[8]
GALACTOKINASE FROM HUMAN ERYTHROCYTES
47
Kinetic Constants. Measurements of initial velocity in the presence or in the absence of reaction products have been used to propose an ordered reaction mechanism for galactokinase4 in which galactose combines with an initial enzyme-MgATP 2- complex followed by the release of galactose 1-phosphate and then MgADP-. Michaelis constants at pH 7.8 are galactose, 0.58 mM, and MgATP 2-, 0.17 mM. Substrate inhibition occurs with galactose concentrations above 1 mM while free Mg 2+ ion is a competitive inhibitor of MgATP 2- with a Ki of 25 raM. Of the reaction products, ADP'-'- (or MgADP-) is a competitive inhibitor of MgATP z- with a Ki of 0.2 raM, and a noncompetitive inhibitor of galactose, while galactose 1-phosphate is a noncompetitive inhibitor with respect to either galactose or MgATP z-. Other Properties. Galactokinase has a pH optimum between 7.5 and 8.0 in Tris or triethanolamine buffers.4 Enzyme activity is stimulated 2- to 4-fold by 5 mM cysteine, glutathione, fl-mercaptoethanol, or DTT and is inhibited by p-chloromercuribenzoate.
[8] Galactokinase from Human Erythrocytes By
KARL-GEORG BLUME a n d ERNEST BEUTLER
a-D-Galactose -I- ATP --* a-D-galactose 1-phosphate A- ADP
As in other tissues from other mammals and in microorganisms, human red cell galactokinase (ATP:u-galactose-l-phosphotransferase, EC 2.7.1.6) catalyzes the first step in the conversion of galactose to glucose: the phosphorylation by ATP of a-D-galactose to a-D-galactose 1-phosphate. In human tissues the galactokinase reaction is unidirectional toward galactose phosphorylation.
Clinical Significance The activity of the enzyme of normal human red cells is high at birth and decreases so that by the age of approximately 4 years it has reached the adult value, approximately one-third of that observed in the newborn. 1 Kinetic alteration of human galactokinase during maturation have been describedJ ,3 and a fetal enzyme has been separated chromatographically from adult enzyme2 Induction of the enzyme in cultured human 1 W. G. Ng, G. N. Donnell, and W. R. Bergren, J. Lab. Clin. Med. 66, 115 (1965). C. K. Mathai and E. Beutler, Enzymologia 33, 224 (1967). ~S. K. Srivastava, K. G. Blume, C. van Loon, and E. Beutler, Arch. Biochem. Biophys. 150, 191 (1972).
48
KINASES
[8]
amniotic cells 4 and fibroblasts 5 has been achieved by the addition of galactose to the growth medium. However, galactose intake does not influence the level of enzyme in human red cells. 6 Blood samples containing a high percentage of reticulocytes have somewhat higher levels of enzyme than the blood of normal subjects. 6 Hereditary deficiency of galactokinase was first discovered in 1965. 7 So far, almost 20 patients with this inborn error of metabolism have since been identified. The disease is transmitted as an autosomal recessive disorder: homozygotes or doubly mutant individuals have a marked galactokinase deficiency, whereas heterozygotes are partially deficient. The gene frequency for galactokinase deficiency is not accurately known, but a survey suggested that as many as 1% of the population studied might be heterozygous, s The disorder is characterized by galactosemia, galactosuria, and cataract formation in early childhood. I t has been suggested that heterozygotes for galactokinase deficiency may also be at risk for cataract formation, but clear-cut evidence for this has not been obtained2 A 30% decrease in the average enzyme activity has been found in Negro populations. 1° This difference in galactokinase activity implies the existence of racially determined enzyme polymorphism. The diagnosis of galactokinase deficiency can be established by measuring the enzyme activity in hemolysates 7 or lysates from cultured human amniotic cells 4 or fibroblasts2 Since normal human galactokinase exhibits rather low specific activity spectrophotometric methods are not satisfactory. However, the use of radioactively labeled galactose permits accurate measurement of galactokinase in human tissue extracts as well" as in hemolysates2
Assay Method P r i n c i p l e . The procedure is a modification 6 of a method which has been described for the determination of galactokinase from E s c h e r i c h i a coli. 11 It depends upon the.incubation of [14C]galactose with A T P and the enzyme. The reaction is stopped by the addition of a large excess of nonradioactive galactose. The product, [14C]galactose 1-phosphate, is
P. F. Benson, S. Blunt, and S. P. Brown, Lancet 1, 106 (1973).
' F. Zacchello, P. F. Benson, S. Brown, P. Croll, F. Gianelli, and T. P. Mann, Nature (London) New Biol. 239, 95 (1972). 6E. Beutler, N. V. Paniker, and F. Trinidad, Biochem. Med. 5, 325 (1971). 7R. Gitzelmann, Lancet 2, 670 (1965). s j. S. Mayes and R. Guthrie, Biochem. Genet. 2, 219 (1968). E. Beutler, F. Matsumoto, W, Kuhl, A. Krill, N. Levy, R. Sparkes, and M. Degnan, N. Engl. J. Med. 288, 1203 (1973). 10T. A. Tedesco, R. Bonow, K. Miller, and W. J. Mellman, Science 178, 176 (1972). 1~j. R. Sherman and J. Adler, J. Biol. Chem. 238, 873 (1963).
[8]
GALACTOKINASE FROM HUMAN ERYTHROCYTES
49
measured as the radioactivity remaining on ion exchange paper after elution of nonphosphorylated galactose from the paper with water. Preparation o/Substrate. Since many commercially available preparations of [l~C]galactose are contaminated with labeled hexoses other than galactose that would cause errors, it is necessary to purify the substrate for galactokinase reaction. The contaminants are phosphorylated by hexokinase and ATP and are then removed on DEAE-cellulose. A small column is prepared by placing a small quantity of glass wool into the outlet of a l-ml syringe. Microgranular DEAE (Whatman DE 52) slurry to give a volume of 0.2-0.3 ml is placed into the barrel. To decrease nonspecific absorption of galactose the column is washed with 0.5 ml of 3.8 mM galactose solution followed by 5 ml of water. [1-~4C]Galactose or [U-l~C]galaetose is diluted to contain approximately 10 t~Ci/ml. Two microliters of 1 M Tris'HC1 buffer pH 8.0, 5 ~l of 0.1 M MgC1, 100 td of hexokinase diluted in water to contain 2 units/ml, and 10 ~l of 60 mM neutralized ATP are added to each milliliter of the [1-~4C]galacrose solution. After 60 min at 37 °, the reaction mixture is passed through the column, which is then washed with sufficient additional water to give five times the original volume. The radioactive galactose solution which is eluted from the column contains approximately 2 t~Ci/ml and is stable when frozen. Preparation o] Reaction Mixture. A partial reaction mixture is prepared by mixing the following reagents in the proportions shown: 1 M Tris" HC1, pH 7.4, 400 td; 100 mM NaF, 100 td; 100 mM MgCl~, 100 td; 7.6 mM galactose, 100 ~l; 2 t~Ci/ml [1-14C]galactose, 200 ~l; 120 mM ATP, 100 ~l. The mixture is incubated at 37 ° for 4 hr and then stored in the frozen state. It is particularly important that freshly prepared galactose solutions not be used in the assay. An equilibrium between a- and B-form of galactose which is the result of mutarotation requires incubation for 4 hr at 37 ° or boiling for 10 min. Preparation of Hemolysates. Blood samples drawn into ACD, 1'-' heparin or EDTA are washed two times in 0.9% saline solution and then hemolysed by the addition of 4 volumes of stabilizing solution containing 7 mM fl-mercaptoethanol and 2.7 mM neutralized EDTA, to 1 volume of packed red cells. Stroma is removed by centrifugation at 5000 g and 4 ° for 15 rain. The hemoglobin content of the supernatant is measured, and galactokinase activity is determined. Uncentrifuged hemolysates or even whole blood with a modified reaction mixture 13 may be used with 1--Acid-citrate-dextrose solution containing 7.3 g of citric acid, 22 g of sodium citrate - 2H20 and 24.5 g of glucose per liter. ~ E. Beutler and F. Matsumoto, J. Lab. Clin. Med. 82, 818 (1973).
50
KINASES
[8]
the same results2 Leukocyte contamination of red cell preparations does not significantly influence the result? Assay Procedure. Hemolysate, 100 ~l, is mixed with 100 ~l of the partial reaction mixture described above. Two 50-t~l aliquots of the reaction mixture are removed immediately, and each is mixed on a spot plate or in a small test tube with 20 ~l of 1 M galactose. Then 50 ~l of each mixture are spotted on a circle of D E A E paper ( W h a t m a n DE-81) 15-20 m m in diameter. One of the spotted papers is dropped immediately into a small cup of water (0 min sample), while the other is permitted to dry (100% sample). The remainder of the reaction mixture is now incubated for 1 hr at 37 °, and another 50-~1 aliquot (60-min sample) is treated in a fashion identical with the 0-min sample. The 0-rain sample and the 60-rain sample are placed, while wet, on a sintered-glass funnel
Reaction mixture: Tris • HC1, 1 M, pH 7.4, 400 ~l NaF, 100 raM, 100 ul MgCl~, 100 mM, 100 ~l Galactose, 7.6 mM, 100 td [1-14C] Galactose, 2 t~Ci/ml, 200 td ATP, 120 mM, 100 td 100 ul
1:4 Hemoiysate I
100 ul j 200 ~1
l
50 ul +20~1 1 M G a l SPOT 50 ~1
DRY
50 ul -t-20td 1 M G a l SPOT 50 td
60-Min sample 50 td +20td 1 M G a l SPOT 50 ~1
WASH
WASH
DRY
DRY
Counting solution: 0.3 g POPOP + 5.0 g PPO/1 liter toluene COUNT AND CALCULATE Fro. 1. The determination of erythrocyte galactokinase using [14C]galactose: Schematic summary [E. Beutler, N. V. Paniker, and F. Trinidad, Biochem. Med. 5, 325 (1971)].
[8]
GALACTOKINASE FROM HUMAN ERYTHROCYTES
51
and washed with 600~00 ml of distilled water. The samples are permitted to dry and are ready for counting; this is accomplished in a scintillation solution consisting of toluene containing 0.03% p-bis-2-(5-phenyloxazolyl)benzene (POPOP) and 0.5% 2,5-diphenyloxazole (PPO). The procedure is summarized in Fig. 1. Linearity. When various dilutions of normal hemolysates are made the percentage of galactose phosphorylated increases in an approximately linear fashion.6 When fairly concentrated hemolysates are used, a considerable degree of quenching is observed: there is approximately 55% quenching with a 50% hemolysate, 20% quenching with a 25% hemolysate, and little or no quenching with a 12% hemolysate. Drying samples at too high temperature produces an increase in the amount of quenching because of the change in color of hemoglobin under these conditions. The phosphorylation of galactose is linear with time over a 2.5-hr period. 6 Definition o/ Unit and Specific Activity. One unit of galactokinase is defined as the amount of enzyme which phosphorylates 1 umole of galactose per minute under the conditions of assay. The red cells of normal human adults have been found to contain 0.0297 ± 0.0049 unit of galactokinase per gram of hemoglobin (mean ± standard deviation). Some differences in apparent activity are observed with different batches of [14C]galactose, probably because of varying degrees of contamination with the keto sugar (tagatose) or the 2-epimer (talose). Purification
Procedure
~4
All procedures are carried out at 4 °. All centrifugations are performed for 20 min at 37,500 g. The following buffers are used during the purification procedure. Buffer A: l0 mM KH2PO4-K2HP04, 7 mM fl-mercaptoethanol, 0.5 mM Na~-EDTA, final pH 7.2; buffer B : 100 mM KH2PO4K2HP04, 7 mM fl-mercaptoethanol, 0.5 mM Na-EDTA, final pH 7.2; buffer C: l0 mM KH2HPO/KoHPO,, 7 mM fl-mercaptoethanol, 0.5 mM Na2-EDTA, final pH 6.7. Preparation of Ilemolysate. One-hundred milliliters of washed red cells are mixed with 400 ml of buffer A and frozen and thawed twice. After ccntrifugation the sediment is discarded. DEAE-Sephadex Chromatography. The hemolyzate is applied to a DEAE-Sephadex A-50 column (2.5 X 35 cm) equilibrated with buffer A. Hemoglobin is eluted with 3.5 liters of buffer A. Galactokinase is then eluted by descending chromatography using a 500-ml linear gradient between buffer A and buffer B at a flow rate of 30 ml/hr. Fractions containing galaetokinase are pooled. 14 K. G. B l u m e and E. Beutler, J. Biol. Chem. 246, 6507 (1971).
52
KINASES
[8]
PURIFICATION PROCEDURE FOR HUMAN RED CELL GALACTOKINASE
Fraction Hemolysate DEAE-Sephadex A 50 eluate Ammonium sulfate, 35-60% precipitate Sephadex G-75 filtrate CM-Sephadex C-50 eluate
Volume (ml) 500 66 1.8 16 18
Specific activity (mU/mg)
Purification (fold)
47.5 1.42
0. 021 4.30
1 204
19.2
9.29
440
Protein (mg/ml)
0.41 0.06
28.2 81.2
1336 3850
Ammonium Sulfate. The DEAE-Sephadex A-50 eluate is fractionated with solid (NH4)2S0,. The precipitate which forms at 35% saturation (20.9 g/100 ml) is discarded. Galactokinase is precipitated between 35 and 60% (16.4 g/100 ml) ammonium sulfate saturation and is redissolved in a small amount of buffer A. Sephadex G-75 Filtration. The ammonium sulfate 35 to 60% precipitate is then ehromatographed through a Sephadex G-75 column (1.5 }( 90 cm), equilibrated with buffer A using ascending chromatography with a flow rate of 8.0 ml/hour. Fractions containing galactokinase are combined. CM-Sephadex Chromatography. The Sephadex G-75 filtrate is passed through a CM-Sephadex C-50 column (1.5 X 30 cm), equilibrated with buffer C, with descending chromatography at a flow rate of 4.2 ml/hr. Whereas contaminating proteins were absorbed, galactokinase is not bound to the cation exchanger. Fractions containing galactokinase are pooled and concentrated by ultrafiltration or by ammonium sulfate precipitation at 60% saturation. The cumulative purification ranges between 3300 and 3900-fold, the yield between 14 and 19%. The table summarizes a typical enzyme preparation including all steps described. Purification of fetal galactokinase from human placenta can be achieved by means of a similar method. 3 Properties
Purity. By analytical disc electrophoresis in polyacrylamide (20 and 50 /zg protein per gel column) a single protein band is found with the purified enzyme when stained with Coomassie blue. Of the enzymes of red cell glyeolysis, hexose monophosphate shunt, glutathione and ga]ac-
[9]
N-ACETYLMANNOSAMINE KINASE
53
rose metabolism only traces of triosephosphate isomerase activity (representing less than 0.1% of the specific activity of triosephosphate isomerase in crude hemolysates) are present in the final preparation. Stability. Intact red cells lose less than 5% of galactokinase activity when stored in acid-citrate-dextrose at 4 ° for 3 weeks2 When glucose to give a final added concentration of about 20 mM is added to blood in EDTA solution, the enzyme is stable for at least 20 days at 4 °, 6 days at 25 °, and 4 days at 30 °. In hemolysates the enzyme is unstable, the activity decreases about 30% in 24 hr. After purification the enzyme preparations lose about 10% of activity within 6 days when stored at 4 ° in 3.0 M ammonium sulfate. Other Properties. The Km of galactokinase preparations from red cells for galactose is 126~-11 ~M ( m e a n ± standard deviation), for ATP 342 + 63 ~M. Identical results have been obtained for galactokinase from crude hemoylsates from adult red cells. Km values of galactokinase from cord blood red cell hemolysates and after partial purification are significantly higher for galactose: 225 +_ 8 ~M. 2 Similar results have been obtained with extracts and purified preparations of galactokinase from human placenta, 3 indicating different kinetic properties of human fetal and adult galactokinase. Both forms of the enzyme can be separated by ion exchange chromatography2 The pH optimum for the enzymic phosphorylation of galactose is pH 7.6 in hemolysates and pH 7.8 after purification. The molecular weight of galactokinase determined by Sephadex G-75 and Sephadex G-200 chromatography is 55,000 in crude extracts and after purification. By sodium dodecyl sulfate-polyacrylamide electrophoresis of 3600- to 3850-fold purified preparations and staining with Coomassie blue the molecular weight of the assumed monomer of red cell galactokinase is found to be 26,000. The isoelectric point of the enzyme determined by isoelectrofocusing in a pH 5-8 stabilized sucrose gradient is pH 5.7. The ultraviolet spectrum of purified galactokinase exhibits a minimum at 251 nm and a maximum at 280 nm.
[9] N-Acetylmannosamine Kinase By SANTIMOY BANERJEE a n d SUDHAMOY GHOSH N-Acetylmannosamine + ATP --~ N-acetylmannosamine-6-P + ADP
N-Acetylmannosamine kinase has been previously purified from rat liver. 1,: The enzyme does not appear to be synthesized in bacteria if they 1 W. Kundig, S. Ghosh, and S. Roseman, J. Biol. Chem. 241, 5619 (1966). 2 W. Kundig and S. Roseman, this series, Vol. 8 [28].
GALACTOKINASE FROM HUMAN ERYTHROCYTES
47
Kinetic Constants. Measurements of initial velocity in the presence or in the absence of reaction products have been used to propose an ordered reaction mechanism for galactokinase4 in which galactose combines with an initial enzyme-MgATP 2- complex followed by the release of galactose 1-phosphate and then MgADP-. Michaelis constants at pH 7.8 are galactose, 0.58 mM, and MgATP 2-, 0.17 mM. Substrate inhibition occurs with galactose concentrations above 1 mM while free Mg 2+ ion is a competitive inhibitor of MgATP 2- with a Ki of 25 raM. Of the reaction products, ADP'-'- (or MgADP-) is a competitive inhibitor of MgATP z- with a Ki of 0.2 raM, and a noncompetitive inhibitor of galactose, while galactose 1-phosphate is a noncompetitive inhibitor with respect to either galactose or MgATP z-. Other Properties. Galactokinase has a pH optimum between 7.5 and 8.0 in Tris or triethanolamine buffers.4 Enzyme activity is stimulated 2- to 4-fold by 5 mM cysteine, glutathione, fl-mercaptoethanol, or DTT and is inhibited by p-chloromercuribenzoate.
[8] Galactokinase from Human Erythrocytes By
KARL-GEORG BLUME a n d ERNEST BEUTLER
a-D-Galactose -I- ATP --* a-D-galactose 1-phosphate A- ADP
As in other tissues from other mammals and in microorganisms, human red cell galactokinase (ATP:u-galactose-l-phosphotransferase, EC 2.7.1.6) catalyzes the first step in the conversion of galactose to glucose: the phosphorylation by ATP of a-D-galactose to a-D-galactose 1-phosphate. In human tissues the galactokinase reaction is unidirectional toward galactose phosphorylation.
Clinical Significance The activity of the enzyme of normal human red cells is high at birth and decreases so that by the age of approximately 4 years it has reached the adult value, approximately one-third of that observed in the newborn. 1 Kinetic alteration of human galactokinase during maturation have been describedJ ,3 and a fetal enzyme has been separated chromatographically from adult enzyme2 Induction of the enzyme in cultured human 1 W. G. Ng, G. N. Donnell, and W. R. Bergren, J. Lab. Clin. Med. 66, 115 (1965). C. K. Mathai and E. Beutler, Enzymologia 33, 224 (1967). ~S. K. Srivastava, K. G. Blume, C. van Loon, and E. Beutler, Arch. Biochem. Biophys. 150, 191 (1972).
48
KINASES
[8]
amniotic cells 4 and fibroblasts 5 has been achieved by the addition of galactose to the growth medium. However, galactose intake does not influence the level of enzyme in human red cells. 6 Blood samples containing a high percentage of reticulocytes have somewhat higher levels of enzyme than the blood of normal subjects. 6 Hereditary deficiency of galactokinase was first discovered in 1965. 7 So far, almost 20 patients with this inborn error of metabolism have since been identified. The disease is transmitted as an autosomal recessive disorder: homozygotes or doubly mutant individuals have a marked galactokinase deficiency, whereas heterozygotes are partially deficient. The gene frequency for galactokinase deficiency is not accurately known, but a survey suggested that as many as 1% of the population studied might be heterozygous, s The disorder is characterized by galactosemia, galactosuria, and cataract formation in early childhood. I t has been suggested that heterozygotes for galactokinase deficiency may also be at risk for cataract formation, but clear-cut evidence for this has not been obtained2 A 30% decrease in the average enzyme activity has been found in Negro populations. 1° This difference in galactokinase activity implies the existence of racially determined enzyme polymorphism. The diagnosis of galactokinase deficiency can be established by measuring the enzyme activity in hemolysates 7 or lysates from cultured human amniotic cells 4 or fibroblasts2 Since normal human galactokinase exhibits rather low specific activity spectrophotometric methods are not satisfactory. However, the use of radioactively labeled galactose permits accurate measurement of galactokinase in human tissue extracts as well" as in hemolysates2
Assay Method P r i n c i p l e . The procedure is a modification 6 of a method which has been described for the determination of galactokinase from E s c h e r i c h i a coli. 11 It depends upon the.incubation of [14C]galactose with A T P and the enzyme. The reaction is stopped by the addition of a large excess of nonradioactive galactose. The product, [14C]galactose 1-phosphate, is
P. F. Benson, S. Blunt, and S. P. Brown, Lancet 1, 106 (1973).
' F. Zacchello, P. F. Benson, S. Brown, P. Croll, F. Gianelli, and T. P. Mann, Nature (London) New Biol. 239, 95 (1972). 6E. Beutler, N. V. Paniker, and F. Trinidad, Biochem. Med. 5, 325 (1971). 7R. Gitzelmann, Lancet 2, 670 (1965). s j. S. Mayes and R. Guthrie, Biochem. Genet. 2, 219 (1968). E. Beutler, F. Matsumoto, W, Kuhl, A. Krill, N. Levy, R. Sparkes, and M. Degnan, N. Engl. J. Med. 288, 1203 (1973). 10T. A. Tedesco, R. Bonow, K. Miller, and W. J. Mellman, Science 178, 176 (1972). 1~j. R. Sherman and J. Adler, J. Biol. Chem. 238, 873 (1963).
[8]
GALACTOKINASE FROM HUMAN ERYTHROCYTES
49
measured as the radioactivity remaining on ion exchange paper after elution of nonphosphorylated galactose from the paper with water. Preparation o/Substrate. Since many commercially available preparations of [l~C]galactose are contaminated with labeled hexoses other than galactose that would cause errors, it is necessary to purify the substrate for galactokinase reaction. The contaminants are phosphorylated by hexokinase and ATP and are then removed on DEAE-cellulose. A small column is prepared by placing a small quantity of glass wool into the outlet of a l-ml syringe. Microgranular DEAE (Whatman DE 52) slurry to give a volume of 0.2-0.3 ml is placed into the barrel. To decrease nonspecific absorption of galactose the column is washed with 0.5 ml of 3.8 mM galactose solution followed by 5 ml of water. [1-~4C]Galactose or [U-l~C]galaetose is diluted to contain approximately 10 t~Ci/ml. Two microliters of 1 M Tris'HC1 buffer pH 8.0, 5 ~l of 0.1 M MgC1, 100 td of hexokinase diluted in water to contain 2 units/ml, and 10 ~l of 60 mM neutralized ATP are added to each milliliter of the [1-~4C]galacrose solution. After 60 min at 37 °, the reaction mixture is passed through the column, which is then washed with sufficient additional water to give five times the original volume. The radioactive galactose solution which is eluted from the column contains approximately 2 t~Ci/ml and is stable when frozen. Preparation o] Reaction Mixture. A partial reaction mixture is prepared by mixing the following reagents in the proportions shown: 1 M Tris" HC1, pH 7.4, 400 td; 100 mM NaF, 100 td; 100 mM MgCl~, 100 td; 7.6 mM galactose, 100 ~l; 2 t~Ci/ml [1-14C]galactose, 200 ~l; 120 mM ATP, 100 ~l. The mixture is incubated at 37 ° for 4 hr and then stored in the frozen state. It is particularly important that freshly prepared galactose solutions not be used in the assay. An equilibrium between a- and B-form of galactose which is the result of mutarotation requires incubation for 4 hr at 37 ° or boiling for 10 min. Preparation of Hemolysates. Blood samples drawn into ACD, 1'-' heparin or EDTA are washed two times in 0.9% saline solution and then hemolysed by the addition of 4 volumes of stabilizing solution containing 7 mM fl-mercaptoethanol and 2.7 mM neutralized EDTA, to 1 volume of packed red cells. Stroma is removed by centrifugation at 5000 g and 4 ° for 15 rain. The hemoglobin content of the supernatant is measured, and galactokinase activity is determined. Uncentrifuged hemolysates or even whole blood with a modified reaction mixture 13 may be used with 1--Acid-citrate-dextrose solution containing 7.3 g of citric acid, 22 g of sodium citrate - 2H20 and 24.5 g of glucose per liter. ~ E. Beutler and F. Matsumoto, J. Lab. Clin. Med. 82, 818 (1973).
50
KINASES
[8]
the same results2 Leukocyte contamination of red cell preparations does not significantly influence the result? Assay Procedure. Hemolysate, 100 ~l, is mixed with 100 ~l of the partial reaction mixture described above. Two 50-t~l aliquots of the reaction mixture are removed immediately, and each is mixed on a spot plate or in a small test tube with 20 ~l of 1 M galactose. Then 50 ~l of each mixture are spotted on a circle of D E A E paper ( W h a t m a n DE-81) 15-20 m m in diameter. One of the spotted papers is dropped immediately into a small cup of water (0 min sample), while the other is permitted to dry (100% sample). The remainder of the reaction mixture is now incubated for 1 hr at 37 °, and another 50-~1 aliquot (60-min sample) is treated in a fashion identical with the 0-min sample. The 0-rain sample and the 60-rain sample are placed, while wet, on a sintered-glass funnel
Reaction mixture: Tris • HC1, 1 M, pH 7.4, 400 ~l NaF, 100 raM, 100 ul MgCl~, 100 mM, 100 ~l Galactose, 7.6 mM, 100 td [1-14C] Galactose, 2 t~Ci/ml, 200 td ATP, 120 mM, 100 td 100 ul
1:4 Hemoiysate I
100 ul j 200 ~1
l
50 ul +20~1 1 M G a l SPOT 50 ~1
DRY
50 ul -t-20td 1 M G a l SPOT 50 td
60-Min sample 50 td +20td 1 M G a l SPOT 50 ~1
WASH
WASH
DRY
DRY
Counting solution: 0.3 g POPOP + 5.0 g PPO/1 liter toluene COUNT AND CALCULATE Fro. 1. The determination of erythrocyte galactokinase using [14C]galactose: Schematic summary [E. Beutler, N. V. Paniker, and F. Trinidad, Biochem. Med. 5, 325 (1971)].
[8]
GALACTOKINASE FROM HUMAN ERYTHROCYTES
51
and washed with 600~00 ml of distilled water. The samples are permitted to dry and are ready for counting; this is accomplished in a scintillation solution consisting of toluene containing 0.03% p-bis-2-(5-phenyloxazolyl)benzene (POPOP) and 0.5% 2,5-diphenyloxazole (PPO). The procedure is summarized in Fig. 1. Linearity. When various dilutions of normal hemolysates are made the percentage of galactose phosphorylated increases in an approximately linear fashion.6 When fairly concentrated hemolysates are used, a considerable degree of quenching is observed: there is approximately 55% quenching with a 50% hemolysate, 20% quenching with a 25% hemolysate, and little or no quenching with a 12% hemolysate. Drying samples at too high temperature produces an increase in the amount of quenching because of the change in color of hemoglobin under these conditions. The phosphorylation of galactose is linear with time over a 2.5-hr period. 6 Definition o/ Unit and Specific Activity. One unit of galactokinase is defined as the amount of enzyme which phosphorylates 1 umole of galactose per minute under the conditions of assay. The red cells of normal human adults have been found to contain 0.0297 ± 0.0049 unit of galactokinase per gram of hemoglobin (mean ± standard deviation). Some differences in apparent activity are observed with different batches of [14C]galactose, probably because of varying degrees of contamination with the keto sugar (tagatose) or the 2-epimer (talose). Purification
Procedure
~4
All procedures are carried out at 4 °. All centrifugations are performed for 20 min at 37,500 g. The following buffers are used during the purification procedure. Buffer A: l0 mM KH2PO4-K2HP04, 7 mM fl-mercaptoethanol, 0.5 mM Na~-EDTA, final pH 7.2; buffer B : 100 mM KH2PO4K2HP04, 7 mM fl-mercaptoethanol, 0.5 mM Na-EDTA, final pH 7.2; buffer C: l0 mM KH2HPO/KoHPO,, 7 mM fl-mercaptoethanol, 0.5 mM Na2-EDTA, final pH 6.7. Preparation of Ilemolysate. One-hundred milliliters of washed red cells are mixed with 400 ml of buffer A and frozen and thawed twice. After ccntrifugation the sediment is discarded. DEAE-Sephadex Chromatography. The hemolyzate is applied to a DEAE-Sephadex A-50 column (2.5 X 35 cm) equilibrated with buffer A. Hemoglobin is eluted with 3.5 liters of buffer A. Galactokinase is then eluted by descending chromatography using a 500-ml linear gradient between buffer A and buffer B at a flow rate of 30 ml/hr. Fractions containing galaetokinase are pooled. 14 K. G. B l u m e and E. Beutler, J. Biol. Chem. 246, 6507 (1971).
52
KINASES
[8]
PURIFICATION PROCEDURE FOR HUMAN RED CELL GALACTOKINASE
Fraction Hemolysate DEAE-Sephadex A 50 eluate Ammonium sulfate, 35-60% precipitate Sephadex G-75 filtrate CM-Sephadex C-50 eluate
Volume (ml) 500 66 1.8 16 18
Specific activity (mU/mg)
Purification (fold)
47.5 1.42
0. 021 4.30
1 204
19.2
9.29
440
Protein (mg/ml)
0.41 0.06
28.2 81.2
1336 3850
Ammonium Sulfate. The DEAE-Sephadex A-50 eluate is fractionated with solid (NH4)2S0,. The precipitate which forms at 35% saturation (20.9 g/100 ml) is discarded. Galactokinase is precipitated between 35 and 60% (16.4 g/100 ml) ammonium sulfate saturation and is redissolved in a small amount of buffer A. Sephadex G-75 Filtration. The ammonium sulfate 35 to 60% precipitate is then ehromatographed through a Sephadex G-75 column (1.5 }( 90 cm), equilibrated with buffer A using ascending chromatography with a flow rate of 8.0 ml/hour. Fractions containing galactokinase are combined. CM-Sephadex Chromatography. The Sephadex G-75 filtrate is passed through a CM-Sephadex C-50 column (1.5 X 30 cm), equilibrated with buffer C, with descending chromatography at a flow rate of 4.2 ml/hr. Whereas contaminating proteins were absorbed, galactokinase is not bound to the cation exchanger. Fractions containing galactokinase are pooled and concentrated by ultrafiltration or by ammonium sulfate precipitation at 60% saturation. The cumulative purification ranges between 3300 and 3900-fold, the yield between 14 and 19%. The table summarizes a typical enzyme preparation including all steps described. Purification of fetal galactokinase from human placenta can be achieved by means of a similar method. 3 Properties
Purity. By analytical disc electrophoresis in polyacrylamide (20 and 50 /zg protein per gel column) a single protein band is found with the purified enzyme when stained with Coomassie blue. Of the enzymes of red cell glyeolysis, hexose monophosphate shunt, glutathione and ga]ac-
[9]
N-ACETYLMANNOSAMINE KINASE
53
rose metabolism only traces of triosephosphate isomerase activity (representing less than 0.1% of the specific activity of triosephosphate isomerase in crude hemolysates) are present in the final preparation. Stability. Intact red cells lose less than 5% of galactokinase activity when stored in acid-citrate-dextrose at 4 ° for 3 weeks2 When glucose to give a final added concentration of about 20 mM is added to blood in EDTA solution, the enzyme is stable for at least 20 days at 4 °, 6 days at 25 °, and 4 days at 30 °. In hemolysates the enzyme is unstable, the activity decreases about 30% in 24 hr. After purification the enzyme preparations lose about 10% of activity within 6 days when stored at 4 ° in 3.0 M ammonium sulfate. Other Properties. The Km of galactokinase preparations from red cells for galactose is 126~-11 ~M ( m e a n ± standard deviation), for ATP 342 + 63 ~M. Identical results have been obtained for galactokinase from crude hemoylsates from adult red cells. Km values of galactokinase from cord blood red cell hemolysates and after partial purification are significantly higher for galactose: 225 +_ 8 ~M. 2 Similar results have been obtained with extracts and purified preparations of galactokinase from human placenta, 3 indicating different kinetic properties of human fetal and adult galactokinase. Both forms of the enzyme can be separated by ion exchange chromatography2 The pH optimum for the enzymic phosphorylation of galactose is pH 7.6 in hemolysates and pH 7.8 after purification. The molecular weight of galactokinase determined by Sephadex G-75 and Sephadex G-200 chromatography is 55,000 in crude extracts and after purification. By sodium dodecyl sulfate-polyacrylamide electrophoresis of 3600- to 3850-fold purified preparations and staining with Coomassie blue the molecular weight of the assumed monomer of red cell galactokinase is found to be 26,000. The isoelectric point of the enzyme determined by isoelectrofocusing in a pH 5-8 stabilized sucrose gradient is pH 5.7. The ultraviolet spectrum of purified galactokinase exhibits a minimum at 251 nm and a maximum at 280 nm.
[9] N-Acetylmannosamine Kinase By SANTIMOY BANERJEE a n d SUDHAMOY GHOSH N-Acetylmannosamine + ATP --~ N-acetylmannosamine-6-P + ADP
N-Acetylmannosamine kinase has been previously purified from rat liver. 1,: The enzyme does not appear to be synthesized in bacteria if they 1 W. Kundig, S. Ghosh, and S. Roseman, J. Biol. Chem. 241, 5619 (1966). 2 W. Kundig and S. Roseman, this series, Vol. 8 [28].
