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[112c] Fructose-1,6-diphosphatase
636
PHOSPI:IATASE$
[112c]
inorganic phosphate (0.01 M). Fe ++, Cu +÷, Zn++, and Hg ++ do not inhibit at concentrations of 0.1 mM in the presence of 1.0 mM Mg ÷+, whereas Pb ++ (0.1 raM) and Ca +* (0.1 raM) inhibit 80-1009, Ag+ (0.1 mM) inhibits 3 0 9 , and fluoride (0.01 M) inhibits 50-60 9 . AMP inhibits enzymatic activity at concentrations of 0.05-0.5 mM. This inhibition is most marked at pH 7.5 in the presence of EDTA. Under these conditions, 0.1 mM AMP produces 80 9 inhibition of enzymatic activity.
[112c] F r u c t o s e - 1 , 6 - d i p h o s p h a t a s e III. Euglena gracilis
By A. A. APP Fructose 1,6-phosphate T H20 -+ D-fructose6-phosphate -{- P~ Assay Method Principle. The colorimetric assay measuring the liberation of inorganic phosphate may be conveniently used? Reagents
Tris buffer, 0.5 M, pH 8.4 MgC12, 0.1 M Na2EDTA, 0.016 M Fruetose-l,6-diphosphate, sodium salt 4 X 10-~ M Procedure. The following reagents are present in a total volume of 1 ml: 100 micromoles Tris (pH 8.4), 10 micromoles MgC12, 2 mieromoles FDP, 1.6 micromoles EDTA, and the enzyme solution to be tested. ~ After incubation at 30 ° (1-3 minutes, depending on the level of activity) the reaction is stopped with 5% trichloroacetic acid and a suitable aliquot of the supernatant assayed for P~.~ Blanks consist of all components except FDP. One unit of fructose diphosphatase activity is defined as the liberation of 1 micromole of P~ per minute. Protein was estimated by absorbency or by the Lowry procedure2 As long as consideration is given to phosphatase activity from other
1E. Racker and E. A. R. Schroeder, Arch. Biochem. Biophys. 74, 326 (1958). A. A. App and A. T. Jagendorf, Biochim. Biophys. Acta 85, 427 (1964). 3H. H. Taussky and E. Shorr, J. Biol. Chem. 20'2, 675 (1953). O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1961). See also Vol. III, p. 448.
[112c]
FRUCTOSE-1,6-DIPHOSPHATASE
637
than alkaline fruetose-l,6-diphosphatase, the assay may be employed with crude Euglena extracts. Purification Proce4ure
Euglena gracilis, strain Z, was grown aseptically in 9-liter carboys for 6-9 days in white light at 25 ° . The cultures were aerated through capillary tubing and grown in a previously described medium consisting of 0.5% proteose peptone, 0.2% yeast extract, and 0.1% sodium acetate2 The cells were harvested in a Sorvall continuous flow centrifuge, washed and resuspended in water, then broken by sonication for 3 minutes with a Raytheon 10-ke oscillator, Model DF 101. The sonicate from 230 g (wet weight) of EugIena was centrifuged for 15 minutes at 20,000 g. The pellet was resuspended in water, the mixture was recentrifuged, and the combined supernatants were dialyzed overnight against water at 4 ° . The dialyzed extract was centrifuged at 20,200 g for 15 minutes and the pH of the supernatant was adjusted to 5.1 with 0.1 M citric acid. This acidified supernatant was held at 60 ° for about 10 minutes, or until heavy precipitation occurred. The precipitate was removed by centrifuging, the supernatant solution was then cooled to 3 ° and acidified to pH 4.35 with more 0.1 M citric acid. The second precipitate was also removed by centrifugation, and the pH of the supernatant solution was raised to 4.95 by addition of sodium citrate. One milliliter of a protamine sulfate solution (5.0 mg/ml, pH 5.5) was then added for every 20 ml of enzyme solution. Precipitation was allowed to occur for 25 minutes at 4 °, and the precipitate was removed by centrifuging at 20,200 g for 15 minutes. The supernatant solution was retained and its pH readjusted to 4.95 if necessary. The next step was chromatography on a DEAE-cellulose column. The cellulose derivative was cleaned as described by Peterson and Sober, formed into a 3.25 X 13.0 em column, and equilibrated at 4 ° with 0.01 M citrate buffer (pH 5.5).6 Approximately 380 ml of the enzyme solution was then passed through the column. Gradient elution of the enzyme was accomplished using a mixing vessel with 1 liter of 0.01 M citrate buffer at pH 5.5 and a reservoir containing 0.01 M citric acid. In this procedure a great deal of protein was removed before the fructose diphosphatase began to appear in the effluent at approximately pH 4.8. The most active fractions from this first run were pooled and rechromatographed with an identical procedure using a 2 X 12.75 cm column and a 700 ml mixing vessel. Elution profiles from this second column showed a close association between fructose diphosphatase activity and the remaining material. G. Brawerman and N. Konigsberg, Biochim. Biophys. Acta 43, 374 (1960). s E. A. Peterson and H. A. Sober, see Vol. V [1].
638
[112c]
PHOSPHATASES PURIFICATION OF FRUCTOSE DIPHOSPHATASE FROM
Fraction Dialyzed, centrifuged Heated, centrifuged pH precipitation, centrifuged Protamine sulfate, centrifuged First DEAE-column Second DEAE-column
Euglena g r a c i l i s a
Specific Fructose activity Total Protein, diphosphatase (units/mg recovery (mg/ml) (units/ml) protein) (%) 20.0 4.0 2.75 2.37 0.130 0.100 b
3.25 2.80 2.08 2.25 3.65 7.2 b
0.16 0.70 0.76 0.95 28.0 72b
100 85 64 69 46 31
a Reproduced from A. A. App and A. T. Jagendorf, Biochim. Biophys. Acta 85, 427 (1964). Best specificactivity. The table shows the yield of enzyme activity and specific activities of the fractions obtained by this purification procedure. Although the highest specific activity found in this experiment was 72, in some experiments specific activities as high as 105-128 were obtained, indicating a maximum purification of 800-fold. Unfortunately, the low total yield of protein at this stage precluded attempts at further purification or even determining the degree of purity by ordinary physical chemical procedures. The highly purified fructose diphosphatase is stable at pH 4.95 in 0.01 M citrate and m a y be stored at --20 ° for months without loss of activity. Properties
pH Optimum and Substrates. The purified enzyme has a pH optimum for activity of 8.25. I t is highly specific in that no hydrolysis of the following phosphorylated sugars at a concentration of 2 micromoles/ml can be detected: fructose l-phosphate, glucose l-phosphate, fructose 6phosphate, glucose 6-phosphate, ribulose 5-phosphate, or DL-glyeerophosphate. Inhibitors and K,~. An effective inhibitor of Euglena fructose diphosphatase is PCMB, giving almost complete inhibition at 5 }( 10-~M. Iodoaeetate has no effect, and sodium fluoride is only weakly inhibitory, requiring 2 }( 10.2 M concentration to even approach 5 0 ~ . A Lineweaver-Burke analysis of activity as a function of substrate concentration showed a Km of 3 X 10-~ M for fructose diphosphate. In these studies the magnesium concentration was varied with the F D P in order to maintain a constant Mg++:substrate ratio. Activators. I t was observed that optimum enzyme activity requires a
[113]
ALKALINE PHOSPHATASE (CRYSTALLINE)
639
distinct ratio of magnesium to substrate rather than a given magnesium concentration.2 Thus differing optimum curves axe obtained for activity versus magnesium concentration at three different FDP levels (2, 5, and 10 micromoles/ml, respectively). However, when these data are recalculated on the basis of the Mg÷÷:FDP ratio, the optimum ratios coincide. The optimum Mg++:FDP ratio appears to be between 20 and 25 moles of Mg ÷÷ per mole of FDP. This optimum ratio was independent of enzyme concentration over a fivefold range. Under certain conditions, EDTA will enhance the activity. The optimum concentration of EDTA for enhancement apparently depends on the Mg ++:FDP ratio.
[ 113 ] A l k a l i n e P h o s p h a t a s e ( C r y s t a l l i n e ) 1
By M. MALAMYand B. L. HORECKER R-O-P -4- H20 -~ P -b ROH Assay Method
principle. The assay is based on the formation of p-nitrophenol in the hydrolysis of p-nitrophenylphosphate. This is measured spectrophotometrically at 420 m~. Reagents p-Nitrophenylphosphate: 1.0 mM in 1 M Tris buffer, pH 8
Procedure. To 1 ml of the buffered p-nitrophenylphosphate solution add the enzyme solution and read the absorption at 420 m~ in a Beckman DU spectrophotometer. Keep the cell compartment at 27 °. Calculate the units of enzyme on the basis of the amount required to liberate micromole of p-nitrophenol in 1 hour using a molar absorptivity coefficient for p-nitrophenol of 1.32 X 104. The specific activity is the number of units per milligram of protein under the conditions of the test. Determine protein by the turbidometric method of Biicher2 One milligram per milliliter of the purified protein gives an absorbance reading of 0.72 at 278 m ~ . ~, 8
1M. H. Malamy and B. L. ttorecker, Biochem. J. 3, 1893 (1964). 2T. Biicher, Biochim. Biophys. Acta 1, 292 (1947). 3D. J. Plocke, C. Levinthal, and B. L. Vallee, Biochemistry 1, 373 (1962).
PHOSPI:IATASE$
[112c]
inorganic phosphate (0.01 M). Fe ++, Cu +÷, Zn++, and Hg ++ do not inhibit at concentrations of 0.1 mM in the presence of 1.0 mM Mg ÷+, whereas Pb ++ (0.1 raM) and Ca +* (0.1 raM) inhibit 80-1009, Ag+ (0.1 mM) inhibits 3 0 9 , and fluoride (0.01 M) inhibits 50-60 9 . AMP inhibits enzymatic activity at concentrations of 0.05-0.5 mM. This inhibition is most marked at pH 7.5 in the presence of EDTA. Under these conditions, 0.1 mM AMP produces 80 9 inhibition of enzymatic activity.
[112c] F r u c t o s e - 1 , 6 - d i p h o s p h a t a s e III. Euglena gracilis
By A. A. APP Fructose 1,6-phosphate T H20 -+ D-fructose6-phosphate -{- P~ Assay Method Principle. The colorimetric assay measuring the liberation of inorganic phosphate may be conveniently used? Reagents
Tris buffer, 0.5 M, pH 8.4 MgC12, 0.1 M Na2EDTA, 0.016 M Fruetose-l,6-diphosphate, sodium salt 4 X 10-~ M Procedure. The following reagents are present in a total volume of 1 ml: 100 micromoles Tris (pH 8.4), 10 micromoles MgC12, 2 mieromoles FDP, 1.6 micromoles EDTA, and the enzyme solution to be tested. ~ After incubation at 30 ° (1-3 minutes, depending on the level of activity) the reaction is stopped with 5% trichloroacetic acid and a suitable aliquot of the supernatant assayed for P~.~ Blanks consist of all components except FDP. One unit of fructose diphosphatase activity is defined as the liberation of 1 micromole of P~ per minute. Protein was estimated by absorbency or by the Lowry procedure2 As long as consideration is given to phosphatase activity from other
1E. Racker and E. A. R. Schroeder, Arch. Biochem. Biophys. 74, 326 (1958). A. A. App and A. T. Jagendorf, Biochim. Biophys. Acta 85, 427 (1964). 3H. H. Taussky and E. Shorr, J. Biol. Chem. 20'2, 675 (1953). O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1961). See also Vol. III, p. 448.
[112c]
FRUCTOSE-1,6-DIPHOSPHATASE
637
than alkaline fruetose-l,6-diphosphatase, the assay may be employed with crude Euglena extracts. Purification Proce4ure
Euglena gracilis, strain Z, was grown aseptically in 9-liter carboys for 6-9 days in white light at 25 ° . The cultures were aerated through capillary tubing and grown in a previously described medium consisting of 0.5% proteose peptone, 0.2% yeast extract, and 0.1% sodium acetate2 The cells were harvested in a Sorvall continuous flow centrifuge, washed and resuspended in water, then broken by sonication for 3 minutes with a Raytheon 10-ke oscillator, Model DF 101. The sonicate from 230 g (wet weight) of EugIena was centrifuged for 15 minutes at 20,000 g. The pellet was resuspended in water, the mixture was recentrifuged, and the combined supernatants were dialyzed overnight against water at 4 ° . The dialyzed extract was centrifuged at 20,200 g for 15 minutes and the pH of the supernatant was adjusted to 5.1 with 0.1 M citric acid. This acidified supernatant was held at 60 ° for about 10 minutes, or until heavy precipitation occurred. The precipitate was removed by centrifuging, the supernatant solution was then cooled to 3 ° and acidified to pH 4.35 with more 0.1 M citric acid. The second precipitate was also removed by centrifugation, and the pH of the supernatant solution was raised to 4.95 by addition of sodium citrate. One milliliter of a protamine sulfate solution (5.0 mg/ml, pH 5.5) was then added for every 20 ml of enzyme solution. Precipitation was allowed to occur for 25 minutes at 4 °, and the precipitate was removed by centrifuging at 20,200 g for 15 minutes. The supernatant solution was retained and its pH readjusted to 4.95 if necessary. The next step was chromatography on a DEAE-cellulose column. The cellulose derivative was cleaned as described by Peterson and Sober, formed into a 3.25 X 13.0 em column, and equilibrated at 4 ° with 0.01 M citrate buffer (pH 5.5).6 Approximately 380 ml of the enzyme solution was then passed through the column. Gradient elution of the enzyme was accomplished using a mixing vessel with 1 liter of 0.01 M citrate buffer at pH 5.5 and a reservoir containing 0.01 M citric acid. In this procedure a great deal of protein was removed before the fructose diphosphatase began to appear in the effluent at approximately pH 4.8. The most active fractions from this first run were pooled and rechromatographed with an identical procedure using a 2 X 12.75 cm column and a 700 ml mixing vessel. Elution profiles from this second column showed a close association between fructose diphosphatase activity and the remaining material. G. Brawerman and N. Konigsberg, Biochim. Biophys. Acta 43, 374 (1960). s E. A. Peterson and H. A. Sober, see Vol. V [1].
638
[112c]
PHOSPHATASES PURIFICATION OF FRUCTOSE DIPHOSPHATASE FROM
Fraction Dialyzed, centrifuged Heated, centrifuged pH precipitation, centrifuged Protamine sulfate, centrifuged First DEAE-column Second DEAE-column
Euglena g r a c i l i s a
Specific Fructose activity Total Protein, diphosphatase (units/mg recovery (mg/ml) (units/ml) protein) (%) 20.0 4.0 2.75 2.37 0.130 0.100 b
3.25 2.80 2.08 2.25 3.65 7.2 b
0.16 0.70 0.76 0.95 28.0 72b
100 85 64 69 46 31
a Reproduced from A. A. App and A. T. Jagendorf, Biochim. Biophys. Acta 85, 427 (1964). Best specificactivity. The table shows the yield of enzyme activity and specific activities of the fractions obtained by this purification procedure. Although the highest specific activity found in this experiment was 72, in some experiments specific activities as high as 105-128 were obtained, indicating a maximum purification of 800-fold. Unfortunately, the low total yield of protein at this stage precluded attempts at further purification or even determining the degree of purity by ordinary physical chemical procedures. The highly purified fructose diphosphatase is stable at pH 4.95 in 0.01 M citrate and m a y be stored at --20 ° for months without loss of activity. Properties
pH Optimum and Substrates. The purified enzyme has a pH optimum for activity of 8.25. I t is highly specific in that no hydrolysis of the following phosphorylated sugars at a concentration of 2 micromoles/ml can be detected: fructose l-phosphate, glucose l-phosphate, fructose 6phosphate, glucose 6-phosphate, ribulose 5-phosphate, or DL-glyeerophosphate. Inhibitors and K,~. An effective inhibitor of Euglena fructose diphosphatase is PCMB, giving almost complete inhibition at 5 }( 10-~M. Iodoaeetate has no effect, and sodium fluoride is only weakly inhibitory, requiring 2 }( 10.2 M concentration to even approach 5 0 ~ . A Lineweaver-Burke analysis of activity as a function of substrate concentration showed a Km of 3 X 10-~ M for fructose diphosphate. In these studies the magnesium concentration was varied with the F D P in order to maintain a constant Mg++:substrate ratio. Activators. I t was observed that optimum enzyme activity requires a
[113]
ALKALINE PHOSPHATASE (CRYSTALLINE)
639
distinct ratio of magnesium to substrate rather than a given magnesium concentration.2 Thus differing optimum curves axe obtained for activity versus magnesium concentration at three different FDP levels (2, 5, and 10 micromoles/ml, respectively). However, when these data are recalculated on the basis of the Mg÷÷:FDP ratio, the optimum ratios coincide. The optimum Mg++:FDP ratio appears to be between 20 and 25 moles of Mg ÷÷ per mole of FDP. This optimum ratio was independent of enzyme concentration over a fivefold range. Under certain conditions, EDTA will enhance the activity. The optimum concentration of EDTA for enhancement apparently depends on the Mg ++:FDP ratio.
[ 113 ] A l k a l i n e P h o s p h a t a s e ( C r y s t a l l i n e ) 1
By M. MALAMYand B. L. HORECKER R-O-P -4- H20 -~ P -b ROH Assay Method
principle. The assay is based on the formation of p-nitrophenol in the hydrolysis of p-nitrophenylphosphate. This is measured spectrophotometrically at 420 m~. Reagents p-Nitrophenylphosphate: 1.0 mM in 1 M Tris buffer, pH 8
Procedure. To 1 ml of the buffered p-nitrophenylphosphate solution add the enzyme solution and read the absorption at 420 m~ in a Beckman DU spectrophotometer. Keep the cell compartment at 27 °. Calculate the units of enzyme on the basis of the amount required to liberate micromole of p-nitrophenol in 1 hour using a molar absorptivity coefficient for p-nitrophenol of 1.32 X 104. The specific activity is the number of units per milligram of protein under the conditions of the test. Determine protein by the turbidometric method of Biicher2 One milligram per milliliter of the purified protein gives an absorbance reading of 0.72 at 278 m ~ . ~, 8
1M. H. Malamy and B. L. ttorecker, Biochem. J. 3, 1893 (1964). 2T. Biicher, Biochim. Biophys. Acta 1, 292 (1947). 3D. J. Plocke, C. Levinthal, and B. L. Vallee, Biochemistry 1, 373 (1962).