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[84] Fructose-1,6-diphosphatase from liver
[84]
FRUCTOSE-1,6-DIPHOSPItATASE FROM LIVER
543
fore becomes an indicator for the presence of microsomes, as in mitochondria prepared from liver or kidney. Homogenates prepared from liver in isotonic sucrose, KC1, NaCl, or in distilled water have the same G-6-Pase activity, but mitochondria prepared from sucrose have no G-6-Pase activity, and those prepared from salt solutions may contain 10 % to 20 % of the total tissue activity.
[84] Fructose-l,6-diphosphatase from Liver FDP + H20 --~ Pi + F-6-P
By R. W. McGILVERY
Assay Method Principle. The method is that of Pogell and McGilvery' in which the P~ liberated from FDP is determined. Reagents 0.05 M FDP. Prepare a solution of the sodium salt at pH 7.4 preferably from the purified cyclohexylammonium salt? 0.05 M boric acid-NaOH buffer, pH 9.5. 0.05 M MgS04. 0.005 M MnC12. 0.05 M cysteine-NaOH, pH 9.5. Prepare this solution daily. 0.1 M trichloroacetic acid. Enzyme. Dilute the solution to be tested to a concentration of 5 to 20 units/ml, with water (see definition below). Crude preparations should be adjusted to pH 9.5 immediately before the assay. Procedure. Place 0.i ml. of FDP, 0.4 ml. of borate buffer, 0.I ml. of MgSO4, and 0.i ml. of MnCl~ in a centrifuge tube. Warm the tube and a container with the enzyme sample in a 38 ° bath for 5 minutes. Then add 0.2 ml. of enzyme to the tube, followed immediately by 0.i ml. of cysteine previously warmed to 38 °. After 20 minutes of incubation add 1 ml. of trichloroacetic acid and determine the Pi liberated, 8 which should not exceed 1.25 micromoles from 5 micromoles of FDP. Definition of Unit and Specific Activity. One unit of the enzyme is de1B. M. Pogell and R. W. McGilvery,J. Biol. Chem. in press. See Vol. III [22]. See Vol. III [147].
544
ENZYMES IN PHOSPHATE METABOLISM
[84]
fined as the amount which will liberate 1 micromole of P~ with linear kinetics in 1 hour under the above conditions. Specific activities are given in units per milligram of protein, estimated as 6.25 × Kjeldahl N for crude preparations and as (1.55E2s0 - 0.76E260) mg./ml, for transparent solutions. Application of Assay Method to Crude Preparations. No exact assay of crude extracts containing nonspecific phosphatases active at pH 9.5 can be made. A rough correction for contaminating activities can be made by substituting F-6-P for F D P in the assay and subtracting the value thus obtained from the FDPase assay. Preparation of the extracts at pH 4.5 (see Purification Procedure) may destroy most of the interfering enzymes, but tests should be made. In most cases, Mn ++ and cysteine should be omitted (see Properties). Purification Procedure Step 1 is essentially according to Gomori, 4 and the remaining steps are those of Pogell and McGilvery.1 Step 1. Preparation of Autolyzed Extract. Homogenize the livers from exsanguinated rabbits (6 to 8 make a convenient amount) for 2 minutes in a Waring blendor with 4 ml. of 0.005 M sodium lactate buffer, pH 3.5, per gram of tissue. Centrifuge the homogenate (specific activity of 1.3 units/rag.) for 30 minutes at 1000 X g. Transfer the supernatant, which should be near pH 4.5, to a flask in a 38 ° bath. Keep the flask in the bath for 8 hours after the temperature of the solution reaches 37 °. Chill the flask to 4 ° in ice, and adjust the solution to pH 7.0 with 1 N NaOH. Remove and discard insoluble materials by centrifugation for 60 minutes at 2500 X g. Step 2. First Ammonium Sulfate Fractionation. Bring the autolysate (specific activity of 20 to 25 units/mg.) to 2.28 M by the slow addition of solid ammonium sulfate and then to 2.75 M by the dropwise addition of 3.89 M ammonium sulfate solution. Remove and discard the precipitate by centrifugation for 20 minutes at 2500 X g. Raise the concentration of ammonium sulfate to 3.00 M by dropwise addition of 3.89 M solution. Collect the precipitated enzyme (45 to 65% from the autolysate, specific activity of 50 to 100 units/mg.) by centrifugation. Step 3. Second Ammonium Sulfate Fractionation. Dissolve the precipitate, and dilute the solution to a protein concentration of 4 to 4.5 mg./ml. with a measured volume of water. Measure the total volume, and calculate the salt concentration of the diluted solution. Remove a slight amount of insoluble material by centrifugation for 1 hour at 18,000 X g. 4 G. Gomori, J. Biol. Chem. 148, 139 (1943).
[84]
FRUCTOSE-1,6-DIPttOSPHATASE FROM LIVER
545
Adjust the supernatant to p H 7.0 with 1 N N a O H , and raise the salt concentration to 1.90 M with solid ammonium sulfate and then to 2.48 M by dropwise addition of 3.89 M solution. Centrifuge for 30 minutes at 2500 X g, and discard the precipitate. Raise the supernatant to 3.00 M ammonium sulfate b y addition of the 3.89 M solution. Collect the precipitate (25 to 35% from the autolyzate, specific activity of 100 to 120 units/mg.) b y centrifugation for 1 hour at 2500 X g. Step ~. Alumina Adsorption. Dissolve the precipitate, and dilute it to a concentration of 5 mg. of protein per milliliter with water. Bring the solution to p H 4.0 with 1 N lactic acid, and add an a m o u n t of C~ alumina 5 suspension containing an a m o u n t of alumina equal to the weight of protein. After 10 minutes of gentle stirring, collect the gel b y centrifugation. Successively wash the gel b y centrifugation with portions of 0.005 M sodium lactate, p H 4.1, and 0.1 M sodium borate, p H 8.5, equal in volume to the protein solution before addition of the alumina. Elute the enzyme b y three extractions of the gel with 0.1 M sodium borate, p H 9.25, at the centrifuge, using a volume one-third t h a t of the original solution before adsorption for each extraction. In occasional preparations, the use of more alumina with an eluting buffer of higher p H will be required. The eluate should contain ca. 2000 units per rabbit liver (10 to 15% of the activity from the autolysate) with a specific activity near 400 units/mg, of protein, representing an increase in specific activity of 300-fold over the original homogenate. SUMMARY OF PURIFICATION PROCEDUREa
Step
Fraction
1. Homogenate Autolysate 2. 2.75-3.00 M (NH4)2SO4 3. 2.48-3.00 M (NH4)~S04 4. Eluate from alumina
Totalb volume, ml.
Total units b
Specific activity, units/mg.
Recovery, %
1980 1320 --75
-122,000 56,000 40,000 16,800
1.3 20-25 50-100 100-120 400
-100 45-65 25-35 10-15
B. M. Pogell and R. W. McGilvery, J. Biol. Chem. in press. b The total values are for a preparation from 400 g. of rabbit liver. The other columns give the ranges encountered in various trials.
Properties Specificity. The enzyme has no effect on G-l-P, G-6-P, F-6-P, PGA, or L-sorbose-l-P. F-1-P and L-sorbose-l,6-di-P are hydrolyzed at rates of 6 See Vol. I [11] for preparation of alumina C~.
546
ENZYMES IN" PHOSPHATE METABOLISM
[85]
0.009 and 0.03, respectively, the rate of F D P hydrolysis, indicating a specificity toward the phosphate bond bearing a 1-relation to a 2,5-furanose ring of D-arabino configuration. Although only one of the P groups of F D P is hydrolyzed, occasional purified enzyme preparations give products having higher acid-labile P contents than does F-6-P. The cause of this anomaly is unknown. Activators and Inhibitors. Mg ++ or Mn ++ is required for activity. In crude extracts, Mg ++ is better than Mn ++, and addition of Mn ++ or cysteine in the presence of Mg ++ causes inhibition. At all stages beyond step 1, addition of Mn ++ or cysteine in the presence of Mg ++ results in stimulation and is necessary in order to demonstrate recovery of the enzyme on fractionation. The individual effects vary from one preparation to another and from step to step. The conditions given in the assay method represent a compromise. Preparations stimulated by 0.0005 M Mn ++ are slightly stimulated by Fe ++ and inhibited by Co ++, Ni ++, Cu ++, and Zn ++ at the same concentration? Fluoride at 0.01 M inhibits 60%. 4 Stability. Preparations carried through step 3 have been stored in the frozen state for 15 months with loss of less than half of the activity. After step 4, the stability is markedly lowered. The enzyme from step 2 can be incubated for 2 hours at 38 ° without substrate or activators at any pH between 4 and 9 with loss of less than half of the activity. Heating the crude extract at pH 4.6 in a 100 ° bath until its temperature rises to 70 ° causes 10 to 15% loss. At 75 ° the loss is about one-third, and at 80 ° it is greater than 90%. pH Optimum. The enzyme is most active under the assay conditions at p H 9.3 to 9.5. Serine, barbital, and borate buffers give equal values.
[85] "5" Nucleotidases 5-AMP + H~O --~ Adenosine + P By LEON A. HEPPEL and R. J. HILMOE " 5 " Nucleotidases 1 are enzymes which hydrolyze phosphate esterified at carbon 5' of the ribose or deoxyribose portions of the nucleotide molecule. Thus far, only mononucleotides have ~,een available as substrates; polynucleotides containing phosphomonoester groups at carbon 5' of the sugar have not been reported. 1j. Reis, Bull. soc. chim. biol. 16, 385 (1934).
FRUCTOSE-1,6-DIPHOSPItATASE FROM LIVER
543
fore becomes an indicator for the presence of microsomes, as in mitochondria prepared from liver or kidney. Homogenates prepared from liver in isotonic sucrose, KC1, NaCl, or in distilled water have the same G-6-Pase activity, but mitochondria prepared from sucrose have no G-6-Pase activity, and those prepared from salt solutions may contain 10 % to 20 % of the total tissue activity.
[84] Fructose-l,6-diphosphatase from Liver FDP + H20 --~ Pi + F-6-P
By R. W. McGILVERY
Assay Method Principle. The method is that of Pogell and McGilvery' in which the P~ liberated from FDP is determined. Reagents 0.05 M FDP. Prepare a solution of the sodium salt at pH 7.4 preferably from the purified cyclohexylammonium salt? 0.05 M boric acid-NaOH buffer, pH 9.5. 0.05 M MgS04. 0.005 M MnC12. 0.05 M cysteine-NaOH, pH 9.5. Prepare this solution daily. 0.1 M trichloroacetic acid. Enzyme. Dilute the solution to be tested to a concentration of 5 to 20 units/ml, with water (see definition below). Crude preparations should be adjusted to pH 9.5 immediately before the assay. Procedure. Place 0.i ml. of FDP, 0.4 ml. of borate buffer, 0.I ml. of MgSO4, and 0.i ml. of MnCl~ in a centrifuge tube. Warm the tube and a container with the enzyme sample in a 38 ° bath for 5 minutes. Then add 0.2 ml. of enzyme to the tube, followed immediately by 0.i ml. of cysteine previously warmed to 38 °. After 20 minutes of incubation add 1 ml. of trichloroacetic acid and determine the Pi liberated, 8 which should not exceed 1.25 micromoles from 5 micromoles of FDP. Definition of Unit and Specific Activity. One unit of the enzyme is de1B. M. Pogell and R. W. McGilvery,J. Biol. Chem. in press. See Vol. III [22]. See Vol. III [147].
544
ENZYMES IN PHOSPHATE METABOLISM
[84]
fined as the amount which will liberate 1 micromole of P~ with linear kinetics in 1 hour under the above conditions. Specific activities are given in units per milligram of protein, estimated as 6.25 × Kjeldahl N for crude preparations and as (1.55E2s0 - 0.76E260) mg./ml, for transparent solutions. Application of Assay Method to Crude Preparations. No exact assay of crude extracts containing nonspecific phosphatases active at pH 9.5 can be made. A rough correction for contaminating activities can be made by substituting F-6-P for F D P in the assay and subtracting the value thus obtained from the FDPase assay. Preparation of the extracts at pH 4.5 (see Purification Procedure) may destroy most of the interfering enzymes, but tests should be made. In most cases, Mn ++ and cysteine should be omitted (see Properties). Purification Procedure Step 1 is essentially according to Gomori, 4 and the remaining steps are those of Pogell and McGilvery.1 Step 1. Preparation of Autolyzed Extract. Homogenize the livers from exsanguinated rabbits (6 to 8 make a convenient amount) for 2 minutes in a Waring blendor with 4 ml. of 0.005 M sodium lactate buffer, pH 3.5, per gram of tissue. Centrifuge the homogenate (specific activity of 1.3 units/rag.) for 30 minutes at 1000 X g. Transfer the supernatant, which should be near pH 4.5, to a flask in a 38 ° bath. Keep the flask in the bath for 8 hours after the temperature of the solution reaches 37 °. Chill the flask to 4 ° in ice, and adjust the solution to pH 7.0 with 1 N NaOH. Remove and discard insoluble materials by centrifugation for 60 minutes at 2500 X g. Step 2. First Ammonium Sulfate Fractionation. Bring the autolysate (specific activity of 20 to 25 units/mg.) to 2.28 M by the slow addition of solid ammonium sulfate and then to 2.75 M by the dropwise addition of 3.89 M ammonium sulfate solution. Remove and discard the precipitate by centrifugation for 20 minutes at 2500 X g. Raise the concentration of ammonium sulfate to 3.00 M by dropwise addition of 3.89 M solution. Collect the precipitated enzyme (45 to 65% from the autolysate, specific activity of 50 to 100 units/mg.) by centrifugation. Step 3. Second Ammonium Sulfate Fractionation. Dissolve the precipitate, and dilute the solution to a protein concentration of 4 to 4.5 mg./ml. with a measured volume of water. Measure the total volume, and calculate the salt concentration of the diluted solution. Remove a slight amount of insoluble material by centrifugation for 1 hour at 18,000 X g. 4 G. Gomori, J. Biol. Chem. 148, 139 (1943).
[84]
FRUCTOSE-1,6-DIPttOSPHATASE FROM LIVER
545
Adjust the supernatant to p H 7.0 with 1 N N a O H , and raise the salt concentration to 1.90 M with solid ammonium sulfate and then to 2.48 M by dropwise addition of 3.89 M solution. Centrifuge for 30 minutes at 2500 X g, and discard the precipitate. Raise the supernatant to 3.00 M ammonium sulfate b y addition of the 3.89 M solution. Collect the precipitate (25 to 35% from the autolyzate, specific activity of 100 to 120 units/mg.) b y centrifugation for 1 hour at 2500 X g. Step ~. Alumina Adsorption. Dissolve the precipitate, and dilute it to a concentration of 5 mg. of protein per milliliter with water. Bring the solution to p H 4.0 with 1 N lactic acid, and add an a m o u n t of C~ alumina 5 suspension containing an a m o u n t of alumina equal to the weight of protein. After 10 minutes of gentle stirring, collect the gel b y centrifugation. Successively wash the gel b y centrifugation with portions of 0.005 M sodium lactate, p H 4.1, and 0.1 M sodium borate, p H 8.5, equal in volume to the protein solution before addition of the alumina. Elute the enzyme b y three extractions of the gel with 0.1 M sodium borate, p H 9.25, at the centrifuge, using a volume one-third t h a t of the original solution before adsorption for each extraction. In occasional preparations, the use of more alumina with an eluting buffer of higher p H will be required. The eluate should contain ca. 2000 units per rabbit liver (10 to 15% of the activity from the autolysate) with a specific activity near 400 units/mg, of protein, representing an increase in specific activity of 300-fold over the original homogenate. SUMMARY OF PURIFICATION PROCEDUREa
Step
Fraction
1. Homogenate Autolysate 2. 2.75-3.00 M (NH4)2SO4 3. 2.48-3.00 M (NH4)~S04 4. Eluate from alumina
Totalb volume, ml.
Total units b
Specific activity, units/mg.
Recovery, %
1980 1320 --75
-122,000 56,000 40,000 16,800
1.3 20-25 50-100 100-120 400
-100 45-65 25-35 10-15
B. M. Pogell and R. W. McGilvery, J. Biol. Chem. in press. b The total values are for a preparation from 400 g. of rabbit liver. The other columns give the ranges encountered in various trials.
Properties Specificity. The enzyme has no effect on G-l-P, G-6-P, F-6-P, PGA, or L-sorbose-l-P. F-1-P and L-sorbose-l,6-di-P are hydrolyzed at rates of 6 See Vol. I [11] for preparation of alumina C~.
546
ENZYMES IN" PHOSPHATE METABOLISM
[85]
0.009 and 0.03, respectively, the rate of F D P hydrolysis, indicating a specificity toward the phosphate bond bearing a 1-relation to a 2,5-furanose ring of D-arabino configuration. Although only one of the P groups of F D P is hydrolyzed, occasional purified enzyme preparations give products having higher acid-labile P contents than does F-6-P. The cause of this anomaly is unknown. Activators and Inhibitors. Mg ++ or Mn ++ is required for activity. In crude extracts, Mg ++ is better than Mn ++, and addition of Mn ++ or cysteine in the presence of Mg ++ causes inhibition. At all stages beyond step 1, addition of Mn ++ or cysteine in the presence of Mg ++ results in stimulation and is necessary in order to demonstrate recovery of the enzyme on fractionation. The individual effects vary from one preparation to another and from step to step. The conditions given in the assay method represent a compromise. Preparations stimulated by 0.0005 M Mn ++ are slightly stimulated by Fe ++ and inhibited by Co ++, Ni ++, Cu ++, and Zn ++ at the same concentration? Fluoride at 0.01 M inhibits 60%. 4 Stability. Preparations carried through step 3 have been stored in the frozen state for 15 months with loss of less than half of the activity. After step 4, the stability is markedly lowered. The enzyme from step 2 can be incubated for 2 hours at 38 ° without substrate or activators at any pH between 4 and 9 with loss of less than half of the activity. Heating the crude extract at pH 4.6 in a 100 ° bath until its temperature rises to 70 ° causes 10 to 15% loss. At 75 ° the loss is about one-third, and at 80 ° it is greater than 90%. pH Optimum. The enzyme is most active under the assay conditions at p H 9.3 to 9.5. Serine, barbital, and borate buffers give equal values.
[85] "5" Nucleotidases 5-AMP + H~O --~ Adenosine + P By LEON A. HEPPEL and R. J. HILMOE " 5 " Nucleotidases 1 are enzymes which hydrolyze phosphate esterified at carbon 5' of the ribose or deoxyribose portions of the nucleotide molecule. Thus far, only mononucleotides have ~,een available as substrates; polynucleotides containing phosphomonoester groups at carbon 5' of the sugar have not been reported. 1j. Reis, Bull. soc. chim. biol. 16, 385 (1934).