[59]
FRUCTOSE-1,6-DIPHOSPHATASE FROM BOVINE LIVER
363
or glucose-l-P occurs when incubations are carried out for 30 rain and assayed for release of inorganic phosphate. Effect o] pH, EDTA, and Metals. Maximal activity takes place at p H 9-9.5. In the presence of E D T A (0.1-0.2 mM), activity at p H 9 is doubled and an equal amount of activity appears at pH 7.5. High coneentrations of E D T A (1 mM) inhibit all activity. F D P a s e has an absolute requirement for a divalent cation best satisfied by 1 m M Mg 2+. Regulation. Unlike most FDPases, ~ the enzyme from P. pallidum is not significantly inhibited by 5'-AMP. Activity, measured at pH 7.5 and pH 9.0, is the same in cell-free extracts derived from four different stages of the developmental cycle of P. pallidum (myxamebae, multicellular aggregate, preculmination stage, and differentiated fruiting body). 6B. L. Horecker, S. Pontremoli, 0. M. Rosen, and S. Rosen, Fed. Proc., Fed. Amer. Soc. Exp. Biol. 25, 1521 (1966).
[59] F r u c t o s e - l , 6 - d i p h o s p h a t a s e
from Bovine Liver
By ARTHUR M. GELLER and WILLIAM L. BYRNE Fructose 1,6-diphosphate + H20--~ fructose 6-phosphate + Pi Fructose-l,6-diphosphatase is a required enzyme in the gluconeogenic pathway. The purified enzyme from bovine liver requires a metal cofactor for activity, and is maximally active at neutral pH. 1-3. Activity is inhibited by high concentrations of the metal cofactor, by high concentrations of the substrate, fructose-l,6-P2, and by 5'-AMP2 ,4
Assay Method Principle. Fructose-l,6-diphosphatase activity is assayed either by measuring the formation of inorganic phosphate using a discontinuous assay 1 or by a continuous spectrophotometric assay which measures the reduction of N A D P ÷ when fructose 6-phosphate formation is coupled with phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. 3 Method I. Phosphate Release Assay. The assay mixture contains 25 1W. L. Byrne, G. T. Rajagopolan, L. D. Griffin, E. H. Ellis, T. M. Harris, P. Hochachka, L. Reid, and A. M. Geller, Arch. Biochem. Biophys. 146, 118 (1971). 2A. M. Geller and W. L. Byrne, Arch. Biochem. Biophys. 153, 526 (1972). '~C. J. Marcus, A. M. Geller, and W. L. Byrne, J. Biol. Chem. 248, 8567 (1973). 4H. J. Cohen, T. M. Harris, A. M. Geller, and W. L. Byrne, Arch. Biochem. Biophys. 146, 144 (1971).
364
PHOSPHATASES
[59]
mM Tris-25 mM histidine, pH 6.5, 5 mM MgS04, 0.1 mM EDTA, 5.0 mM fructose 1,6-diphosphate, and enzyme in a final volume of 2.0 ml at 37 °. The Tris-histidine buffer can be used between pH 6.0 and 9.5. At 0-, 15-, and 30-min intervals, 0.5-ml aliquots are withdrawn, pipetted into 1.0 ml of 10% trichloroacetic acid, and centrifuged if necessary. The deproteinized samples are analyzed for inorganic phosphate by the method of Fiske and SubbaRow2
Method II. Spectrophotometric Assay Fructose 1,6-diphosphate -~ H~O--* fructose 6-phosphate + Pi (1) Fructose 6-phosphate ~ glucose 6-phosphate (2) Glucose 6-phosphate ~ NADP+ ~ 6-phospho-~-gluconolactone + NADPH + H+ (3) In this assay fructose-l,6-diphosphatase (reaction 1) is coupled with phosphoglucose isomerase (reaction 2) and glucose-6-phosphate dehydrogenase (reaction 3). The reaction is followed by measuring NADPH formation at 340 nm. The standard assay mixture contains 8.4 ~M Fru-l,6-P2, and phosphoglucose isomerase and glucose-6-phosphate dehydrogenase in excess, in a final volume of 1.0 ml. Solutions containing Tris, histidine, and EDTA are prepared and adjusted to the desired pH at 37°; the temperature at which the assay is carried out. Since NH4 ÷ has been shown to stimulate fructose-l,6-diphosphatase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase are diluted in 10 mM Tris.HC1, pH 7.5, and dialyzed at 4 ° for 15 hr against 500 volumes of the same buffer to remove (NH4)~S04. Reactions are always initiated by the addition of enzyme, because it has been observed that there is a difference in the initial rate when the assay is started with substrate or magnesium. In order to use the coupled assay at micromolar substrate concentrations, very dilute enzyme solutions are required. A typical assay of the purified enzyme uses about 27 ng of protein. The diluted enzyme is unstable, so it is stored at a concentration of 2.7 ~g/ml in bovine serum albumin (10 mg/ml) and 5.0 mM MgS04. The dilution into the final assay is generally 100-fold. Units. One unit of fructose-l,6-diphosphatase activity is defined as that amount of enzyme which causes the liberation of 1.0/~mole of inorganic phosphate per minute at 37 ° . Specific activity is expressed as units per milligram of protein. Purification Procedure
All purification steps are carried out at 6 °, unless otherwise stated, however, the pH for all solutions is adjusted at room temperature. The 5 C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925).
[59[
365
FRUCTOSE-1,6-DIPHOSPHATASE FROM BOVINE LIVER PURIFICATION OF FRUCTOSE-1,6-DIPHOSPHATASE FROM BOVINE LIVER
Fraction 1. 2. 3. 4. 5.
Homogenateb Methanol b Ammonium sulfate DEAE-Sephadex Cellex-P
Protein (mg/ml)
Activitya (units/ml)
Total activity (units)
Specific activity (units/rag)
12 31.5 42 1.5 3.2
0.1 11.5 21.8 6.8 57.7
35,000 15,167 5,300 490 138
0. 083 0.37 0.52 4.5 18.0
a Activity was measured by measuring the release of inorganic phosphate at pH 6.5 at 37°. b These two steps are carried out at the New England Enzyme Center, Boston, Massachusetts. livers used generally weight about 15 pounds. Fresh or frozen liver (cut up into small pieces) give equivalent results. Protein determinations are carried out by the method of L o w r y et al., 6 and bovine serum albumin is used as the color standard. Bovine fructose-l,6-diphosphatase, when assayed as described in Method I, is normally maximally active at p H 6.5, with less activity at p H 9.0. Generally the ratio of activity at 6.5 to the activity at p H 9.0 is approximately 2.0. Under some circumstances this ratio decreases during purification. This m a y be due to proteolysis, since most hepatic fructose-l,6-diphosphatases are very sensitive to the action of proteolytic enzymes/,s During the purification procedure, each step is monitored by determining the p H 6.5 to p H 9.0 activity ratio and by measuring the percentage inhibition by 0.1 m M 5 ' - A M P 2 Typical results are summarized in the table. M e t h a n o l F r a c t i o n a t i o n . Two hundred grams of liver are homogenized in 1 liter of 50 m M sodium acetate, p H 5.0, 3 m M MgS04, and 1.0 m M E D T A . The homogenate is then centrifuged for 10 rain at 20,000 g at 0 °, and the precipitate is discarded. Methanol (precooled to --18 °) is added to the supernatant until the mixture is 35% methanol ( v / v ) . This solution is stirred for 10 rain at - - 1 8 °, and then centrifuged at 20,000 g at - - 1 8 ° for 10 rain. The precipitate is discarded. The supernatant is again placed in a bath at - - 1 8 °, and sufficient methanol is added to bring the methanol concentration to 60% ( v / v ) . The solution is recentriO. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). ~A. M. Geller, G. T. Rajagopolan, E. H. Ellis, and W. L. Byrne, Arch. Biochem. Biophys. 146, 134 (1971). s K. Nakashima and B. L. Horecker, Arch. Biochem. Biophys. 146, 153 (1960). 9 R. A. Arneson, Dissertation, Duke University, Durham, North Carolina, 1971.
366
e~OSem~TASES
[59]
fuged at 20,000 g for 10 min at --18 °. The precipitate is taken up in 10 mM Tris, pH 7.9, 3.0 mM MgS04, and 1.0 mM EDTA, and then dialyzed against the same buffer. The methanol fractionation step described above is for laboratory scale purification. This step has been scaled up at the New England Enzyme Center, Tufts University, Boston, Massachusetts, so that the homogentate of an entire liver can be processed at one time. The yield for this step is low (between 30 and 45%), but the overall savings in effort compensates for the poor yield. Ammonium Sulfate Fractionation. The dialyzed methanol preparation is diluted to 7.0 units/ml with 10 mM Tris, pH 7.9, 3.0 mM MgS04 and 1.0 mM EDTA. Ammonium sulfate is added slowly until 0.50 saturation (add 31.0 g/100 ml) is reached. The solution is stirred for 20 min, centrifuged at 10,000 g for 15 min, and the precipitate is discarded. The supernatant is brought to 0.65 saturation (add 9.7 g/100 ml) with ammonium sulfate, stirred for 20 min, and centrifuged again at 10,000 g for 15 min. The precipitate is dissolved in 50 mM Tris, pH 8.0. (The buffer volume should be about 10% of the volume of the dialyzed methanol preparation.) This solution is then dialyzed against 10 mM Tris, pH 7.8, 0.5 mM EDTA, 1.0 mM Fru-l,6-P~ (calcium salt), and 80 mM NaC1. DEAE-Sephadex Step. Before use DEAE-Sephadex was washed with 0.5 N NaOH, 0.5 N HC1, and distilled water. The resin was poured at room temperature into a 4.5 )< 67 column, and then allowed to cool at 6 °. The resin was then washed with 10 mM Tris, pH 7.8, 0.5 mM EDTA, 1 mM Fru-l,6-P._, (calcium salt), and 80 mM NaC1 until the pH of the effluent was 7.8. Before being placed on the column the dialyzed enzyme was incubated at 37 ° for 60 min and then centrifuged at 10,000 g for 10 rain. This step is necessary in order to keep materials from precipitating during the time the enzyme was on the column. The sample is placed on the column, and washed into the column with 150 ml of the buffer with which the column is equilibrated. When the buffer level reaches the top of the column bed, an additional 200 ml of buffer is added. The sample is then eluted with 4 liters of a 80 mM to 0.13 M NaC1 linear gradient made up in 10 mM Tris, pH 7.8, 0.5 mM EDTA, and 1 mM Fru-l,6-P~ (Ca 2+ salt). The height of the 4-liter reservoir is adjusted to give the smallest hydrostatic pressure that will allow flow of liquid through the column, since higher hydrostatic pressure decreased the flow rate due to compression of the DEAE-Sephadex. The eluent is collected in 20-ml samples. Fructose-l,6-diphosphatase is eluted from the column at concentrations of NaC1 between 0.11 M and 0.12 M. About 45% of the protein put on the column comes off before the
[59]
FRUCTOSE-1,6-DIPttOSPHATASE FROM BOVINE LIVER
367
fructose-l,6-diphosphatase peak. Approximately 4% to 5% of the total protein is present in those tubes which contain fructose-l,6-diphosphatase activity. The tubes having a specific activity of about 3.3 units/ml or higher are combined and dialyzed against 10 mM Tris, pH 7.3, 0.1 mM EDTA, 70 mM NaC1, and 1 mM MgSO4. CeUex-P Step. Cellex-P (phosphoeellulose from Bio-Rad Laboratories) was washed with 0.5 M NaOH and distilled water, poured into a 4.5 X 90 em eolumn, and equilibrated with 10 mM Tris, pH 7.3, 0.1 mM EDTA, and 70 mM NaC1. For optimal results only 0.45 mg of protein may be placed on the column per milliliter of column bed. Just prior to placing the enzyme on the column, enough 0.1 M EDTA is added to the dialyzed sample to make the final eoneentration 2 mM, thus making the Mg 2+ unavailable to the enzyme. The presence of 1.0 mM Mg 2÷ during dialysis is required to ensure that none of the Fru-l,6-P2 used in the DEAE-Sephadex proeedure remains. It is important to eliminate the Fru-l,6-P2 since it influenees the binding of the enzyme to the column, and, as indieated below, Fru-l,6-P2 is used as a specific eluting agent. After the enzyme is placed on the column, the column is washed with 2500 ml of 10 mM Tris, pH 7.3, 0.1 mM EDTA, and 70 mM NaC1. About 14% of the protein and none of the fruetose-l,6-diphosphatase comes off in this wash solution. Then the column is connected to a reservoir containing 2000 ml of 10 mM Tris, pH 7.3, 0.1 mM EDTA, 70 mM NaC1, 1 mM Fru-l,6-P., (Ca ~÷ salt), and 1 mM AMP. Fruetose-l,6-diphosphatase is eluted from the column in a sharp symmetrical peak just after the void volume; this peak eontains about 16% of the protein put onto the column. The tubes eontaining fruetose-l,6-diphosphatase ae*ivity are combined, lyophilized, and then taken up in 10 mM Tris, pH 8.0, 3 mM MgSO4, and 1 mM EDTA. The sample is then dialyzed against this same buffer and stored at --15 ° .
Properties Molecular Weight. The molecular weight of bovine liver fructose 1,6diphosphatase as determined by sedimentation equilibrium and sedimentation velocity is approximately 130,000.1 The sedimentation coefficient is concentration-dependent, giving an extrapolated value of 7.61 S for S2o.w. The carboxy-terminal amino acid has been found to be lysine. No amino terminal residues have been detected. Purified enzyme gives a single band on gel eleetrophoresis, s Kinetics. Bovine fructose-l,6-diphosphatase has a Km for Fru-l,6-P2 of about 1.7 ~M (at 5 mM MgSO4). 3 High concentrations of Fru-l,6-P2
368
PI~OSP~IAT~.SES
[59]
are inhibitory. Maximal activity is observed at about 10 ~M, and higher concentrations inhibit. A maximum of 50% inhibition is observed. The enzyme also requires a metal cofactor, either magnesium or manganese. The concentration of metal ion required for half-maximal activity at pH 6.5 is 0.26 mM for magnesium and 30 ~M for manganese. High concentrations of metal cofactor are inhibitory. fl-Glycerophosphate is a substrate for bovine fructose-l,6-diphosphatase at pH 9.0, with a Km of 10 mM, but it is not a substrate at pH 6.5.4 5'-AMP is a specific allosteric inhibitor of fructose-l,6-diphosphatase, with a Ki of 60 ~M. The Ki is temperature dependent, decreasing with decreasing temperature. pH Optimum. The pH optimum of bovine hepatic fructose-l,6-diphosphatase is between pH 6.5 and 7.5. The shape of the pH-activity curve is dependent upon the concentration of both the substrate, Fru-l,6-P~, and the metal cofactor2 Activators and Inhibitors. Proteolytic enzymes such as Nagarse, papain, and Pronase stimulate activity 2- to 3-fold at pH 9.0, with a simultaneous loss of activity at pH 6.5. 7 Reagents such as N-ethylmaleimide, 1-fluoro-2,4-dinitrobenzene, 5,5'-dithiobis-(2-nitrobenzoic acid), and p-mercuribenzoate have similar, although not identical, effects on activity. After incubation with these latter reagents, fructose-l,6-diphosphatase activity is inhibited up to 90% at neutral pH (pH 6.5-7.0) and slightly increased at alkaline pH (pH 9.0), when magnesium is the metal cofactor in the assay. However, when magnanese is used as the metal cofactor, activity is increased up to 3-fold at alkaline pH, with less activation at neutral pH. Iodoacetate inhibits at both neutral and alkaline pH. 2 Glutaraldehyde treatment inhibits activity at both pH 6.5 and 9.0, shifts the pH optimum to about pH 9.0, and desensitizes the enzyme to inhibition by 5'-AMP and Fru-l,6-P2. TM Stability. Concentrated solutions of the enzyme stored at pH 7.5-8.0 in 10 mM Tris, 1.0 mM EDTA, 3.0 MgSO4, or 20 mM fl-glycerophosphate-l.0 mM Fru-l,6-P2 may be stored for many months at --20% Bovine fructose 1,6-diphosphatase is unstable after incubation at 50 °, losing about 50% of its activity within 15 min. Fru-l,6-P2, Mg 2÷, and Mn 2÷ protect against the loss of activity, while 5'-AMP has no protective effect. 10v. J. Aloyo, A. M. Geller, C. J. Marcus, and W. L. Byrne, Biochem. Biophys. Acta 289, 242 (1972).
FRUCTOSE-1,6-DIPHOSPHATASE FROM BOVINE LIVER
363
or glucose-l-P occurs when incubations are carried out for 30 rain and assayed for release of inorganic phosphate. Effect o] pH, EDTA, and Metals. Maximal activity takes place at p H 9-9.5. In the presence of E D T A (0.1-0.2 mM), activity at p H 9 is doubled and an equal amount of activity appears at pH 7.5. High coneentrations of E D T A (1 mM) inhibit all activity. F D P a s e has an absolute requirement for a divalent cation best satisfied by 1 m M Mg 2+. Regulation. Unlike most FDPases, ~ the enzyme from P. pallidum is not significantly inhibited by 5'-AMP. Activity, measured at pH 7.5 and pH 9.0, is the same in cell-free extracts derived from four different stages of the developmental cycle of P. pallidum (myxamebae, multicellular aggregate, preculmination stage, and differentiated fruiting body). 6B. L. Horecker, S. Pontremoli, 0. M. Rosen, and S. Rosen, Fed. Proc., Fed. Amer. Soc. Exp. Biol. 25, 1521 (1966).
[59] F r u c t o s e - l , 6 - d i p h o s p h a t a s e
from Bovine Liver
By ARTHUR M. GELLER and WILLIAM L. BYRNE Fructose 1,6-diphosphate + H20--~ fructose 6-phosphate + Pi Fructose-l,6-diphosphatase is a required enzyme in the gluconeogenic pathway. The purified enzyme from bovine liver requires a metal cofactor for activity, and is maximally active at neutral pH. 1-3. Activity is inhibited by high concentrations of the metal cofactor, by high concentrations of the substrate, fructose-l,6-P2, and by 5'-AMP2 ,4
Assay Method Principle. Fructose-l,6-diphosphatase activity is assayed either by measuring the formation of inorganic phosphate using a discontinuous assay 1 or by a continuous spectrophotometric assay which measures the reduction of N A D P ÷ when fructose 6-phosphate formation is coupled with phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. 3 Method I. Phosphate Release Assay. The assay mixture contains 25 1W. L. Byrne, G. T. Rajagopolan, L. D. Griffin, E. H. Ellis, T. M. Harris, P. Hochachka, L. Reid, and A. M. Geller, Arch. Biochem. Biophys. 146, 118 (1971). 2A. M. Geller and W. L. Byrne, Arch. Biochem. Biophys. 153, 526 (1972). '~C. J. Marcus, A. M. Geller, and W. L. Byrne, J. Biol. Chem. 248, 8567 (1973). 4H. J. Cohen, T. M. Harris, A. M. Geller, and W. L. Byrne, Arch. Biochem. Biophys. 146, 144 (1971).
364
PHOSPHATASES
[59]
mM Tris-25 mM histidine, pH 6.5, 5 mM MgS04, 0.1 mM EDTA, 5.0 mM fructose 1,6-diphosphate, and enzyme in a final volume of 2.0 ml at 37 °. The Tris-histidine buffer can be used between pH 6.0 and 9.5. At 0-, 15-, and 30-min intervals, 0.5-ml aliquots are withdrawn, pipetted into 1.0 ml of 10% trichloroacetic acid, and centrifuged if necessary. The deproteinized samples are analyzed for inorganic phosphate by the method of Fiske and SubbaRow2
Method II. Spectrophotometric Assay Fructose 1,6-diphosphate -~ H~O--* fructose 6-phosphate + Pi (1) Fructose 6-phosphate ~ glucose 6-phosphate (2) Glucose 6-phosphate ~ NADP+ ~ 6-phospho-~-gluconolactone + NADPH + H+ (3) In this assay fructose-l,6-diphosphatase (reaction 1) is coupled with phosphoglucose isomerase (reaction 2) and glucose-6-phosphate dehydrogenase (reaction 3). The reaction is followed by measuring NADPH formation at 340 nm. The standard assay mixture contains 8.4 ~M Fru-l,6-P2, and phosphoglucose isomerase and glucose-6-phosphate dehydrogenase in excess, in a final volume of 1.0 ml. Solutions containing Tris, histidine, and EDTA are prepared and adjusted to the desired pH at 37°; the temperature at which the assay is carried out. Since NH4 ÷ has been shown to stimulate fructose-l,6-diphosphatase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase are diluted in 10 mM Tris.HC1, pH 7.5, and dialyzed at 4 ° for 15 hr against 500 volumes of the same buffer to remove (NH4)~S04. Reactions are always initiated by the addition of enzyme, because it has been observed that there is a difference in the initial rate when the assay is started with substrate or magnesium. In order to use the coupled assay at micromolar substrate concentrations, very dilute enzyme solutions are required. A typical assay of the purified enzyme uses about 27 ng of protein. The diluted enzyme is unstable, so it is stored at a concentration of 2.7 ~g/ml in bovine serum albumin (10 mg/ml) and 5.0 mM MgS04. The dilution into the final assay is generally 100-fold. Units. One unit of fructose-l,6-diphosphatase activity is defined as that amount of enzyme which causes the liberation of 1.0/~mole of inorganic phosphate per minute at 37 ° . Specific activity is expressed as units per milligram of protein. Purification Procedure
All purification steps are carried out at 6 °, unless otherwise stated, however, the pH for all solutions is adjusted at room temperature. The 5 C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925).
[59[
365
FRUCTOSE-1,6-DIPHOSPHATASE FROM BOVINE LIVER PURIFICATION OF FRUCTOSE-1,6-DIPHOSPHATASE FROM BOVINE LIVER
Fraction 1. 2. 3. 4. 5.
Homogenateb Methanol b Ammonium sulfate DEAE-Sephadex Cellex-P
Protein (mg/ml)
Activitya (units/ml)
Total activity (units)
Specific activity (units/rag)
12 31.5 42 1.5 3.2
0.1 11.5 21.8 6.8 57.7
35,000 15,167 5,300 490 138
0. 083 0.37 0.52 4.5 18.0
a Activity was measured by measuring the release of inorganic phosphate at pH 6.5 at 37°. b These two steps are carried out at the New England Enzyme Center, Boston, Massachusetts. livers used generally weight about 15 pounds. Fresh or frozen liver (cut up into small pieces) give equivalent results. Protein determinations are carried out by the method of L o w r y et al., 6 and bovine serum albumin is used as the color standard. Bovine fructose-l,6-diphosphatase, when assayed as described in Method I, is normally maximally active at p H 6.5, with less activity at p H 9.0. Generally the ratio of activity at 6.5 to the activity at p H 9.0 is approximately 2.0. Under some circumstances this ratio decreases during purification. This m a y be due to proteolysis, since most hepatic fructose-l,6-diphosphatases are very sensitive to the action of proteolytic enzymes/,s During the purification procedure, each step is monitored by determining the p H 6.5 to p H 9.0 activity ratio and by measuring the percentage inhibition by 0.1 m M 5 ' - A M P 2 Typical results are summarized in the table. M e t h a n o l F r a c t i o n a t i o n . Two hundred grams of liver are homogenized in 1 liter of 50 m M sodium acetate, p H 5.0, 3 m M MgS04, and 1.0 m M E D T A . The homogenate is then centrifuged for 10 rain at 20,000 g at 0 °, and the precipitate is discarded. Methanol (precooled to --18 °) is added to the supernatant until the mixture is 35% methanol ( v / v ) . This solution is stirred for 10 rain at - - 1 8 °, and then centrifuged at 20,000 g at - - 1 8 ° for 10 rain. The precipitate is discarded. The supernatant is again placed in a bath at - - 1 8 °, and sufficient methanol is added to bring the methanol concentration to 60% ( v / v ) . The solution is recentriO. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). ~A. M. Geller, G. T. Rajagopolan, E. H. Ellis, and W. L. Byrne, Arch. Biochem. Biophys. 146, 134 (1971). s K. Nakashima and B. L. Horecker, Arch. Biochem. Biophys. 146, 153 (1960). 9 R. A. Arneson, Dissertation, Duke University, Durham, North Carolina, 1971.
366
e~OSem~TASES
[59]
fuged at 20,000 g for 10 min at --18 °. The precipitate is taken up in 10 mM Tris, pH 7.9, 3.0 mM MgS04, and 1.0 mM EDTA, and then dialyzed against the same buffer. The methanol fractionation step described above is for laboratory scale purification. This step has been scaled up at the New England Enzyme Center, Tufts University, Boston, Massachusetts, so that the homogentate of an entire liver can be processed at one time. The yield for this step is low (between 30 and 45%), but the overall savings in effort compensates for the poor yield. Ammonium Sulfate Fractionation. The dialyzed methanol preparation is diluted to 7.0 units/ml with 10 mM Tris, pH 7.9, 3.0 mM MgS04 and 1.0 mM EDTA. Ammonium sulfate is added slowly until 0.50 saturation (add 31.0 g/100 ml) is reached. The solution is stirred for 20 min, centrifuged at 10,000 g for 15 min, and the precipitate is discarded. The supernatant is brought to 0.65 saturation (add 9.7 g/100 ml) with ammonium sulfate, stirred for 20 min, and centrifuged again at 10,000 g for 15 min. The precipitate is dissolved in 50 mM Tris, pH 8.0. (The buffer volume should be about 10% of the volume of the dialyzed methanol preparation.) This solution is then dialyzed against 10 mM Tris, pH 7.8, 0.5 mM EDTA, 1.0 mM Fru-l,6-P~ (calcium salt), and 80 mM NaC1. DEAE-Sephadex Step. Before use DEAE-Sephadex was washed with 0.5 N NaOH, 0.5 N HC1, and distilled water. The resin was poured at room temperature into a 4.5 )< 67 column, and then allowed to cool at 6 °. The resin was then washed with 10 mM Tris, pH 7.8, 0.5 mM EDTA, 1 mM Fru-l,6-P._, (calcium salt), and 80 mM NaC1 until the pH of the effluent was 7.8. Before being placed on the column the dialyzed enzyme was incubated at 37 ° for 60 min and then centrifuged at 10,000 g for 10 rain. This step is necessary in order to keep materials from precipitating during the time the enzyme was on the column. The sample is placed on the column, and washed into the column with 150 ml of the buffer with which the column is equilibrated. When the buffer level reaches the top of the column bed, an additional 200 ml of buffer is added. The sample is then eluted with 4 liters of a 80 mM to 0.13 M NaC1 linear gradient made up in 10 mM Tris, pH 7.8, 0.5 mM EDTA, and 1 mM Fru-l,6-P~ (Ca 2+ salt). The height of the 4-liter reservoir is adjusted to give the smallest hydrostatic pressure that will allow flow of liquid through the column, since higher hydrostatic pressure decreased the flow rate due to compression of the DEAE-Sephadex. The eluent is collected in 20-ml samples. Fructose-l,6-diphosphatase is eluted from the column at concentrations of NaC1 between 0.11 M and 0.12 M. About 45% of the protein put on the column comes off before the
[59]
FRUCTOSE-1,6-DIPttOSPHATASE FROM BOVINE LIVER
367
fructose-l,6-diphosphatase peak. Approximately 4% to 5% of the total protein is present in those tubes which contain fructose-l,6-diphosphatase activity. The tubes having a specific activity of about 3.3 units/ml or higher are combined and dialyzed against 10 mM Tris, pH 7.3, 0.1 mM EDTA, 70 mM NaC1, and 1 mM MgSO4. CeUex-P Step. Cellex-P (phosphoeellulose from Bio-Rad Laboratories) was washed with 0.5 M NaOH and distilled water, poured into a 4.5 X 90 em eolumn, and equilibrated with 10 mM Tris, pH 7.3, 0.1 mM EDTA, and 70 mM NaC1. For optimal results only 0.45 mg of protein may be placed on the column per milliliter of column bed. Just prior to placing the enzyme on the column, enough 0.1 M EDTA is added to the dialyzed sample to make the final eoneentration 2 mM, thus making the Mg 2+ unavailable to the enzyme. The presence of 1.0 mM Mg 2÷ during dialysis is required to ensure that none of the Fru-l,6-P2 used in the DEAE-Sephadex proeedure remains. It is important to eliminate the Fru-l,6-P2 since it influenees the binding of the enzyme to the column, and, as indieated below, Fru-l,6-P2 is used as a specific eluting agent. After the enzyme is placed on the column, the column is washed with 2500 ml of 10 mM Tris, pH 7.3, 0.1 mM EDTA, and 70 mM NaC1. About 14% of the protein and none of the fruetose-l,6-diphosphatase comes off in this wash solution. Then the column is connected to a reservoir containing 2000 ml of 10 mM Tris, pH 7.3, 0.1 mM EDTA, 70 mM NaC1, 1 mM Fru-l,6-P., (Ca ~÷ salt), and 1 mM AMP. Fruetose-l,6-diphosphatase is eluted from the column in a sharp symmetrical peak just after the void volume; this peak eontains about 16% of the protein put onto the column. The tubes eontaining fruetose-l,6-diphosphatase ae*ivity are combined, lyophilized, and then taken up in 10 mM Tris, pH 8.0, 3 mM MgSO4, and 1 mM EDTA. The sample is then dialyzed against this same buffer and stored at --15 ° .
Properties Molecular Weight. The molecular weight of bovine liver fructose 1,6diphosphatase as determined by sedimentation equilibrium and sedimentation velocity is approximately 130,000.1 The sedimentation coefficient is concentration-dependent, giving an extrapolated value of 7.61 S for S2o.w. The carboxy-terminal amino acid has been found to be lysine. No amino terminal residues have been detected. Purified enzyme gives a single band on gel eleetrophoresis, s Kinetics. Bovine fructose-l,6-diphosphatase has a Km for Fru-l,6-P2 of about 1.7 ~M (at 5 mM MgSO4). 3 High concentrations of Fru-l,6-P2
368
PI~OSP~IAT~.SES
[59]
are inhibitory. Maximal activity is observed at about 10 ~M, and higher concentrations inhibit. A maximum of 50% inhibition is observed. The enzyme also requires a metal cofactor, either magnesium or manganese. The concentration of metal ion required for half-maximal activity at pH 6.5 is 0.26 mM for magnesium and 30 ~M for manganese. High concentrations of metal cofactor are inhibitory. fl-Glycerophosphate is a substrate for bovine fructose-l,6-diphosphatase at pH 9.0, with a Km of 10 mM, but it is not a substrate at pH 6.5.4 5'-AMP is a specific allosteric inhibitor of fructose-l,6-diphosphatase, with a Ki of 60 ~M. The Ki is temperature dependent, decreasing with decreasing temperature. pH Optimum. The pH optimum of bovine hepatic fructose-l,6-diphosphatase is between pH 6.5 and 7.5. The shape of the pH-activity curve is dependent upon the concentration of both the substrate, Fru-l,6-P~, and the metal cofactor2 Activators and Inhibitors. Proteolytic enzymes such as Nagarse, papain, and Pronase stimulate activity 2- to 3-fold at pH 9.0, with a simultaneous loss of activity at pH 6.5. 7 Reagents such as N-ethylmaleimide, 1-fluoro-2,4-dinitrobenzene, 5,5'-dithiobis-(2-nitrobenzoic acid), and p-mercuribenzoate have similar, although not identical, effects on activity. After incubation with these latter reagents, fructose-l,6-diphosphatase activity is inhibited up to 90% at neutral pH (pH 6.5-7.0) and slightly increased at alkaline pH (pH 9.0), when magnesium is the metal cofactor in the assay. However, when magnanese is used as the metal cofactor, activity is increased up to 3-fold at alkaline pH, with less activation at neutral pH. Iodoacetate inhibits at both neutral and alkaline pH. 2 Glutaraldehyde treatment inhibits activity at both pH 6.5 and 9.0, shifts the pH optimum to about pH 9.0, and desensitizes the enzyme to inhibition by 5'-AMP and Fru-l,6-P2. TM Stability. Concentrated solutions of the enzyme stored at pH 7.5-8.0 in 10 mM Tris, 1.0 mM EDTA, 3.0 MgSO4, or 20 mM fl-glycerophosphate-l.0 mM Fru-l,6-P2 may be stored for many months at --20% Bovine fructose 1,6-diphosphatase is unstable after incubation at 50 °, losing about 50% of its activity within 15 min. Fru-l,6-P2, Mg 2÷, and Mn 2÷ protect against the loss of activity, while 5'-AMP has no protective effect. 10v. J. Aloyo, A. M. Geller, C. J. Marcus, and W. L. Byrne, Biochem. Biophys. Acta 289, 242 (1972).
