buffer A, of Matrex Blue A (48 X 1.6 cm) at 30 ml * h-l. To remove loosely bound material the column was washed overnight with 25 mM NaCl in buffer A and the enzyme eluted with 1 mM NAD+ and 100 mM NaCl in buffer A at 12 ml. h-‘. Active fractions were pooled, concentrated, brought up to 1 M (NH&SO,, and then applied to the TSK Phenyl-5PW (75 X 8 mm) column. The enzyme was eluted with a linear gradient starting from 1 M (NH&SO, and 0% propylene glycol and changing to 0.25 M (NH,)sSOI and 15% (w/v) propylene glycol (both in buffer A) in 20 ml, followed with 10 ml 20% propylene glycol at a flow rate of 60 ml * h-i. Active fractions were pooled, filtered as before, and then applied to a Mono Q HR 5/5 column, The column was washed with 2 ml (O-O.25 M NaCl) and the enzyme eluted with a linear salt gradient (0.25-0.65 M NaCl) in 23 ml at 60 ml. h-i, all in buffer A. Pure enzyme was found in fractions
5.2
5.4
I
I
5.6
5.8
6.0
LdJw FIG. 6. The M, of the native enzyme was determined using Sephacryl S400 in 0.2 M NaCl in buffer A. M, markers are thyroglobulin, 669 kDa; ferritin, 450 kDa; beef liver GDH, 350 kDa; and catalase, 240 kDa. Purified D. discoideum GDH (7).
Glutamate dehydrogenase assay. NAD-dependent GDH was assayed by monitoring the oxidation of NADH at 30°C with a Hitachi recording spectrophotometer as described by Mazo (26). The assay was commenced with the addition of enzyme to the reaction mixture containing 0.2 mM NADH, 2.5 mM cu-ketoglutarate, and 50 mM NH&l in 100 mM TrisHCl (pH 7.5). One unit (U) of enzyme activity is defined as the amount of enzyme required to oxidize 1 pmol of substrate * min-‘. For the pH activity profile, a mixed buffer containing 50 mM bis-Tris-HCl and 50 mM Tris-HCl was used from pH 6.5-8.5. Protein determinations. Protein assays were performed using the Bradford method (27) and bovine serum albumin was used to construct the standard curve. Gel electrophoresis. SDS-polyacrylamide gel electrophoresis was performed essentially as described by Laemmli (28) using the Bio-Rad Mini-Protean gel electrophoresis apparatus as described by the manufacturer. Determination of native M,. The M, of the native enzyme was estimated using Sephacryl S400 (50 X 1 cm). Purified enzyme and standards were made up in buffer A containing 0.2 M NaCl. Samples were
r
5
0
20
10
Fraction
FIG. 4. The elution profile activity (0) and salt gradient details.
30
uee-e-ao.o 40
50 Velocity
number
of NAD-GDH from (---). See Experimental
Mono Q. Enzyme Procedures for
FIG. 6. Eadie-Hofstee plot for cY-ketoglutarate: enzyme activity plotted against enzyme activity over a-ketoglutarate concentration with 0.1 mM AMP (0), r = 0.98, or without AMP (O), r = 0.96.
228
PAMULA
AND
WHELDRAKE
r
1.5 r E ‘i
.r E a
1.2
0.9
5 ; .$ ‘G
0.6
g
0.3
I
0.0 6.0
6.5
7.0
Velocity
FIG. ‘7. Eadie-Hofstee plot for NADH: enzyme activity enzyme activity over NADH concentration with 0.1 = 0.98, or without AMP (O), r = 0.99.
RESULTS
of Glutamate Dehydrogenase
After overnight activation of the crude supernatant at 4°C the first step in the purification involved DE-52 (Table I). After eluting from DE-52 the desalted eluate was applied to a Matrex Blue A column. The column was washed with 25 mM NaCl to remove loosely bound material and the enzyme was eluted with 1 mM NAD+ and 100 mM NaCl (Fig. 1). This step resulted in a 60-fold purification. Examination of a sample by polyacrylamide gel electrophoresis from this step showsthree major bands (Fig. 2). The active fractions were then applied, in the presence of (NHJ2S04, to a hydrophobic interaction col-
FIG. 9. pH activity profile of glutamate mental Procedures for details.
6.5
9.0
mM
plotted against AMP (O), r
dehydrogenase.
See Experi-
umn (Fig. 3) attached to the Pharmacia FPLC system. Elution of the enzyme from this resin resulted in the removal of at least one major contaminant (Fig. 2) and the enzyme was purified approximately 2-fold. The active fractions were desalted before being applied to and eluted from the Mono Q column (Fig. 4), again using the FPLC. This final step resulted in a single band on SDS-polyacrylamide gel electrophoresis (Fig. 2). The enzyme was purified llOl-fold with a yield of 23.4% (Table I). The subunit molecular weight of the purified enzyme was 54 kDa, as determined by SDS-polyacrylamide gel electrophoresis (Fig. 2), while the native M, was 356 kDa by Sephacryl S400 (Fig. 5). On this basis the structure of the native enzyme is probably hexameric, which is similar to that found in most organisms (29). Eadie-Hofstee plots for a-ketoglutarate, NADH, and NH: are shown in Figs. 6-8. The K,,, values are 0.36 + 0.03 mM for a-ketoglutarate (Fig. 6), 16.0 + 0.1 I.LM for NADH (Fig. 7), and 34.5 f 2.7 mM for NH: (Fig. 8). The pH
0.3
Velocity
FIG. 8. Eadie-Hofstee plot for NH&l: enzyme activity enzyme activity over NH&l concentration with 0.1 = 0.93, or without AMP (O), r = 0.96.
6.0
PH
plotted against mM AMP (O), r
applied directly to the column in 0.5 ml and developed at a 10 ml * h-’ using the same buffer. The elution peak was found by measuring activity (GDH) or absorbance at 280 nm.
Purification
1
7.5
0.6
0.9
Concentration
FIG. 10. The effect of various (0) on NAD-GDH activity.
concentrations
1.2
1.5
(mM)
of AMP
(0) and ADP
NAD-DEPENDENT
GLUTAMATE
DEHYDROGENASE
activity profile shows the purified enzyme has an optimum of pH 7.25-7.5 (Fig. 9). Effect of AMP
and ADP on Glutamate
Dehydrogenase
The purified enzyme was tested with a number of nucleotides known to be allosteric modifiers of mammalian GDHs to determine their effect on enzymatic activity. ADP had a slight stimulatory effect, 25% at 0.1 mM, whereas AMP was highly stimulatory, 102% at 0.1 mM, and dAMP stimulated GDH activity 67% at 0.1 mM. Other nucleotides tested had no effect (data not shown). Since both AMP and ADP had a stimulatory effect on the purified enzyme, these nucleotides were tested over a range of concentrations. The enzyme is stimulated by low concentrations of AMP but at concentrations above 0.3 mM, AMP slightly inhibits the reaction (Fig. 10). By contrast ADP is not nearly as stimulatory at the same concentrations as AMP (Fig. 10). In view of the possible regulatory role of AMP the K,s were reexamined in the presence of 0.1 mM AMP. ‘The K, for cY-ketoglutarate was 2.3 + 0.1 mM (Fig. 6), 71.4 f 5.5 pM for NADH (Fig. 7), and 27.9 + 3.6 mM for NH: (Fig. 8). Coenzyme Specificity
of NAD-GDH
from D. discoideum
Previous workers (30) have partially purified two forms of GDH from D. dkcoideum, a mitochondrial enzyme that can utilize both NAD+ and NADP+, and an extramitochondrial enzyme that is NAD+ specific. Our purified NAD-dependent GDH showed some activity when NADPH was substituted for NADH in the assay mixture (data not shown). The level of activity was just above background and some doubt exists as to whether NADPH can substitute for NADH in the amination of cY-ketoglutarate. DISCUSSION After overnight incubation the crude extract was fractionated using a DE-52 column which resulted in a 3-fold purification and a considerable reduction of protein (Table I). The next step in the purification, the use of the Matrex Blue A resin, was the most effective, resulting in a 60fold purification, and examination of the eluate by polyacrylamide gel electrophoresis (Fig. 2) shows three major bands. The use of the hydrophobic column caused a 32% loss of enzyme activity (Table I) but was effective in the removal of one major contaminant. The last step, a Mono Q column, also caused an appreciable loss (64%) of enzyme activity, but was required to remove the remaining contaminants (Fig. 2). These losses are probably due to a combination of the instability of the enzyme and a combination of high dilution and/or absorptive losses. Despite this the final yield was 0.4 mg of pure NAD-GDH (23.4%) and a llOl-fold purification was achieved in approximately 5 days.
FROM
Dictyostelium
229
dixoideum
AMP has two interesting effects, it increases both V,, and the K,,, for a-ketoglutarate and NADH (six- and fourfold, respectively) when assayed in the direction of L-glutamate formation. Hence, the affinity of the NADdependent GDH for two of its substrates decreases. During the vegetative stage, slime molds are actively feeding and macromolecules are freely available for metabolism so that it is expected that the AMP concentration is low. Since starvation is the cue for differentiation it is expected that the level of AMP would increase. The increase in AMP concentration and hence V,, would have the effect of increasing the rate of formation of both a-ketoglutarate and NADH, thereby increasing the cell’s energy balance. Late in morphogenesis, the total level of GDH decreases. This presumably reflects a decrease in protein catabolism during late morphogenesis (31, 12) and hence a general decrease in the availability of amino acids. It has been shown that the concentrations of glutamate (32) and glutamine (33) increase substantially during morphogenesis. In addition, there is a general slow down in the TCA cycle late in morphogenesis (34, 35). The reasons for the accumulation of glutamate and glutamine are not known but a decrease in GDH would be expected to be a precondition for such an event. The regulation exerted by AMP over NAD-GDH makes good metabolic sense and is in agreement with what is known about slime mold metabolism. It would be of interest to see if the NAD-dependent GDH enzyme is sensitive to AMP throughout morphogenesis and what effect, if any, activation of this enzyme has on its ability to respond to AMP. We are in the process of pursuing these points. REFERENCES 1. Sussman, 61,426-435. 2. Loomis,
M.,
and Bradley,
W. F. (1988)
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