PRELIMINARY COMMUNICATION
/ . Biochem., 77, 1117-1121 (1975)
Phosphorylation of D-Glucosamine by Rat Liver Glucokinase Michihiko OGUCHI, Yoko MIYATAKE, Junko AYABE, and Nobu AKAMATSU Department of Biochemistry, St. Marianna University School of Medicine, Takatsu-ku, Kawasaki, Kanagawa 213 Received for publication, February 12, 1975
D-Glucosamine was found to be phosphorylated by a rat liver extract in the presence of a high concentration of glucose, which was formerly believed to be a strong competitive inhibitor of this reaction. Results suggested that glucosamine may be phosphorylated by high Km hexokinase, i.e. glucokinase [EC 2. 7.1. 2]. The enzyme involved was separated from specific N-acetyl-D-glucosamine kinase [EC 2. 7.1. 59]. The phosphorylation was not inhibited by a physiological level of glucose or glucose 6-phosphate, which strongly inhibited low Km hexokinase. The apparent Km of glucokinase for glucosamine was estimated as 8 mM, which is ten times that of low Km hexokinase.
Glucosamine is phosphorylated by low Km hexokinase [EC 2. 7.1.1] and it is reported to have a carcinostatic action by causing ATP depletion (7, 2). Glucosamine is metabolized in rat liver and after its administration there is a rapid increase in UDP-N-acetylhexosamine in the liver. However, it has been suggested that it is not phosphorylated, because this is prevented by glucose, which has an inhibition constant (0.1 mM), of almost one-hundredth of the glucose content of liver tissue (3). Nevertheless the possibility that glucosamine is phosphorylated in the liver has been suggested from in vivo and in vitro experiments (4, 5). Thus, phosphorylation of glucosamine was reinvestigated and in the present study, it was demonstrated that the liver contains a kinase which phosphorylates glucosamine even in the presence of the normal cellular level of glucose. Results suggested that the kinase responsible for the phosphorylation is hepatic glucokinase [EC 2.7.1.2]. Vol. 77, No. 5, 1975
Livers were obtained from male albino Wistar strain rats weighing 100—150 g and homogenized with 9 volumes of 0.25 M sucrose containing 1 mM EDTA (pH 7.0) in a PotterElvehjem homogenizer. All procedures were performed at 0—4°C. The crude homogenate was centrifuged at 105,000 Xg for 1 hr. The resulting supernatant was then passed through a Sephadex G-25 column (1.0x20 cm) using buffer consisting of 10 mM potassium phosphate, 1 mM EDTA, and 0.5 mM dithiothreitol, at pH 7.0. The preparation was used immediately after elution. For enzyme purification, the livers (34 g) were homogenized with two volumes of 0.25 M sucrose containing 1 mM EDTA, and then centrifuged for 40 min at 30,000 x g. The resulting supernatant was then brought to 30% saturation (17.6 g/dl) of ammonium sulfate by addition of solid ammonium sulfate. The supernatant obtained by centrifugation at 10,000Xg for 30 min was then brought to 70% ammonium sulfate
1117
1118
PRELIMINARY COMMUNICATION
saturation (27.3 g/dl). The resulting precipitate was collected by centrifugation and dissolved in a small amount of potassium phosphate buffer as described above. The enzyme solution was desalted using a Sephadex G-25 column (3.0x40 cm) and applied to a DEAEcellulose column. Subsequent procedures are specified later. Protein was measured by the method of Lowry et al. (6), using crystalline bovine serum albumin as a standard. Phosphorylation of glucosamine was carried out with the high speed supernatant, which contained more than 80% of the a 25mM GlcN
a.0.20
10
20 30 ScrnM
A.1mM GlcN
So.i-
control
0 10 Time (mln)
20
30
Fig. 1. Effects of glucose and glucose-6-P on glucosamine phosphorylation. The reaction mixture for assay of glucosamine phosphorylation was as follows; O.lmmole Tris-HCl buffer (pH 8.0), 10//moles ATP, 20 //moles MgCl,, 5 //moles EDTA (pH 7.0), 0.5 //mole dithiothreitol, 10 //moles NaF, 0.1 mmole KC1, various concentrations of D-glucosamine and enzyme solution (1.4 mg as protein) in a total volume of 1.0 ml. The reaction was carried out at 37° and then stopped by boiling the mixture for 2min. After diluting the mixture with 4 ml of water, the denaturated protein was removed by centrifugation. The resulting supernatant was applied to a Dowex-1 formate (200-400 mesh) column (0.6x3.5 cm). Glucosamine was washed out with 10 ml of water, and then glucosamine-6-P was eluted with 5 ml of 0.01 M formic acid. The eluate was evaporated to dryness in vacuo at 30°, and the residue was dissolved in 0.6 ml of water, and assayed for glucosamine-6-P by the method of Lewy and McAllan (7). A and B; 1 and 25 mM glucosamine, respectively. C; inhibitory effects of various concentrations of glucose. The incubation time was 20 min.
activity. Figure 1 shows results on the phosphorylation of glucosamine and the effects of intracellular levels of glucose and glucose-6-P.1 The reaction proceeded linearly for 30 min. Using 1 mM glucosamine, the activity was inhibited 60% by 5 mM glucose or 1 mM glucose6-P, as shown in Fig. 1A, while with 25 mM glucosamine these concentrations caused 30% inhibition (Fig. IB). Figure 1C shows the percentage inhibitions with various concentrations of glucose: inhibitions of less than 85 and 35% were noted with 1 and 25 mM glucosamine, respectively, even when the glucose concentration was increased to 40 mM. The results suggest that even in the presence of a physiological level of glucose, glucosamine is phosphorylated in the liver to yield as much as 10 //moles of glucosamine-6-P per hour per gram wet liver weight with 25 mM glucosamine as substrate. Rat liver contains various isozymes of hexokinase, so it seemed interesting to study whether phosphorylation of glucosamine was preferentially catalyzed by one of these isozymes. The typical pattern of liver hexokinase on DEAE-cellulose column chromatography is illustrated in Fig. 2. When glucosamine was used as substrate, the activity was found in 4 major peaks. Glucose phosphorylation was assayed with concentrations of 0.1 and 10 mM glucose as substrate for low Km hexokinase and glucokinase, respectively. The low Km hexokinase was separated into 3 peaks, which were designated as Types I, II, and III in order of elution by an increasing concentration of KC1 (8). It can be seen that the first 3 peaks of activity for phosphorylation of glucosamine, in order from the left, corresponded to peaks of low Km hexokinase. However, the fourth paak did not correspond to low Km hexokinase but to high Km hexokinase, i.e. glucokinase. The activity of Nacetylglucosamine kinase [EC 2.7.1.59] is also shown in the figure. The enzyme was eluted after glucokinase, although in largely overlapped the latter. The difference between the peaks of activity for phosphorylation of glucosamine and N-acetylglucosamine confirms 1
Abbreviation: glucose-6-P, D-glucose 6-phosphate. / . Biochem.
D-GLUCOSAMINE METABOLISM
1119
,,300
30
0.1 mM Glc 10 mM Glc K)mM GlcN 10mM GlcNAc
I c
1
"c E
uO
h 10 LJf5
S
75
1
c
30
A0
50 60 70 Fraction Number
80
90
Fig. 2. ' DEAE-cellulose column chromatography of hexokinases from rat liver. The enzyme obtained as described in the text was adsorbed on a column (2.5x40cm) equilibrated with 10 mM potassium phosphate, 5mM EDTA (pH 7.0), and 0.5 mM dithiothreitol, pH 7.0. An enzyme concentration of 1.19 g of protein in a volume of 70 ml was used, following the procedure of Grossbard and Schimke (fl). Enzyme was eluted with a linear gradient of KC1 prepared by placing 500 ml of the above phosphate buffer in the mixing chamber and 500 ml of 0.6 M KC1 in the same buffer in the reservoir chamber. Fractions of 10 ml were collected. Phosphorylation of glucosamine was assayed as noted in the legend of Fig. 1, except that 10 mM glucosamine was used. Glucose phosphorylation was assayed by a slight modification of the method of Parry and Walker (10). The reaction mixture contained 60//moles Tris-HCl buffer (pH 8.0), 6 //moles ATP, 12 //moles MgCl,, 0.6 //mole NADP+, 0.4 unit of gIucose-6-P dehydrogenase [EC 1.1.1.49], 60 //moles KC1 and 0.1 or 10 mM glucose for low and high Km hexokinases, respectively, and the enzyme in a final volume of 0.6 ml. The increase in optical density at 340 nm at 25° was recorded in a Shimadzu UV-200 spectrophotometer. For assay of N-acetylglucosamine kinase, the reaction mixture was the same as for glucosamine phosphorylation except that N-acetylglucosamine (10//moles), wa3 used as substrate instead of glucosamine. The N-acetylglucosamine-6-P formed was then separated from free N-acetylglucosamine on a Dowex-1 formate column, as described in the legend of Fig. 1, except that the phosphorylated product was eluted with 3 N formic acid (5 ml). After evaporation, N-acetylglucosamine-6-P was determined by the method of Reissig et al. ( / / ) . The Roman numerals in the figure designate the Types of hexokinase (5).
the report offSaeki, Suzuki, and Tsuiki (9) that these activities are due to different enzymes. The effects of glucose and glucose-6-P on phosphorylation of glucosamine were studied using each of the fractions of hexokinase obtained by DEAE-cellulose chromatography as shown in Table I. Phosphorylation of glucosamine with the Type I, II, and III Vol. 77, No. 5, 1975
enzymes was strongly inhibited by glucose or glucose-6-P. These inhibitors had similar effects on all three low Km hexokinases, except that Type I hexokinase was much the most strongly inhibited by glucose-6-P. In contrast to the low Km hexokinases, Type IV hexokinase, i.e. glucokinase was not inhibited by 5 mM glucose or 1 mM glucose-6-P, as shown in the table. Thus, the phosphoryla-
1120
PRELIMINARY COMMUNICATION"
TABLE I. The effects of glucose and glucose-6-P on glucosamine phosphorylation with hexokinase isozymes from rat liver. Glucosamine phosphorylation by hexokinase Types I, II, III, and IV was measured as described in the legend of Fig. 1. The assays were carried out both in the presence and absence of glucose or glucose-6-P with an incubation time of 20 min and activities in the presence of the inhibitors, are expressed as percentages of those with glucosamine alone. Hexokinase type Substrate
Addition I
II
III
IV
% of control activity 1 mM glucosamine
25 mM glucosamine
5mM glucose 1 mM glucose-6-P 5mM glucose 1 mM glucose-6-P
tion of glucosamine by glucokinase is considerably different from that by the low Km hexokinases. The Michaelis constant of glucokinase for glucosamine was calculated from LineweaverBurk plots to be 8mM, which was much higher than that of hexokinase Type I (0.5 mM). The Km value of glucokinase for glucose was also reported to be about 10 mM (12), whereas the Km value of Type I hexokinase for glucose is 0.05 mM (8), which is about one-tenth of that for glucosamine. Phosphorylation of glucosamine by glucokinase was studied further using preparations obtained by chromatography on a Sephadex G-100 column, hydroxylapatite gel and DEAESephadex A-50 column. On gel filtration on a Sephadex G-100 column the activity for phosphorylation of glucosamine was eluted in the same position as that of glucokinase between bovine serum albumin (mol. wt., 69,000) and ovalbumin (mol. wt., 40,000), whereas Type I hexokinase was eluted soon after the void volume, and its molecular weight has been estimated to be 96,000 (12). Similar results were obtained with chromatography on hydroxylapatite gel and a DEAESephadex column. The activity for phosphorylation of glucosamine was consistently associated with glucokinase activity. However, the enzyme was unstable especially when highly purified. Further purification of the enzyme is now in progress. Glucokinase has unique properties differing
4 5
6
5
43
42
33 2
40 35
41 48
—
104 126
from those of low Km hexokinase. Namely, it has a high Km value for glucose, which approximates to the normal cellular level of glucose and, it is not inhibited by a physiological level of glucose-6-P (13). These characters may explain why phosphorylation of glucosamine is not inhibited by glucose or glucose-6-P. However, it was reported that glucosamine is not phosphorylated by glucokinase but is a competitive inhibitor of glucose phosphorylation (13). The reaction product, glucosamine-6-P, was not determined in the previous work, so it is difficult to compare the previous results with ours. However, the pool of free glucosamine in rat liver is very small compared with the Km value of glucokinase for glucosamine (8 mM), so the physiological significance of the phosphorylation of glucosamine by this enzyme is questionable. The antitumor effect of glucosamine due to ATP depletion has been studied in various experimental hepatomas but the therapeutic effect of glucosamine is still uncertain. Scarcely any glucokinase was found in rapid growing, malignant hepatoma cells, but appreciable activity was detected in certain strains of slow growing hepatoma and Yoshida sarcoma (14). Therefore, the carcinostatic effect of glucosamine may depend on the extent of possible deviation of hexokinase in the neoplastic tissue.
/ . Biochem.
D-GLUCOSAMINE METABOLISM
REFERENCES 1. Quastel, J.H. & Cantero, A. (1953) Nature 171, 252-254 2. Bekesi, J.G. & Winzler, J.R. (1969) / . Biol. Chtm. 244, 5663-5668 3. McGarrahan, J.F. & Maley, F. (1962) / . Biol. Chem. 237, 2458-2465 4. Tesoriere, G., Vento, R., & Cacioppo, F. (1970) Ital. J. Biochem. 19, 40-53 5. Lange, C.J. & Kohn, P. (1961) / . Biol. Chem. 236, 1-5 6. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall. R.J. (1951) / . Biol. Chem. 193, 265-275 7. Lewy, G.A. & McAllan, A. (1959) Biochem. J. 73, 127-132
Vol. 77, No. 5, 1975
1121 8. Grossbard, L. & Schimke, R.T. (1966) / . Biol. Chem. 241, 3546-3560 9. Saeki.H., Suzuki, R., & Tsuiki, S. (1971) Tohoktt J. exp. Med. 105, 87-98 10. Parry, M.J. & Walker, D.G. (1966) Biochem. / . 99, 266-274 11. Reissig, J.L., Strominger, J., & Leloir, L.F. (1955) / . Biol. Chem. 217, 959-966 12. Pilkis, S.J. (1972) Arch. Biochem. Biophys. 149, 349-360 13. Salas, J., Salas, M., Vinuela, E., & Sols, A. (1965) / . Biol. Chem. 240, 1014-1018 14. Sato, S., Matsushima, T., & Sugimura, T. (1969) Cancer Res. 29, 1437-1446
/ . Biochem., 77, 1117-1121 (1975)
Phosphorylation of D-Glucosamine by Rat Liver Glucokinase Michihiko OGUCHI, Yoko MIYATAKE, Junko AYABE, and Nobu AKAMATSU Department of Biochemistry, St. Marianna University School of Medicine, Takatsu-ku, Kawasaki, Kanagawa 213 Received for publication, February 12, 1975
D-Glucosamine was found to be phosphorylated by a rat liver extract in the presence of a high concentration of glucose, which was formerly believed to be a strong competitive inhibitor of this reaction. Results suggested that glucosamine may be phosphorylated by high Km hexokinase, i.e. glucokinase [EC 2. 7.1. 2]. The enzyme involved was separated from specific N-acetyl-D-glucosamine kinase [EC 2. 7.1. 59]. The phosphorylation was not inhibited by a physiological level of glucose or glucose 6-phosphate, which strongly inhibited low Km hexokinase. The apparent Km of glucokinase for glucosamine was estimated as 8 mM, which is ten times that of low Km hexokinase.
Glucosamine is phosphorylated by low Km hexokinase [EC 2. 7.1.1] and it is reported to have a carcinostatic action by causing ATP depletion (7, 2). Glucosamine is metabolized in rat liver and after its administration there is a rapid increase in UDP-N-acetylhexosamine in the liver. However, it has been suggested that it is not phosphorylated, because this is prevented by glucose, which has an inhibition constant (0.1 mM), of almost one-hundredth of the glucose content of liver tissue (3). Nevertheless the possibility that glucosamine is phosphorylated in the liver has been suggested from in vivo and in vitro experiments (4, 5). Thus, phosphorylation of glucosamine was reinvestigated and in the present study, it was demonstrated that the liver contains a kinase which phosphorylates glucosamine even in the presence of the normal cellular level of glucose. Results suggested that the kinase responsible for the phosphorylation is hepatic glucokinase [EC 2.7.1.2]. Vol. 77, No. 5, 1975
Livers were obtained from male albino Wistar strain rats weighing 100—150 g and homogenized with 9 volumes of 0.25 M sucrose containing 1 mM EDTA (pH 7.0) in a PotterElvehjem homogenizer. All procedures were performed at 0—4°C. The crude homogenate was centrifuged at 105,000 Xg for 1 hr. The resulting supernatant was then passed through a Sephadex G-25 column (1.0x20 cm) using buffer consisting of 10 mM potassium phosphate, 1 mM EDTA, and 0.5 mM dithiothreitol, at pH 7.0. The preparation was used immediately after elution. For enzyme purification, the livers (34 g) were homogenized with two volumes of 0.25 M sucrose containing 1 mM EDTA, and then centrifuged for 40 min at 30,000 x g. The resulting supernatant was then brought to 30% saturation (17.6 g/dl) of ammonium sulfate by addition of solid ammonium sulfate. The supernatant obtained by centrifugation at 10,000Xg for 30 min was then brought to 70% ammonium sulfate
1117
1118
PRELIMINARY COMMUNICATION
saturation (27.3 g/dl). The resulting precipitate was collected by centrifugation and dissolved in a small amount of potassium phosphate buffer as described above. The enzyme solution was desalted using a Sephadex G-25 column (3.0x40 cm) and applied to a DEAEcellulose column. Subsequent procedures are specified later. Protein was measured by the method of Lowry et al. (6), using crystalline bovine serum albumin as a standard. Phosphorylation of glucosamine was carried out with the high speed supernatant, which contained more than 80% of the a 25mM GlcN
a.0.20
10
20 30 ScrnM
A.1mM GlcN
So.i-
control
0 10 Time (mln)
20
30
Fig. 1. Effects of glucose and glucose-6-P on glucosamine phosphorylation. The reaction mixture for assay of glucosamine phosphorylation was as follows; O.lmmole Tris-HCl buffer (pH 8.0), 10//moles ATP, 20 //moles MgCl,, 5 //moles EDTA (pH 7.0), 0.5 //mole dithiothreitol, 10 //moles NaF, 0.1 mmole KC1, various concentrations of D-glucosamine and enzyme solution (1.4 mg as protein) in a total volume of 1.0 ml. The reaction was carried out at 37° and then stopped by boiling the mixture for 2min. After diluting the mixture with 4 ml of water, the denaturated protein was removed by centrifugation. The resulting supernatant was applied to a Dowex-1 formate (200-400 mesh) column (0.6x3.5 cm). Glucosamine was washed out with 10 ml of water, and then glucosamine-6-P was eluted with 5 ml of 0.01 M formic acid. The eluate was evaporated to dryness in vacuo at 30°, and the residue was dissolved in 0.6 ml of water, and assayed for glucosamine-6-P by the method of Lewy and McAllan (7). A and B; 1 and 25 mM glucosamine, respectively. C; inhibitory effects of various concentrations of glucose. The incubation time was 20 min.
activity. Figure 1 shows results on the phosphorylation of glucosamine and the effects of intracellular levels of glucose and glucose-6-P.1 The reaction proceeded linearly for 30 min. Using 1 mM glucosamine, the activity was inhibited 60% by 5 mM glucose or 1 mM glucose6-P, as shown in Fig. 1A, while with 25 mM glucosamine these concentrations caused 30% inhibition (Fig. IB). Figure 1C shows the percentage inhibitions with various concentrations of glucose: inhibitions of less than 85 and 35% were noted with 1 and 25 mM glucosamine, respectively, even when the glucose concentration was increased to 40 mM. The results suggest that even in the presence of a physiological level of glucose, glucosamine is phosphorylated in the liver to yield as much as 10 //moles of glucosamine-6-P per hour per gram wet liver weight with 25 mM glucosamine as substrate. Rat liver contains various isozymes of hexokinase, so it seemed interesting to study whether phosphorylation of glucosamine was preferentially catalyzed by one of these isozymes. The typical pattern of liver hexokinase on DEAE-cellulose column chromatography is illustrated in Fig. 2. When glucosamine was used as substrate, the activity was found in 4 major peaks. Glucose phosphorylation was assayed with concentrations of 0.1 and 10 mM glucose as substrate for low Km hexokinase and glucokinase, respectively. The low Km hexokinase was separated into 3 peaks, which were designated as Types I, II, and III in order of elution by an increasing concentration of KC1 (8). It can be seen that the first 3 peaks of activity for phosphorylation of glucosamine, in order from the left, corresponded to peaks of low Km hexokinase. However, the fourth paak did not correspond to low Km hexokinase but to high Km hexokinase, i.e. glucokinase. The activity of Nacetylglucosamine kinase [EC 2.7.1.59] is also shown in the figure. The enzyme was eluted after glucokinase, although in largely overlapped the latter. The difference between the peaks of activity for phosphorylation of glucosamine and N-acetylglucosamine confirms 1
Abbreviation: glucose-6-P, D-glucose 6-phosphate. / . Biochem.
D-GLUCOSAMINE METABOLISM
1119
,,300
30
0.1 mM Glc 10 mM Glc K)mM GlcN 10mM GlcNAc
I c
1
"c E
uO
h 10 LJf5
S
75
1
c
30
A0
50 60 70 Fraction Number
80
90
Fig. 2. ' DEAE-cellulose column chromatography of hexokinases from rat liver. The enzyme obtained as described in the text was adsorbed on a column (2.5x40cm) equilibrated with 10 mM potassium phosphate, 5mM EDTA (pH 7.0), and 0.5 mM dithiothreitol, pH 7.0. An enzyme concentration of 1.19 g of protein in a volume of 70 ml was used, following the procedure of Grossbard and Schimke (fl). Enzyme was eluted with a linear gradient of KC1 prepared by placing 500 ml of the above phosphate buffer in the mixing chamber and 500 ml of 0.6 M KC1 in the same buffer in the reservoir chamber. Fractions of 10 ml were collected. Phosphorylation of glucosamine was assayed as noted in the legend of Fig. 1, except that 10 mM glucosamine was used. Glucose phosphorylation was assayed by a slight modification of the method of Parry and Walker (10). The reaction mixture contained 60//moles Tris-HCl buffer (pH 8.0), 6 //moles ATP, 12 //moles MgCl,, 0.6 //mole NADP+, 0.4 unit of gIucose-6-P dehydrogenase [EC 1.1.1.49], 60 //moles KC1 and 0.1 or 10 mM glucose for low and high Km hexokinases, respectively, and the enzyme in a final volume of 0.6 ml. The increase in optical density at 340 nm at 25° was recorded in a Shimadzu UV-200 spectrophotometer. For assay of N-acetylglucosamine kinase, the reaction mixture was the same as for glucosamine phosphorylation except that N-acetylglucosamine (10//moles), wa3 used as substrate instead of glucosamine. The N-acetylglucosamine-6-P formed was then separated from free N-acetylglucosamine on a Dowex-1 formate column, as described in the legend of Fig. 1, except that the phosphorylated product was eluted with 3 N formic acid (5 ml). After evaporation, N-acetylglucosamine-6-P was determined by the method of Reissig et al. ( / / ) . The Roman numerals in the figure designate the Types of hexokinase (5).
the report offSaeki, Suzuki, and Tsuiki (9) that these activities are due to different enzymes. The effects of glucose and glucose-6-P on phosphorylation of glucosamine were studied using each of the fractions of hexokinase obtained by DEAE-cellulose chromatography as shown in Table I. Phosphorylation of glucosamine with the Type I, II, and III Vol. 77, No. 5, 1975
enzymes was strongly inhibited by glucose or glucose-6-P. These inhibitors had similar effects on all three low Km hexokinases, except that Type I hexokinase was much the most strongly inhibited by glucose-6-P. In contrast to the low Km hexokinases, Type IV hexokinase, i.e. glucokinase was not inhibited by 5 mM glucose or 1 mM glucose-6-P, as shown in the table. Thus, the phosphoryla-
1120
PRELIMINARY COMMUNICATION"
TABLE I. The effects of glucose and glucose-6-P on glucosamine phosphorylation with hexokinase isozymes from rat liver. Glucosamine phosphorylation by hexokinase Types I, II, III, and IV was measured as described in the legend of Fig. 1. The assays were carried out both in the presence and absence of glucose or glucose-6-P with an incubation time of 20 min and activities in the presence of the inhibitors, are expressed as percentages of those with glucosamine alone. Hexokinase type Substrate
Addition I
II
III
IV
% of control activity 1 mM glucosamine
25 mM glucosamine
5mM glucose 1 mM glucose-6-P 5mM glucose 1 mM glucose-6-P
tion of glucosamine by glucokinase is considerably different from that by the low Km hexokinases. The Michaelis constant of glucokinase for glucosamine was calculated from LineweaverBurk plots to be 8mM, which was much higher than that of hexokinase Type I (0.5 mM). The Km value of glucokinase for glucose was also reported to be about 10 mM (12), whereas the Km value of Type I hexokinase for glucose is 0.05 mM (8), which is about one-tenth of that for glucosamine. Phosphorylation of glucosamine by glucokinase was studied further using preparations obtained by chromatography on a Sephadex G-100 column, hydroxylapatite gel and DEAESephadex A-50 column. On gel filtration on a Sephadex G-100 column the activity for phosphorylation of glucosamine was eluted in the same position as that of glucokinase between bovine serum albumin (mol. wt., 69,000) and ovalbumin (mol. wt., 40,000), whereas Type I hexokinase was eluted soon after the void volume, and its molecular weight has been estimated to be 96,000 (12). Similar results were obtained with chromatography on hydroxylapatite gel and a DEAESephadex column. The activity for phosphorylation of glucosamine was consistently associated with glucokinase activity. However, the enzyme was unstable especially when highly purified. Further purification of the enzyme is now in progress. Glucokinase has unique properties differing
4 5
6
5
43
42
33 2
40 35
41 48
—
104 126
from those of low Km hexokinase. Namely, it has a high Km value for glucose, which approximates to the normal cellular level of glucose and, it is not inhibited by a physiological level of glucose-6-P (13). These characters may explain why phosphorylation of glucosamine is not inhibited by glucose or glucose-6-P. However, it was reported that glucosamine is not phosphorylated by glucokinase but is a competitive inhibitor of glucose phosphorylation (13). The reaction product, glucosamine-6-P, was not determined in the previous work, so it is difficult to compare the previous results with ours. However, the pool of free glucosamine in rat liver is very small compared with the Km value of glucokinase for glucosamine (8 mM), so the physiological significance of the phosphorylation of glucosamine by this enzyme is questionable. The antitumor effect of glucosamine due to ATP depletion has been studied in various experimental hepatomas but the therapeutic effect of glucosamine is still uncertain. Scarcely any glucokinase was found in rapid growing, malignant hepatoma cells, but appreciable activity was detected in certain strains of slow growing hepatoma and Yoshida sarcoma (14). Therefore, the carcinostatic effect of glucosamine may depend on the extent of possible deviation of hexokinase in the neoplastic tissue.
/ . Biochem.
D-GLUCOSAMINE METABOLISM
REFERENCES 1. Quastel, J.H. & Cantero, A. (1953) Nature 171, 252-254 2. Bekesi, J.G. & Winzler, J.R. (1969) / . Biol. Chtm. 244, 5663-5668 3. McGarrahan, J.F. & Maley, F. (1962) / . Biol. Chem. 237, 2458-2465 4. Tesoriere, G., Vento, R., & Cacioppo, F. (1970) Ital. J. Biochem. 19, 40-53 5. Lange, C.J. & Kohn, P. (1961) / . Biol. Chem. 236, 1-5 6. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall. R.J. (1951) / . Biol. Chem. 193, 265-275 7. Lewy, G.A. & McAllan, A. (1959) Biochem. J. 73, 127-132
Vol. 77, No. 5, 1975
1121 8. Grossbard, L. & Schimke, R.T. (1966) / . Biol. Chem. 241, 3546-3560 9. Saeki.H., Suzuki, R., & Tsuiki, S. (1971) Tohoktt J. exp. Med. 105, 87-98 10. Parry, M.J. & Walker, D.G. (1966) Biochem. / . 99, 266-274 11. Reissig, J.L., Strominger, J., & Leloir, L.F. (1955) / . Biol. Chem. 217, 959-966 12. Pilkis, S.J. (1972) Arch. Biochem. Biophys. 149, 349-360 13. Salas, J., Salas, M., Vinuela, E., & Sols, A. (1965) / . Biol. Chem. 240, 1014-1018 14. Sato, S., Matsushima, T., & Sugimura, T. (1969) Cancer Res. 29, 1437-1446
