JOURNAL
OF
BACTERIOLOGY, Feb. 1978, p. 1039-1041
Vol. 133, No. 2
0021-9193/78/0133-1039$02.00/0 Copyright © 1978 American Society for Microbiology
Printedin U.S.A.
Multiple Forms of Glucokinase from Dictyostelium discoideum K. A. KILLICK* AND B. E. WRIGHT Department of Developmental Biology, Boston Biomedical Research Institute, Boston, Massachusetts 02114
Received for publication 15 September 1977
A single form of glucokinase with an apparent Km value equal to 0.12 mM glucose was detectable in extracts prepared from aggregating cells, whereas kinetic and electrophoretic evidence indicated the presence of this form as well as a second glucose-phosphorylating enzyme with a Km value of about 0.01 mM glucose in extracts from culminating cells.
Although starvation imposes upon the differentiating cellular slime mold, Dictyostelium discoideum, the characteristics of a metabolically closed system, alterations in the levels of end product saccharides may be achieved by permitting development to proceed in the presence of exogenous glucose (9). The major consequences of this perturbation are five- to sixfold elevations in trehalose and alkali-soluble glycogen content, which stem from an increased rate of glucose 6-phosphate synthesis catalyzed by glucokinase (ATP:D-glucose-6-phosphotransferase, EC 2.7.1.2) (9). The latter glucose-phosphorylating enzyme has been partially purified from aggregating cells and characterized as a glucokinase based on substrate specificity studies (1). Therefore, for convenience, each isozyme discussed in this paper may be referred to as a glucokinase, realizing that the enzymes present in culninating cells have not yet been purified and characterized. During a computer simulation analysis of the effect of exogenous glucose on saccharide accumulation, it was assumed that the specific activity of glucokinase was unaffected in cells exposed to exogenous glucose. One prediction of this study was the existence in vivo of a glucose pool, at a level of about 0.01 mM, or about 1/100 of that determined for the whole organism (8). The present investigation was undertaken to determine whether exogenous glucose affects the specific activity of glucokinase and whether multiple forms of the enzyme exist, possibly associated with the two postulated glucose pools. D. discoideum NC-4, ATCC 24697, was grown on nutrient agar with Escherichia coli as the bacterial associate (5). To initiate development, the cells were washed free of bacteria and were then spread onto sterile sheets of unbuffered 2.5% Noble agar. Where indicated in the text, these sheets were supplemented with 25 mM
glucose. Following incubation of the organism for either 10 h (late aggregation) or 22 h (late
culmination) at 220C, extracts were prepared
from washed cells in 50 mM tris(hydroxymethyl)aminomethane (pH 8.0) buffer that contained 0.1 mM ethylenediaminetetraacetate (1) (standard buffer) by a single freeze-thaw cycle (4), followed by centrifugation (15 min, 33,000 x g). Glucokinase activity was assayed at 230C according to the methods of Baumann (1), and one unit of enzymatic activity is defined as that amount of enzyme that catalyzes the synthesis of 1 ,umol of product per min. The specific activity is expressed as units per milligram of acidprecipitable protein. Protein was measured by a modification of the method of Lowry et al. (6), using bovine serum albumin as the standard. Samples of enzyme were partially purified for polyacrylamide gel electrophoresis and kinetic studies by first adding streptomycin sulfate, with stirring, to cell-free extracts to a final concentration of 2% (wt/vol). After being stirred (30 min) on ice, the solution was centrifuged (15 min, 33,000 x g), and glucokinase activity was concentrated from the recovered supernatant by the addition of solid ammonium sulfate to 60% saturation. After being stirred (30 min) on ice, the solution was centrifuged (15 min, 33,000 x g), and the pelleted enzyme was solubilized with a minimal amount of standard buffer. After overnight dialysis (400) against standard buffer, samples were centrifuged (15 min, 33,000 x g). This procedure resulted in recoveries of 70 to 80% of the glucokinase activity originally present in the cell-free extracts. Samples of enzyme were subjected to discontinuous gel electrophoresis (40C) in 15% polyacrylamide gels, using the tris(hydroxymethyl)aminomethane-glycine buffer system described by Canalco (2). After electrophoresis, the gels were sectioned by hand with a razor
1039
1040
NOTES
blade into 1.0-mm segments. Each gel slice was placed into 0.5 ml of standard buffer, emulsified with a glass rod, and left at 40C overnight. Samples of gel eluate were then assayed for glucokinase activity with the standard system described above. Recovery of glucokinase activity following electrophoresis was 70 to 73%. The specific activity of glucokinase was measured at the aggregation and culmination stages of development in extracts prepared from cells, which had undergone morphogenesis in either the presence or the absence of glucose. The results from a typical experiment are shown in Table 1. Enzyme specific activity increased 1.4fold during this developmental period, as shown in earlier studies (3), and was unaffected by perturbing morphogenesis with exogenous glucose. The variation in three experiments was less than 10%. Hence, the assumption that glucose would not have a significant effect on the specific enzyme activity was valid (9). To determine whether multiple forms of glucokinase exist during nonperturbed morphogenesis, kinetic and electrophoretic studies on the enzyme from the aggregation and culmination stages of development were undertaken. Using the Lineweaver-Burk double-reciprocal plot (Fig. 1), kinetic evidence demonstrated that extracts from aggregating cells contained a single form of the enzyme. The apparent Km for glucose was 0.12 mM (variation within 10% in subsequent determinations), and polyacrylamide gel electrophoresis revealed the presence of one peak of enzymatic activity with an Rm value of 0.33 (Fig. 2, I). Using similar methods of kinetic analysis, evidence was obtained demonstrating that preparations of the culmination enzyme contained at least two glucose-phosphorylating enzymes (Fig. 3) having apparent Km values of 0.10 mM (form 1) and 0.02 mM (form 2) glucose. These apparent Km values (Fig. 3) were determined after extrapolation of the straight-line portions of each curve. Since each point represents the sum of two velocities, the extrapolated intercepts do not yield the correct values for the constants (7). When the true values of the TABLE 1. Specific activity of glucokinase during development Glucoseb Sp act Stage of developmenta 0.095 Aggregation + 0.090 Aggregation 0.106 Culmination + Culmination 0.122 aAggregation, 10 to 12 h, and culmination, 20 to 22 h, of development. b Presence or absence of 25 mM exogenous glucose in 2.5% unbuffered Noble agar.
J. BACTF,RIOL.
0
20
40
60
80
100
[glucoseY' mM-1 FIG. 1. Effect of glucose concentration on the activity of glucokinase from aggregating cells of D. discoideum. Glucokinase activity was assayed in the presence of 2 mM ATP, 13 mM Mg2", and varying glucose concentrations with enzyme prepared from cells at the aggregation stage of development (i.e., 10 to 12 h). I
700
.C
E 600-
t
~~~~~~B
0
co500 10 0
o4001-000-
10 20 30 0 l0 20 30 DISTANCE FROM ORIGIN (mm) FIG. 2. Polyacrylamide discontinuous gel electrophoresis of glucokinase. Enzyme was partially purified from cells at the aggregation (I) and culmination (II) stages of development. Samples were then subjected to electrophoresis, and glucokinase activity was assayed after gel sectioning and extrusion as described in the text. The arrows indicate the positions of the tracking dye. 0
kinetic constants were determined by successive
approximations (7), apparent Km values of 0.01 mM and 0.12 mM glucose (with an approximate 10% variation) were obtained for the low and high Km forms of the enzyme, respectively. Calculation of the relative amounts of each form of the enzyme from their V., values indicated that 10 to 20% of the total glucokinase activity was present as the low Km form. Since these kinetic results could have been due to a multisite enzyme having substrate bind-
VOL. 133, 1978
NOTES T
' 0.4 E
O,
0.3
E
.1
' 0.2
.1
d, 0.1
o
20
40
60
80
[glucoseY' mM-1 FIG. 3. Effect of glucose concentration
loo
on
the
ac-
tivity of glucokinase from culminating cells of D. discoideum. Glucokinase activity was assayed in the presence of 2 mM ATP, 13 mM Mg2", and varying glucose concentrations with enzyme prepared from cells during the culmination process (i.e., 21 to 23 h of development).
ing sites of different affinities as opposed to resulting from the presence of multiple enzymes (7), direct physical evidence for multiple glucosephosphorylating enzymes was sought using polyacrylamide gel electrophoresis. Assay of gel sections following electrophoresis indicated that enzymatic activity was associated with two fractions, which differed in their Rm values (Fig. 2, II). The Rm for the enzyme associated with fraction A was 0.20, whereas that associated with fraction B was 0.33. Of the glucokinase activity applied to the gels, average values of 15 and 85% were recovered as the A- and B-electrophoretic fractions of the enzyme, respectively. From a comparison of (i) the Rm values for the enzymes from the aggregation and culmination stages of development, (ii) the percentage of the activity recovered as the A and B fractions (i.e., average values of 15 and 85%, respectively) following electrophoresis, and (iii) the relative amounts of the low and high Km forms of the enzyme calculated from their V,. values, it may be concluded that fraction B is present at both aggregation and culmination and that the two glucose-phosphorylating enzymes having apparent Km values of 0.01 and 0.12 mM glucose correspond to the electrophoretically distinct A and B fractions of the enzyme, respectively. The relationship between the low Km glucosephosphorylating enzyme from culminating organisms
and the perturbable glucose pool
re-
cently simulated by computer (9) remains to be
1041
determined. Since the predicted concentration of glucose was 0.01 mM or lower, maintenance of this concentration might require the associated glucose-phosphorylating enzyme to have an apparent Km value of the order of 0.01 mM (e.g., forn 2). Recently, Wilson and Rutherford (J. B. Wilson and C. L. Rutherford, J. Gen. Physiol., in press) have measured the distribution of glucose between the two cell types (i.e., spore and stalk) of the culminating organism and have found that glucose is localized almost exclusively in the stalk cells. The concentration in the spore cells was 0.01 mM or lower. This suggests that the metabolically perturbable glucose pool postulated by Wright et al. (9) may be localized specifically in the spore cells. It would be extremely interesting to now know whether the two glucose-phosphorylating enzymes show a cell-type-specific distribution between the spore and stalk cells of the culminating organism, kinetically compatible with the glucose levels in these tissues. This investiation was supported by Public Health Service research grants AG00433 and AG00260 from the National Institute of Aging. We express our appreciation to E. Coe for his constructive comments on this manuscript. LITERATURE CITED 1. Baumann, P. 1969. Glucokinase of Dictyostelium discoideum. Biochemistry 8:5011-5015. 2. Canalco. 1968. Canalco disc electrophoresis manual. Canalco, Rockville, Md. 3. Cleland, S. V., and E. L. Coe. 1968. Activities of glycolytic enzymes during the early stages of differentiation in the cellular slime mold. Biochim. Biophys. Acta 156:44-50. 4. Killick, K. A., and B. E. Wright. 1975. Trehalose synthesis during differentiation in Dictyostelium discoideum. Preparation, stabilization and asay of trehalose6-phosphate synthetase. Arch. Biochem. Biophys. 170:6343. 5. Liddel, G. U., and B. E. Wright. 1961. The effect of glucose on respiration of the differentiating slime mold. Dev. Biol. 3:265-276. 6. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 7. Segel, I. H. 1975. Enzyme kinetics: behavior and analysis of rapid equilibrium and steady state enzyme systems, p. 64-72. John Wiley & Sons, New York. 8. Wright, B. E. 1973. Critical variables in differentiation. Prentice-Hall, Inc., Englewood Cliff, N.J. 9. Wright, B. E., A. Tai, and K. A. Killick. 1977. Fourth expansion and glucose perturbation of the Dictyostelium kinetic model. Eur. J. Biochem. 74:217-225.
OF
BACTERIOLOGY, Feb. 1978, p. 1039-1041
Vol. 133, No. 2
0021-9193/78/0133-1039$02.00/0 Copyright © 1978 American Society for Microbiology
Printedin U.S.A.
Multiple Forms of Glucokinase from Dictyostelium discoideum K. A. KILLICK* AND B. E. WRIGHT Department of Developmental Biology, Boston Biomedical Research Institute, Boston, Massachusetts 02114
Received for publication 15 September 1977
A single form of glucokinase with an apparent Km value equal to 0.12 mM glucose was detectable in extracts prepared from aggregating cells, whereas kinetic and electrophoretic evidence indicated the presence of this form as well as a second glucose-phosphorylating enzyme with a Km value of about 0.01 mM glucose in extracts from culminating cells.
Although starvation imposes upon the differentiating cellular slime mold, Dictyostelium discoideum, the characteristics of a metabolically closed system, alterations in the levels of end product saccharides may be achieved by permitting development to proceed in the presence of exogenous glucose (9). The major consequences of this perturbation are five- to sixfold elevations in trehalose and alkali-soluble glycogen content, which stem from an increased rate of glucose 6-phosphate synthesis catalyzed by glucokinase (ATP:D-glucose-6-phosphotransferase, EC 2.7.1.2) (9). The latter glucose-phosphorylating enzyme has been partially purified from aggregating cells and characterized as a glucokinase based on substrate specificity studies (1). Therefore, for convenience, each isozyme discussed in this paper may be referred to as a glucokinase, realizing that the enzymes present in culninating cells have not yet been purified and characterized. During a computer simulation analysis of the effect of exogenous glucose on saccharide accumulation, it was assumed that the specific activity of glucokinase was unaffected in cells exposed to exogenous glucose. One prediction of this study was the existence in vivo of a glucose pool, at a level of about 0.01 mM, or about 1/100 of that determined for the whole organism (8). The present investigation was undertaken to determine whether exogenous glucose affects the specific activity of glucokinase and whether multiple forms of the enzyme exist, possibly associated with the two postulated glucose pools. D. discoideum NC-4, ATCC 24697, was grown on nutrient agar with Escherichia coli as the bacterial associate (5). To initiate development, the cells were washed free of bacteria and were then spread onto sterile sheets of unbuffered 2.5% Noble agar. Where indicated in the text, these sheets were supplemented with 25 mM
glucose. Following incubation of the organism for either 10 h (late aggregation) or 22 h (late
culmination) at 220C, extracts were prepared
from washed cells in 50 mM tris(hydroxymethyl)aminomethane (pH 8.0) buffer that contained 0.1 mM ethylenediaminetetraacetate (1) (standard buffer) by a single freeze-thaw cycle (4), followed by centrifugation (15 min, 33,000 x g). Glucokinase activity was assayed at 230C according to the methods of Baumann (1), and one unit of enzymatic activity is defined as that amount of enzyme that catalyzes the synthesis of 1 ,umol of product per min. The specific activity is expressed as units per milligram of acidprecipitable protein. Protein was measured by a modification of the method of Lowry et al. (6), using bovine serum albumin as the standard. Samples of enzyme were partially purified for polyacrylamide gel electrophoresis and kinetic studies by first adding streptomycin sulfate, with stirring, to cell-free extracts to a final concentration of 2% (wt/vol). After being stirred (30 min) on ice, the solution was centrifuged (15 min, 33,000 x g), and glucokinase activity was concentrated from the recovered supernatant by the addition of solid ammonium sulfate to 60% saturation. After being stirred (30 min) on ice, the solution was centrifuged (15 min, 33,000 x g), and the pelleted enzyme was solubilized with a minimal amount of standard buffer. After overnight dialysis (400) against standard buffer, samples were centrifuged (15 min, 33,000 x g). This procedure resulted in recoveries of 70 to 80% of the glucokinase activity originally present in the cell-free extracts. Samples of enzyme were subjected to discontinuous gel electrophoresis (40C) in 15% polyacrylamide gels, using the tris(hydroxymethyl)aminomethane-glycine buffer system described by Canalco (2). After electrophoresis, the gels were sectioned by hand with a razor
1039
1040
NOTES
blade into 1.0-mm segments. Each gel slice was placed into 0.5 ml of standard buffer, emulsified with a glass rod, and left at 40C overnight. Samples of gel eluate were then assayed for glucokinase activity with the standard system described above. Recovery of glucokinase activity following electrophoresis was 70 to 73%. The specific activity of glucokinase was measured at the aggregation and culmination stages of development in extracts prepared from cells, which had undergone morphogenesis in either the presence or the absence of glucose. The results from a typical experiment are shown in Table 1. Enzyme specific activity increased 1.4fold during this developmental period, as shown in earlier studies (3), and was unaffected by perturbing morphogenesis with exogenous glucose. The variation in three experiments was less than 10%. Hence, the assumption that glucose would not have a significant effect on the specific enzyme activity was valid (9). To determine whether multiple forms of glucokinase exist during nonperturbed morphogenesis, kinetic and electrophoretic studies on the enzyme from the aggregation and culmination stages of development were undertaken. Using the Lineweaver-Burk double-reciprocal plot (Fig. 1), kinetic evidence demonstrated that extracts from aggregating cells contained a single form of the enzyme. The apparent Km for glucose was 0.12 mM (variation within 10% in subsequent determinations), and polyacrylamide gel electrophoresis revealed the presence of one peak of enzymatic activity with an Rm value of 0.33 (Fig. 2, I). Using similar methods of kinetic analysis, evidence was obtained demonstrating that preparations of the culmination enzyme contained at least two glucose-phosphorylating enzymes (Fig. 3) having apparent Km values of 0.10 mM (form 1) and 0.02 mM (form 2) glucose. These apparent Km values (Fig. 3) were determined after extrapolation of the straight-line portions of each curve. Since each point represents the sum of two velocities, the extrapolated intercepts do not yield the correct values for the constants (7). When the true values of the TABLE 1. Specific activity of glucokinase during development Glucoseb Sp act Stage of developmenta 0.095 Aggregation + 0.090 Aggregation 0.106 Culmination + Culmination 0.122 aAggregation, 10 to 12 h, and culmination, 20 to 22 h, of development. b Presence or absence of 25 mM exogenous glucose in 2.5% unbuffered Noble agar.
J. BACTF,RIOL.
0
20
40
60
80
100
[glucoseY' mM-1 FIG. 1. Effect of glucose concentration on the activity of glucokinase from aggregating cells of D. discoideum. Glucokinase activity was assayed in the presence of 2 mM ATP, 13 mM Mg2", and varying glucose concentrations with enzyme prepared from cells at the aggregation stage of development (i.e., 10 to 12 h). I
700
.C
E 600-
t
~~~~~~B
0
co500 10 0
o4001-000-
10 20 30 0 l0 20 30 DISTANCE FROM ORIGIN (mm) FIG. 2. Polyacrylamide discontinuous gel electrophoresis of glucokinase. Enzyme was partially purified from cells at the aggregation (I) and culmination (II) stages of development. Samples were then subjected to electrophoresis, and glucokinase activity was assayed after gel sectioning and extrusion as described in the text. The arrows indicate the positions of the tracking dye. 0
kinetic constants were determined by successive
approximations (7), apparent Km values of 0.01 mM and 0.12 mM glucose (with an approximate 10% variation) were obtained for the low and high Km forms of the enzyme, respectively. Calculation of the relative amounts of each form of the enzyme from their V., values indicated that 10 to 20% of the total glucokinase activity was present as the low Km form. Since these kinetic results could have been due to a multisite enzyme having substrate bind-
VOL. 133, 1978
NOTES T
' 0.4 E
O,
0.3
E
.1
' 0.2
.1
d, 0.1
o
20
40
60
80
[glucoseY' mM-1 FIG. 3. Effect of glucose concentration
loo
on
the
ac-
tivity of glucokinase from culminating cells of D. discoideum. Glucokinase activity was assayed in the presence of 2 mM ATP, 13 mM Mg2", and varying glucose concentrations with enzyme prepared from cells during the culmination process (i.e., 21 to 23 h of development).
ing sites of different affinities as opposed to resulting from the presence of multiple enzymes (7), direct physical evidence for multiple glucosephosphorylating enzymes was sought using polyacrylamide gel electrophoresis. Assay of gel sections following electrophoresis indicated that enzymatic activity was associated with two fractions, which differed in their Rm values (Fig. 2, II). The Rm for the enzyme associated with fraction A was 0.20, whereas that associated with fraction B was 0.33. Of the glucokinase activity applied to the gels, average values of 15 and 85% were recovered as the A- and B-electrophoretic fractions of the enzyme, respectively. From a comparison of (i) the Rm values for the enzymes from the aggregation and culmination stages of development, (ii) the percentage of the activity recovered as the A and B fractions (i.e., average values of 15 and 85%, respectively) following electrophoresis, and (iii) the relative amounts of the low and high Km forms of the enzyme calculated from their V,. values, it may be concluded that fraction B is present at both aggregation and culmination and that the two glucose-phosphorylating enzymes having apparent Km values of 0.01 and 0.12 mM glucose correspond to the electrophoretically distinct A and B fractions of the enzyme, respectively. The relationship between the low Km glucosephosphorylating enzyme from culminating organisms
and the perturbable glucose pool
re-
cently simulated by computer (9) remains to be
1041
determined. Since the predicted concentration of glucose was 0.01 mM or lower, maintenance of this concentration might require the associated glucose-phosphorylating enzyme to have an apparent Km value of the order of 0.01 mM (e.g., forn 2). Recently, Wilson and Rutherford (J. B. Wilson and C. L. Rutherford, J. Gen. Physiol., in press) have measured the distribution of glucose between the two cell types (i.e., spore and stalk) of the culminating organism and have found that glucose is localized almost exclusively in the stalk cells. The concentration in the spore cells was 0.01 mM or lower. This suggests that the metabolically perturbable glucose pool postulated by Wright et al. (9) may be localized specifically in the spore cells. It would be extremely interesting to now know whether the two glucose-phosphorylating enzymes show a cell-type-specific distribution between the spore and stalk cells of the culminating organism, kinetically compatible with the glucose levels in these tissues. This investiation was supported by Public Health Service research grants AG00433 and AG00260 from the National Institute of Aging. We express our appreciation to E. Coe for his constructive comments on this manuscript. LITERATURE CITED 1. Baumann, P. 1969. Glucokinase of Dictyostelium discoideum. Biochemistry 8:5011-5015. 2. Canalco. 1968. Canalco disc electrophoresis manual. Canalco, Rockville, Md. 3. Cleland, S. V., and E. L. Coe. 1968. Activities of glycolytic enzymes during the early stages of differentiation in the cellular slime mold. Biochim. Biophys. Acta 156:44-50. 4. Killick, K. A., and B. E. Wright. 1975. Trehalose synthesis during differentiation in Dictyostelium discoideum. Preparation, stabilization and asay of trehalose6-phosphate synthetase. Arch. Biochem. Biophys. 170:6343. 5. Liddel, G. U., and B. E. Wright. 1961. The effect of glucose on respiration of the differentiating slime mold. Dev. Biol. 3:265-276. 6. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 7. Segel, I. H. 1975. Enzyme kinetics: behavior and analysis of rapid equilibrium and steady state enzyme systems, p. 64-72. John Wiley & Sons, New York. 8. Wright, B. E. 1973. Critical variables in differentiation. Prentice-Hall, Inc., Englewood Cliff, N.J. 9. Wright, B. E., A. Tai, and K. A. Killick. 1977. Fourth expansion and glucose perturbation of the Dictyostelium kinetic model. Eur. J. Biochem. 74:217-225.
