- Email: [email protected]
[56] Fructose-1,6-bisphosphatase from chicken and rabbit muscle
340
PHOSPHATASES
[56]
e n z y m e retains about 43% of the specific activity of the native enzyme when assayed at p H 7.5 by measuring the release of Pi according to the method of Tashima and Yoshimura. 1 Interaction with Turkey Liver Aldolase. As o b s e r v e d with rabbit liver FBPase and aldolase/8 the ability of turkey liver F B P a s e or turkey liver aldolase to penetrate into the gel phase of Ultrogel AcA-34 ( L K B ) decreases significantly when these two enzymes are simultaneously present in the reaction mixture. This phenomenon is, however, not observed when Y - A M P is also present in the mixture. This suggests that turkey liver FBPase is incapable of forming a complex with turkey liver aldolase in the presence of 5'-AMP. is j. S. MacGregor, V. N. Singh, S. Davoust, E. Melloni, S. Pontremoli, and B. L. Horecker, Proc. Natl. Acad. Sci. U.S.A. 77, 3889 (1980).
[56] F r u c t o s e - l , 6 - b i s p h o s p h a t a s e Rabbit
from Chicken
and
Muscle
By JOHN S. MACGREGOR, A. E. ANNAMALAI, A. VAN Tot_, W. J. BLACK, 1 and B. L. HORECKER Fructose 1,6-bisphosphate + H20 ~ fructose 6-phosphate + Pi Assay Method P~qnciple. Fructose 6-phosphate produced in the reaction is converted to glucose 6-phosphate by phosphohexoisomerase. In the presence of glucose-6-phosphate dehydrogenase this product reduces NADP. The production of N A D P H is measured spectrophotometrically at 340 nm. 2 Reagents
Sodium fructose 1,6-bisphosphate, 5.0 m M Diethanolamine, 0.2 M - t r i e t h a n o l a m i n e , 0.2 M, adjusted to p H 7.5 with HCI ( D E A - T E A buffer) MgCI2, 0.5 M A m m o n i u m sulfate [(NH4)2SO4], 0.4 M EDTA, 10 mM, adjusted to p H 7.5 with N a O H i Deceased. 2 B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948).
METHODS IN ENZYMOLOGY, VOL. 90
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181990-6
[56]
CHICKEN AND RABBIT MUSCLE
FRU-I,6-P2ASE
341
NADP, 2 mM Glucose-6-phosphate dehydrogenase (Boehringer-Mannheim Corp.), crystalline suspension in (NH4)2SO4, 5 mg/ml, diluted 1:10 in D E A - T E A buffer Phosphohexoisomerase (Boehringer-Mannheim Corp.), crystalline suspension in (NH4hSO~, 2 mg/ml, diluted 1:5 in D E A - T E A buffer Phosphoenolpyruvate, 50 mM ATP, 50 mM Adenylate kinase (Boehringer-Mannheim Corp.), 0.5 mg/ml Pyruvate kinase (Boehringer-Mannheim Corp.), 1.0 mg/ml Procedure. Place into a quartz cell (1-cm light path) in a final volume of 1 ml: 0.1 ml of T E A - D E A buffer, 0.01 ml of MgCI2, 0.1 ml of EDTA, 0.01 ml of NADP, 0.01 ml each of glucose-6-phosphate dehydrogenase and phosphohexose isomerase, and the aliquot of enzyme to be measured. Record the absorbance at 340 nm at 1-min intervals until no further increase occurs. Add fructose bisphosphate (0.02 ml), and record the absorbance at 340 nm. In the initial purification steps for muscle fructose-1,6-bisphospbatases (Fru-P2ases) it is necessary to include an AMP-removing system in the assay. This consists of 0.01 ml each of phosphoenolpyruvate, ATP, adenylate kinase, and pyruvate kinase. Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount that converts 1 tzmol of fructose bisphosphate to fructose 6-phosphate in 1 rain at room temperature. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Bficher3 standardized with rabbit muscle aldolase. For the last steps protein is determined by analysis with fluorescamine after alkaline hydrolysis 4 with bovine serum albumin as the standard, or by dividing the absorbance at 280 nm by 0.81 or 0.70 for chicken muscle or rabbit muscle Fru-P2ases, respectively. Purification Procedure, Chicken Muscle Fru-P2ase All operations are carried out at 0-4 ° unless otherwise specified. The results of a typical purification procedure in which the enzyme is assayed in the presence of either Mg 2+ or Mn 2+ are presented in Table I. Evtract of Fresh Muscle. Two Cornish Cross hens are killed by exsanguination, and their breast muscles are rapidly excised, placed on ice, and processed immediately. The muscle (248 g) is rinsed in ice-cold :~ T. Bficher, Biochim. Biophys. Acta 1, 292 (1947). 4 N. Nakai, C. Y. Lai, and B. L. Horecker, A,ml. Biochem. 58, 563 (1974).
342
PHOSPHATASES
[56]
TABLE 1 PURIFICATION OF CHICKEN BREAST MUSCLE FRUCTOSE-1,6-BISPHOSPHATASE Total activity (units)"
Fraction Extract Heated fraction Blue D e x t r a n - S e p h a r o s e eluate
Specific activity ~
Volume (ml)
0.2 m M MnClz
5 mM MgCI2
0.2 m M MnCI2
5 mM MgCI~
Recovery (%)
949 852 12
2031 1823 1358
1034 818 611
0.29 3.6 46.0
0.15 1.6 22.4
100 90 67
'~ The e n z y m e was a s s a y e d spectrophotometrically at p H 7.5 in the presence of 40 m M (NH4)2SO4 as described in A s s a y Methods. Protein was determined by the turbidity method of Biicher 3 except in the last step, where fluorescamine was used after alkaline hydrolysis, a
homogenizing buffer (10 mM Tris-HC1, pH 7.2, containing 1 mM EDTA), cut into small pieces, and extracted with four volumes of homogenizing buffer in a large Waring blender at low speed for three 15-sec intervals. The homogenate is centrifuged at 20,000 g for 40 min, and the supernatant solution is filtered through four layers of cheesecloth (Extract). Heat Treatment. The extract is adjusted to pH 7.2 with 2 N NaOH, transferred to a 3-liter Erlenmeyer flask, and heated with constant stirring in a water bath initially at 85°. The temperature of the solution should reach 67° within approximately 5 min and is maintained at that temperature for an additional 3 min, after which it is cooled in a salt-ice bath to 15° within 6 min. The heat-treated extract is centrifuged at 40,000g for 30 min, and the supernatant solution is collected by filtration through glass wool (Heated fraction). Chromatography on Blue Dextran-Sepharose. The heated fraction is dialyzed overnight against four volumes of saturated (NH4)2SO4 solution. The precipitated protein is collected by centrifugation, dissolved in 25 ml of 10 mM Tris-HC1, pH 7.5, containing 1 mM EDTA and dialyzed for a total of 2 hr against 1 liter of the same buffer, replaced after 1 hr with 1 liter of fresh buffer. The enzyme solution, diluted to 500 ml with l0 mM Tris buffer, pH 7.5, is treated with 100 g (equivalent dry weight) of Blue Dextran-Sepharose (Blue Sepharose CL-6B, Pharmacia Fine Chemicals) that has been equilibrated with the same buffer. The solution is stirred in the cold for 30 min, at which time assay of an aliquot of the supernatant should show that >90% of the enzyme is bound to the adsorbent. The suspension is filtered through a coarse sintered-glass funnel using a slight vacuum, and the adsorbent is washed on the funnel with 4 liters of 10 mM
[56]
CHICKEN AND RABBIT MUSCLE FRU-1,6-P2ASE
343
TABLE II PURIFICATION OF RABBIT MUSCLE ERUCTOSE-I,6-BISPHOSPHATASE
Step
Volume (ml)
Total activity (units)
Specific activity (units/ml)
Yield (';÷)
Extract Heated fraction Phosphocellulose eluate
2500 2440 40
1300 1098 513
0.06" 0.68 22.1
100 84 40
" See Table I. Tris-HC1 buffer, pH 7.5, until the absorbance of the filtrate measured at 280 nm is less than 0.01. The Sepharose is then transferred to a column, 3.3 × 16 cm, and washed with 500 ml of 10 mM Tris-HC1, pH 7.5, containing 1 mM AMP. The e n z y m e is eluted with the same buffer, containing 1 m M AMP and 1 m M Fru-P2, at a flow rate of 15 ml/hr (Blue D e x t r a n Sepharose eluate). Ammonium Su~lte Precipitation. Pooled fractions containing the activity are dialyzed overnight against four volumes of saturated (NH4)2SO4 solution. The precipitated protein is stored at 4 ° as a suspension in 80% saturated (NH4hSO4. HomogeneiO,. The homogeneity of the enzyme is based on the following criteria. It emerges from the Blue Dextran column as a sharp peak with constant specific activity; a single band is observed in SDS-slab gel electrophoresis; the specific activity is high, comparable to that reported by Annamalai et al.5 for the homogeneous chicken enzyme isolated by chromatography on a column of phosphocellulose P-11. The enzyme can be crystallized from (NH4)2SO4 solutions, but the specific activity is not significantly altered? Purification Procedure, Rabbit Muscle Fru-Pease All procedures were performed at 0 - 4 ° unless otherwise stated. The results of a typical purification procedure are presented in Table II. Extraction. A rabbit is killed by exsanguination, and the hind leg and back muscles (652 g) are removed, cut into small pieces, and homogenized in 2500 ml o f 10 m M Tris-HC1, p H 7.5, containing 1 mM EDTA (homogenization buffer) in a large Waring blender run at high speed for two l-rain intervals. The homogenate is centrifuged at 20,000g for 40 min, and the residue is discarded. The supernatant solution, pH 6.5, is adjusted to pH 7.1 with 13.8 ml of 2 N K O H (Extract). 5 A. E. Annamalai, O. Tsolas, and B. L. Horecker, Arch. Biochem. Biophys. 183, 48 (1977).
344
PHOSPHATASES
[56]
Heat Treatment. The extract is transferred to four 2-liter Erlenmeyer flasks and heated with constant stirring in a water bath initially at 85 °. After approximately 6 min, when the temperature of the enzyme solution has reached 67 ° , the flasks are removed and rapidly cooled in an ice bath to 15°. The suspension is centrifuged at 20,000 g for 30 min, and the supernatant solution is collected by filtration through glass wool (Heated fraction). Chromatography on Phosphocelhdose. Phosphocellulose (Whatman, P-11) is prepared by removing the fines by decantation from water, followed by alternate washings with 0.5 N N a O H and 0.5 N HC1, then with water until neutral, followed by equilibration in 0.1 M sodium malonate buffer, pH 5.8, containing 1 mM EDTA. After equilibration in malonate buffer the exchanger is filtered and stored as a moist cake at 4 °. The heated fraction is diluted fivefold in ice-cold 1 mM EDTA, and the pH is adjusted to 5.8 by the addition of 1 M malonic acid. Phosphocellulose preequilibrated in malonate buffer is added (125 g of moist cake) to the diluted enzyme solution with constant stirring until more than 90% of the Fru-P2ase activity is adsorbed. The pH of the slurry is maintained at 5.8 during the addition ofphosphocellulose by the addition of 1 M malonic acid. The resin is collected on a Biichner funnel using a gentle vacuum and washed with an additional 2 liters of malonate buffer at a flow rate of about 100 ml/hr, until the absorbance of the effluent at 280 nm is less than 0.02. Fru-P2ase activity is then eluted at a flow rate of 30 ml/hr with 0.1 M sodium malonate, pH 5.8, containing 1 mM EDTA and 2 mM Fru-Pz. Fractions containing enzyme with high specific activity are pooled and precipitated by dialysis against (NH4)2SO4, as previously described for the chicken muscle Fru-P2ase. Properties
Molecular Weight and Subunit Structure. The chicken and rabbit muscle Fru-Pzases were found to have similar molecular weights of 143,000144,000, and each contains four apparently identical subunits, M,. = 36,000 e a c h ? '6 The NH2 terminus is blocked. 7 Catalytic Properties. In the presence of 0.2 mM Mn 2+, and 0.1 mM EDTA or a mixture of 1 mM citrate plus 1 mM histidine, the pH optimum is 7.1. In the absence of chelators, the optimum is shifted to pH 8.0. 5,6 6 W. J. Black, A. van Tol, J. Fernando, and B. L. Horecker, Arch. Biochem. Biophys. 151, 591 (1972). 7 B. L. Horecker, J. S. MacGregor, and S. Pontremoli, i , "Science and Scientists, E s s a y s by Biochemists, Biologists and C h e m i s t s " (M. K a g e y a m a , ed.), p. 107. Jpn. Sci. Soc. Press, Tokyo, 1981.
[57]
RABBIT L I V E R F R U C T O S E - 1 , 6 - B I S P H O S P H A T A S E
345
Rabbit and chicken muscle Fru-P2ases are very sensitive to inhibition by AMP with Ki values of 0.45 and 0.32 raM, respectively. 5. The activities are increased severalfold by the addition of 40 mM (NH4hSO4, and both enzymes are more active when assayed with 0.2 mM MnC12, as compared to 5 mM MgClz. In the presence of 40 mM NH4 +, the substrate-concentration curve is hyperbolic with Km -~ 10 mM. 5,6
[57] F r u c t o s e - l , 6 - b i s p h o s p h a t a s e f r o m R a b b i t L i v e r (Neutral Form) By B. L. HORECKER,W. C. McGREGOR, S. TRANIELLO,E. MELLONI, and S. PONTREMOEI
Fructose 1,6-bisphosphate + H20 ~
D - f r u c t o s e 6 - p h o s p h a t e + P~
Assay Method Pdnciple. Fructose 6-phosphate produced in the reaction is converted
to glucose 6-phosphate by phosphohexoisomerase. In the presence of glucose-6-phosphate dehydrogenase this product reduces NADP. The production of NADPH is measured spectrophotometrically at 340 nm. Reagents
Sodium fructose 1,6-bisphosphate, 0.01 M Diethanolamine, 0.2 M-triethanolamine, 0.2 M, adjusted to pH 7.5 with HC! (DEA-TEA buffer) MgClz, 0.2 M NADP, 0.02 M Glucose-6-phosphate dehydrogenase (Boehringer-Mannheim Corp.), crystalline suspension in (NH4hSO4, 5 mg/ml Phosphohexoisomerase (Boehringer-Mannheim Corp.), crystalline suspension in (NH4)2SO4, 10 mg/ml EDTA, 10 mM, adjusted to pH 7.5 with NaOH (NH4)zSO4, 1 M P r o c e d , re. Place in a quartz cell ( 1-cm light path) in a final volume of 1 ml: 0.1 ml of DEA-TEA buffer, 0.01 ml of MgC12, 0.01 ml of EDTA, 0.04 ml of (NH4)zSO4, 0.01 ml of NADP, 1 /,l each of glucose-6-phosphate dehydrogenase and phosphohexoisomerase, and the aliquot of enzyme to be measured. Record the absorbance at 340 nm for several minutes until
METHODS IN ENZYMOLOGY. VOL. 90
Copyright ~ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-18199(}-6
PHOSPHATASES
[56]
e n z y m e retains about 43% of the specific activity of the native enzyme when assayed at p H 7.5 by measuring the release of Pi according to the method of Tashima and Yoshimura. 1 Interaction with Turkey Liver Aldolase. As o b s e r v e d with rabbit liver FBPase and aldolase/8 the ability of turkey liver F B P a s e or turkey liver aldolase to penetrate into the gel phase of Ultrogel AcA-34 ( L K B ) decreases significantly when these two enzymes are simultaneously present in the reaction mixture. This phenomenon is, however, not observed when Y - A M P is also present in the mixture. This suggests that turkey liver FBPase is incapable of forming a complex with turkey liver aldolase in the presence of 5'-AMP. is j. S. MacGregor, V. N. Singh, S. Davoust, E. Melloni, S. Pontremoli, and B. L. Horecker, Proc. Natl. Acad. Sci. U.S.A. 77, 3889 (1980).
[56] F r u c t o s e - l , 6 - b i s p h o s p h a t a s e Rabbit
from Chicken
and
Muscle
By JOHN S. MACGREGOR, A. E. ANNAMALAI, A. VAN Tot_, W. J. BLACK, 1 and B. L. HORECKER Fructose 1,6-bisphosphate + H20 ~ fructose 6-phosphate + Pi Assay Method P~qnciple. Fructose 6-phosphate produced in the reaction is converted to glucose 6-phosphate by phosphohexoisomerase. In the presence of glucose-6-phosphate dehydrogenase this product reduces NADP. The production of N A D P H is measured spectrophotometrically at 340 nm. 2 Reagents
Sodium fructose 1,6-bisphosphate, 5.0 m M Diethanolamine, 0.2 M - t r i e t h a n o l a m i n e , 0.2 M, adjusted to p H 7.5 with HCI ( D E A - T E A buffer) MgCI2, 0.5 M A m m o n i u m sulfate [(NH4)2SO4], 0.4 M EDTA, 10 mM, adjusted to p H 7.5 with N a O H i Deceased. 2 B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948).
METHODS IN ENZYMOLOGY, VOL. 90
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181990-6
[56]
CHICKEN AND RABBIT MUSCLE
FRU-I,6-P2ASE
341
NADP, 2 mM Glucose-6-phosphate dehydrogenase (Boehringer-Mannheim Corp.), crystalline suspension in (NH4)2SO4, 5 mg/ml, diluted 1:10 in D E A - T E A buffer Phosphohexoisomerase (Boehringer-Mannheim Corp.), crystalline suspension in (NH4hSO~, 2 mg/ml, diluted 1:5 in D E A - T E A buffer Phosphoenolpyruvate, 50 mM ATP, 50 mM Adenylate kinase (Boehringer-Mannheim Corp.), 0.5 mg/ml Pyruvate kinase (Boehringer-Mannheim Corp.), 1.0 mg/ml Procedure. Place into a quartz cell (1-cm light path) in a final volume of 1 ml: 0.1 ml of T E A - D E A buffer, 0.01 ml of MgCI2, 0.1 ml of EDTA, 0.01 ml of NADP, 0.01 ml each of glucose-6-phosphate dehydrogenase and phosphohexose isomerase, and the aliquot of enzyme to be measured. Record the absorbance at 340 nm at 1-min intervals until no further increase occurs. Add fructose bisphosphate (0.02 ml), and record the absorbance at 340 nm. In the initial purification steps for muscle fructose-1,6-bisphospbatases (Fru-P2ases) it is necessary to include an AMP-removing system in the assay. This consists of 0.01 ml each of phosphoenolpyruvate, ATP, adenylate kinase, and pyruvate kinase. Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount that converts 1 tzmol of fructose bisphosphate to fructose 6-phosphate in 1 rain at room temperature. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Bficher3 standardized with rabbit muscle aldolase. For the last steps protein is determined by analysis with fluorescamine after alkaline hydrolysis 4 with bovine serum albumin as the standard, or by dividing the absorbance at 280 nm by 0.81 or 0.70 for chicken muscle or rabbit muscle Fru-P2ases, respectively. Purification Procedure, Chicken Muscle Fru-P2ase All operations are carried out at 0-4 ° unless otherwise specified. The results of a typical purification procedure in which the enzyme is assayed in the presence of either Mg 2+ or Mn 2+ are presented in Table I. Evtract of Fresh Muscle. Two Cornish Cross hens are killed by exsanguination, and their breast muscles are rapidly excised, placed on ice, and processed immediately. The muscle (248 g) is rinsed in ice-cold :~ T. Bficher, Biochim. Biophys. Acta 1, 292 (1947). 4 N. Nakai, C. Y. Lai, and B. L. Horecker, A,ml. Biochem. 58, 563 (1974).
342
PHOSPHATASES
[56]
TABLE 1 PURIFICATION OF CHICKEN BREAST MUSCLE FRUCTOSE-1,6-BISPHOSPHATASE Total activity (units)"
Fraction Extract Heated fraction Blue D e x t r a n - S e p h a r o s e eluate
Specific activity ~
Volume (ml)
0.2 m M MnClz
5 mM MgCI2
0.2 m M MnCI2
5 mM MgCI~
Recovery (%)
949 852 12
2031 1823 1358
1034 818 611
0.29 3.6 46.0
0.15 1.6 22.4
100 90 67
'~ The e n z y m e was a s s a y e d spectrophotometrically at p H 7.5 in the presence of 40 m M (NH4)2SO4 as described in A s s a y Methods. Protein was determined by the turbidity method of Biicher 3 except in the last step, where fluorescamine was used after alkaline hydrolysis, a
homogenizing buffer (10 mM Tris-HC1, pH 7.2, containing 1 mM EDTA), cut into small pieces, and extracted with four volumes of homogenizing buffer in a large Waring blender at low speed for three 15-sec intervals. The homogenate is centrifuged at 20,000 g for 40 min, and the supernatant solution is filtered through four layers of cheesecloth (Extract). Heat Treatment. The extract is adjusted to pH 7.2 with 2 N NaOH, transferred to a 3-liter Erlenmeyer flask, and heated with constant stirring in a water bath initially at 85°. The temperature of the solution should reach 67° within approximately 5 min and is maintained at that temperature for an additional 3 min, after which it is cooled in a salt-ice bath to 15° within 6 min. The heat-treated extract is centrifuged at 40,000g for 30 min, and the supernatant solution is collected by filtration through glass wool (Heated fraction). Chromatography on Blue Dextran-Sepharose. The heated fraction is dialyzed overnight against four volumes of saturated (NH4)2SO4 solution. The precipitated protein is collected by centrifugation, dissolved in 25 ml of 10 mM Tris-HC1, pH 7.5, containing 1 mM EDTA and dialyzed for a total of 2 hr against 1 liter of the same buffer, replaced after 1 hr with 1 liter of fresh buffer. The enzyme solution, diluted to 500 ml with l0 mM Tris buffer, pH 7.5, is treated with 100 g (equivalent dry weight) of Blue Dextran-Sepharose (Blue Sepharose CL-6B, Pharmacia Fine Chemicals) that has been equilibrated with the same buffer. The solution is stirred in the cold for 30 min, at which time assay of an aliquot of the supernatant should show that >90% of the enzyme is bound to the adsorbent. The suspension is filtered through a coarse sintered-glass funnel using a slight vacuum, and the adsorbent is washed on the funnel with 4 liters of 10 mM
[56]
CHICKEN AND RABBIT MUSCLE FRU-1,6-P2ASE
343
TABLE II PURIFICATION OF RABBIT MUSCLE ERUCTOSE-I,6-BISPHOSPHATASE
Step
Volume (ml)
Total activity (units)
Specific activity (units/ml)
Yield (';÷)
Extract Heated fraction Phosphocellulose eluate
2500 2440 40
1300 1098 513
0.06" 0.68 22.1
100 84 40
" See Table I. Tris-HC1 buffer, pH 7.5, until the absorbance of the filtrate measured at 280 nm is less than 0.01. The Sepharose is then transferred to a column, 3.3 × 16 cm, and washed with 500 ml of 10 mM Tris-HC1, pH 7.5, containing 1 mM AMP. The e n z y m e is eluted with the same buffer, containing 1 m M AMP and 1 m M Fru-P2, at a flow rate of 15 ml/hr (Blue D e x t r a n Sepharose eluate). Ammonium Su~lte Precipitation. Pooled fractions containing the activity are dialyzed overnight against four volumes of saturated (NH4)2SO4 solution. The precipitated protein is stored at 4 ° as a suspension in 80% saturated (NH4hSO4. HomogeneiO,. The homogeneity of the enzyme is based on the following criteria. It emerges from the Blue Dextran column as a sharp peak with constant specific activity; a single band is observed in SDS-slab gel electrophoresis; the specific activity is high, comparable to that reported by Annamalai et al.5 for the homogeneous chicken enzyme isolated by chromatography on a column of phosphocellulose P-11. The enzyme can be crystallized from (NH4)2SO4 solutions, but the specific activity is not significantly altered? Purification Procedure, Rabbit Muscle Fru-Pease All procedures were performed at 0 - 4 ° unless otherwise stated. The results of a typical purification procedure are presented in Table II. Extraction. A rabbit is killed by exsanguination, and the hind leg and back muscles (652 g) are removed, cut into small pieces, and homogenized in 2500 ml o f 10 m M Tris-HC1, p H 7.5, containing 1 mM EDTA (homogenization buffer) in a large Waring blender run at high speed for two l-rain intervals. The homogenate is centrifuged at 20,000g for 40 min, and the residue is discarded. The supernatant solution, pH 6.5, is adjusted to pH 7.1 with 13.8 ml of 2 N K O H (Extract). 5 A. E. Annamalai, O. Tsolas, and B. L. Horecker, Arch. Biochem. Biophys. 183, 48 (1977).
344
PHOSPHATASES
[56]
Heat Treatment. The extract is transferred to four 2-liter Erlenmeyer flasks and heated with constant stirring in a water bath initially at 85 °. After approximately 6 min, when the temperature of the enzyme solution has reached 67 ° , the flasks are removed and rapidly cooled in an ice bath to 15°. The suspension is centrifuged at 20,000 g for 30 min, and the supernatant solution is collected by filtration through glass wool (Heated fraction). Chromatography on Phosphocelhdose. Phosphocellulose (Whatman, P-11) is prepared by removing the fines by decantation from water, followed by alternate washings with 0.5 N N a O H and 0.5 N HC1, then with water until neutral, followed by equilibration in 0.1 M sodium malonate buffer, pH 5.8, containing 1 mM EDTA. After equilibration in malonate buffer the exchanger is filtered and stored as a moist cake at 4 °. The heated fraction is diluted fivefold in ice-cold 1 mM EDTA, and the pH is adjusted to 5.8 by the addition of 1 M malonic acid. Phosphocellulose preequilibrated in malonate buffer is added (125 g of moist cake) to the diluted enzyme solution with constant stirring until more than 90% of the Fru-P2ase activity is adsorbed. The pH of the slurry is maintained at 5.8 during the addition ofphosphocellulose by the addition of 1 M malonic acid. The resin is collected on a Biichner funnel using a gentle vacuum and washed with an additional 2 liters of malonate buffer at a flow rate of about 100 ml/hr, until the absorbance of the effluent at 280 nm is less than 0.02. Fru-P2ase activity is then eluted at a flow rate of 30 ml/hr with 0.1 M sodium malonate, pH 5.8, containing 1 mM EDTA and 2 mM Fru-Pz. Fractions containing enzyme with high specific activity are pooled and precipitated by dialysis against (NH4)2SO4, as previously described for the chicken muscle Fru-P2ase. Properties
Molecular Weight and Subunit Structure. The chicken and rabbit muscle Fru-Pzases were found to have similar molecular weights of 143,000144,000, and each contains four apparently identical subunits, M,. = 36,000 e a c h ? '6 The NH2 terminus is blocked. 7 Catalytic Properties. In the presence of 0.2 mM Mn 2+, and 0.1 mM EDTA or a mixture of 1 mM citrate plus 1 mM histidine, the pH optimum is 7.1. In the absence of chelators, the optimum is shifted to pH 8.0. 5,6 6 W. J. Black, A. van Tol, J. Fernando, and B. L. Horecker, Arch. Biochem. Biophys. 151, 591 (1972). 7 B. L. Horecker, J. S. MacGregor, and S. Pontremoli, i , "Science and Scientists, E s s a y s by Biochemists, Biologists and C h e m i s t s " (M. K a g e y a m a , ed.), p. 107. Jpn. Sci. Soc. Press, Tokyo, 1981.
[57]
RABBIT L I V E R F R U C T O S E - 1 , 6 - B I S P H O S P H A T A S E
345
Rabbit and chicken muscle Fru-P2ases are very sensitive to inhibition by AMP with Ki values of 0.45 and 0.32 raM, respectively. 5. The activities are increased severalfold by the addition of 40 mM (NH4hSO4, and both enzymes are more active when assayed with 0.2 mM MnC12, as compared to 5 mM MgClz. In the presence of 40 mM NH4 +, the substrate-concentration curve is hyperbolic with Km -~ 10 mM. 5,6
[57] F r u c t o s e - l , 6 - b i s p h o s p h a t a s e f r o m R a b b i t L i v e r (Neutral Form) By B. L. HORECKER,W. C. McGREGOR, S. TRANIELLO,E. MELLONI, and S. PONTREMOEI
Fructose 1,6-bisphosphate + H20 ~
D - f r u c t o s e 6 - p h o s p h a t e + P~
Assay Method Pdnciple. Fructose 6-phosphate produced in the reaction is converted
to glucose 6-phosphate by phosphohexoisomerase. In the presence of glucose-6-phosphate dehydrogenase this product reduces NADP. The production of NADPH is measured spectrophotometrically at 340 nm. Reagents
Sodium fructose 1,6-bisphosphate, 0.01 M Diethanolamine, 0.2 M-triethanolamine, 0.2 M, adjusted to pH 7.5 with HC! (DEA-TEA buffer) MgClz, 0.2 M NADP, 0.02 M Glucose-6-phosphate dehydrogenase (Boehringer-Mannheim Corp.), crystalline suspension in (NH4hSO4, 5 mg/ml Phosphohexoisomerase (Boehringer-Mannheim Corp.), crystalline suspension in (NH4)2SO4, 10 mg/ml EDTA, 10 mM, adjusted to pH 7.5 with NaOH (NH4)zSO4, 1 M P r o c e d , re. Place in a quartz cell ( 1-cm light path) in a final volume of 1 ml: 0.1 ml of DEA-TEA buffer, 0.01 ml of MgC12, 0.01 ml of EDTA, 0.04 ml of (NH4)zSO4, 0.01 ml of NADP, 1 /,l each of glucose-6-phosphate dehydrogenase and phosphohexoisomerase, and the aliquot of enzyme to be measured. Record the absorbance at 340 nm for several minutes until
METHODS IN ENZYMOLOGY. VOL. 90
Copyright ~ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-18199(}-6