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[14] Assay for d -ribose-5-phosphate ketol isomerase and d -ribulose-5-phosphate 3-epimerase
[14]
PENTOSE-PHOSPHATE
63
I S O M E R A S E AND E P I M E R A S E
1-phosphate is converted to nanomoles on the basis of the specific activity of the [UL-14C]fructose used. Appropriate expression of activity would then be nanomoles per milliliter per minute or nanomoles per milligram of protein per minute. C o m m e n t . The usual fructokinase assays do not directly measure the specific product fructose 1-phosphate, 7,8 and appear to be accurate for fructokinase activity only when the enzyme is in a semipurified state." I n a crude homogenate or in a crude 105,000 g supernatant, any fructokinase assay is inaccurate owing to the activity of nonspecific hexokinases which m a y contribute 30% of the total fructose phosphorylating activity depending on physiological factors such as diet. 1°-~: The assay for fructokinase described here appears to overcome this problem. 7See H. G. Hers, this series, Vol. 1 [34]. 8R. C. Adelman, P. D. Spolter, and S. J. Weinhouse, J. Biol. Chem. 241, 5467 (1966). R. C. Adelman, F. J. Ballard, and S. J. Weinhouse, J. Biol. Chem. 242, 3360 (1967). lo M. M. Weiser, H. Quill, and Isselbacher, K. J. J. Biol. Chem. 246, 2331 (1971). 11M. M. Weiser, H. Quill, and Isselbacher, K. J. Amer. J. Physiol. 221, 844 (1971). 12R. J. Grand and S. Jaksina, Gastroenterology 64~ 429 (1973).
[14] A s s a y f o r D - R i b o s e - 5 - p h o s p h a t e D-Ribulose- 5-phosphate
Ketol Isomerase
and
3-Epimerase
B y TERRY WOOD
D-Ribose 5-phosphate (
' D-ribulose 5-phosphate
isotn~rase
(
~ D-xylulose 5-phosphate
(1)
epimerase
The assay for the isomerase is based upon the increase in absorbance observed at 290 nm when ribose-5-phosphate ketol isomerase acts upon ribose 5-phosphate to form ribulose 5-phosphate. 1,2 When the reaction has reached equilibrium, the addition of epimerase gives rise to a further increase in absorbance as xylulose 5-phosphate is formed. 2 In the presence of an excess of the isomerase, the second rate of absorbance increase is proportional to the activity of the epimerase. Reagents
Triethanolamine. HC1 buffer, 100 m M , p H 7.4 D-Ribose 5-phosphate, disodium salt, 100 mM. Store at 2 ° D-Ribose-5-phosphate ketol isomerase, 200 units/ml, 2.7 m g / m l 1F. C. Knowles and N. G. Pon, J. Amer. Chem. Soc. 90, 6536 (1968). 2 T. Wood, Anal. Biochem. 33, 297 (1970).
64
ENZYME ASSAY PROCEDURES
[14]
Assay o] Ribose-5-phosphate Ketol Isomerase The reaction mixture contains 1.78 ml 50 mM triethanolamine.HC1 buffer and 0.20 ml 100 mM ribose 5-phosphate (20 t~moles). It is placed in the cell compartment at 37 ° of a double-beam spectrophotometer connected to a recorder and read against a control cuvette containing an equal volume of buffer. When temperature equilibrium is reached, 0.02 ml of a suitable dilution of isomerase is added to both cuvettes, and the increase in absorbance at 290 nm is recorded. The initial velocity is linear over at least 5 min for up to 0.2 unit of isomerase.
Assay o] Ribulose-5-phosphate 3-Epimerase The assay mixture is set up as before, and 0.01 ml (2 units) of the isomerase is added. The absorbance increases rapidly at first and then levels off after 12-15 min. If small amounts of epimerase are present in the isomerase the recorder trace will show a slight upward slope. A suitable dilution of epimerase in 0.01 ml is added, and the further increase in absorbance at 290 nm is recorded (Fig. 1). The initial velocity is linear over 5 min for up to 0.2 unit of epimerase.
Calculation o] Isomerase and Epimerase Activity Careful determination of the absorption coefficient for the conversion of ribose 5-phosphate to ribulose 5-phosphate gave a value of 72. 2 Ribulose 5-phosphate and xylulose 5-phosphate differ only in their configuration about carbon-3, and addition of epimerase does not change the absorbance of pure ribulose 5-phosphate. Consequently, the same absorption coefficient is used in calculating the activity of both enzymes.
A
290
EPIMERASE ISOMERASE
0.2
0
5
10
15
TIME (min)
FIG. 1. A s s a y of D-ribulose-5-phosphate 3-epimerase.
[14]
PENTOSE-PHOSPHATE
I S O M E R A S E AND E P I M E R A S E
65
If y = initial velocity in absorbance units per minute, then for a 2.00-ml volume: Rate in mieromoles/min = y X 2.00/0.072 Notes
1. The commercially available sodium salt can give an absorbance less than 0.04 at 290 nm. Ketopentose phosphates in the ribose 5-phosphate may be destroyed by treatment with alkali if desired2 2. The commercial spinach enzyme (75 units/mg) is very suitable. Once prepared, the solution may be stored at --20 ° and thawed when required. Repeated freezing and thawing does not appear to affect the enzyme and may be beneficial by inactivating traces of contaminating ribulose-5-phosphate 3-epimerase. The yeast enzyme (230 units/mg) contains less epimerase impurity but is inactivated by repeated freezing and thawing. 3. The chromophore has its absorption peak at 278-280 nm. A wavelength of 290 nm is used to reduce interference from proteins and nucleic acids. The exact structure of the absorbing chromophore is not certain, but the absorption coefficient is in the range of values for simple straightchain ketose phosphates. ~ After approximately 65 min at 37 ° in 50 mM triethanolamine.HC1 buffer, pH 7.4, the absorbance of the isomerase product begins to increase spontaneously and nearly doubles over the next 4 hr. This increase occurs even after ultrafiltration and is independent of the presence of enzyme2 For this reason the addition of epimerase after equilibrium has been reached should not be long delayed. In 50 mM sodium phosphate buffer the absorbance began to increase spontaneously after only 20 min, and it is recommended that the use of this buffer be avoided, although it is employed in the isomerase assay at 280 nm described by Knowles et al2 4. The validity of the isomerase assay has been checked against the phloroglucinol2 and cysteine-carbazole~ colorimetric methods and that of the assay for epimerase against the spectrophotometric method at 340 nm. 2 5. A number of substances such as EDTA, EGTA, 8-hydroxyquinoline, dithiothreitol, and mercaptoethanol, interfere by reacting with the product of the isgmerase reaction to give chromophores of greater absorb3F. Dickens and D. H. Williamson,Biochem. J. 64, 567 (1956). G. R. Gray and R. Barker, Biochemistry 9, 2454 (1970). T. Wood, unpublished results, 1972. ~F. C. Knowles, M. K. Pon, and N. G. Pon, Anal. Biochem. 29, 40 (1969).
66
ENZYME ASSAY PROCEDURES
[15]
ance at 290 nm. Thus, 10 t~l of 1 mM E D T A can mimic the reaction of 0.15 unit of epimerase. Consequently, the absence of artifacts should be checked by testing, in the assay, the buffers used to dissolve the enzymes. 6. The method for isomerase gave values close to those obtained by the 340 nm assay with undialyzed extracts of rat muscle, heart, kidney, intestine, brain, lung, spleen, and uterus, but spuriously high values were obtained with extracts of rat liver and rat blood hemolysates2
Applications The isomerase assay has been used to follow the purification of the enzyme from spinach 7 and other sources, 8 in the determination of Michaelis constants, 7,s and for the assay of isomerase activity in animal tissues, s,9 The epimerase assay has been used to follow the purification of the enzyme and determine its Michaelis constant, s 7 F. C. Knowles and N. G. Pon, Anal. Biochem. 24, 305 (1968). 8 M. E. Kiely, A. L. Stuart, and T. Wood, Biochim. Biophys. Acta 293, 534 (1973). F. C. Kauffman, J. Neurochem. 19, 1 (1972).
[15] F r u c t o s e - d i p h o s p h a t e A l d o l a s e , P y r u v a t e K i n a s e , a n d Pyridine Nucleotide-Linked Activities after Electrophoresis
By
WALTER A. SUSOR, EDWARD PENHOET, a n d WILLIAM J. RUTTER
A number of methods have been used to localize specific enzyme activities after electrophoresis. The widely used method of choice of most enzymes that can be coupled to the reduction of NAD or NADP is the coupled reduction of a tetrazolium dye to form an insoluble pigment. This method as applied to the detection of fructose-diphosphate aldolase 1 is described in detail below. There are in addition several enzymes which are more readily coupled to the oxidation of NADH or NADPH. These can be assayed by the disappearance of fluorescence or UV absorbance of the reduced nucleotides. The second method presented here was designed for the detection of pyruvate kinase, ~ an enzyme that can be coupled to the oxidation of 1 E. Penhoet, T. l~ajkumar, and W. J. Rutter, Proc. Nat. Acad. Sci. U.S. 56, 1275 (1966). 2 W. A. Susor and W. J. Rutter, Anal. Biochem. 43, 147 (1971).
PENTOSE-PHOSPHATE
63
I S O M E R A S E AND E P I M E R A S E
1-phosphate is converted to nanomoles on the basis of the specific activity of the [UL-14C]fructose used. Appropriate expression of activity would then be nanomoles per milliliter per minute or nanomoles per milligram of protein per minute. C o m m e n t . The usual fructokinase assays do not directly measure the specific product fructose 1-phosphate, 7,8 and appear to be accurate for fructokinase activity only when the enzyme is in a semipurified state." I n a crude homogenate or in a crude 105,000 g supernatant, any fructokinase assay is inaccurate owing to the activity of nonspecific hexokinases which m a y contribute 30% of the total fructose phosphorylating activity depending on physiological factors such as diet. 1°-~: The assay for fructokinase described here appears to overcome this problem. 7See H. G. Hers, this series, Vol. 1 [34]. 8R. C. Adelman, P. D. Spolter, and S. J. Weinhouse, J. Biol. Chem. 241, 5467 (1966). R. C. Adelman, F. J. Ballard, and S. J. Weinhouse, J. Biol. Chem. 242, 3360 (1967). lo M. M. Weiser, H. Quill, and Isselbacher, K. J. J. Biol. Chem. 246, 2331 (1971). 11M. M. Weiser, H. Quill, and Isselbacher, K. J. Amer. J. Physiol. 221, 844 (1971). 12R. J. Grand and S. Jaksina, Gastroenterology 64~ 429 (1973).
[14] A s s a y f o r D - R i b o s e - 5 - p h o s p h a t e D-Ribulose- 5-phosphate
Ketol Isomerase
and
3-Epimerase
B y TERRY WOOD
D-Ribose 5-phosphate (
' D-ribulose 5-phosphate
isotn~rase
(
~ D-xylulose 5-phosphate
(1)
epimerase
The assay for the isomerase is based upon the increase in absorbance observed at 290 nm when ribose-5-phosphate ketol isomerase acts upon ribose 5-phosphate to form ribulose 5-phosphate. 1,2 When the reaction has reached equilibrium, the addition of epimerase gives rise to a further increase in absorbance as xylulose 5-phosphate is formed. 2 In the presence of an excess of the isomerase, the second rate of absorbance increase is proportional to the activity of the epimerase. Reagents
Triethanolamine. HC1 buffer, 100 m M , p H 7.4 D-Ribose 5-phosphate, disodium salt, 100 mM. Store at 2 ° D-Ribose-5-phosphate ketol isomerase, 200 units/ml, 2.7 m g / m l 1F. C. Knowles and N. G. Pon, J. Amer. Chem. Soc. 90, 6536 (1968). 2 T. Wood, Anal. Biochem. 33, 297 (1970).
64
ENZYME ASSAY PROCEDURES
[14]
Assay o] Ribose-5-phosphate Ketol Isomerase The reaction mixture contains 1.78 ml 50 mM triethanolamine.HC1 buffer and 0.20 ml 100 mM ribose 5-phosphate (20 t~moles). It is placed in the cell compartment at 37 ° of a double-beam spectrophotometer connected to a recorder and read against a control cuvette containing an equal volume of buffer. When temperature equilibrium is reached, 0.02 ml of a suitable dilution of isomerase is added to both cuvettes, and the increase in absorbance at 290 nm is recorded. The initial velocity is linear over at least 5 min for up to 0.2 unit of isomerase.
Assay o] Ribulose-5-phosphate 3-Epimerase The assay mixture is set up as before, and 0.01 ml (2 units) of the isomerase is added. The absorbance increases rapidly at first and then levels off after 12-15 min. If small amounts of epimerase are present in the isomerase the recorder trace will show a slight upward slope. A suitable dilution of epimerase in 0.01 ml is added, and the further increase in absorbance at 290 nm is recorded (Fig. 1). The initial velocity is linear over 5 min for up to 0.2 unit of epimerase.
Calculation o] Isomerase and Epimerase Activity Careful determination of the absorption coefficient for the conversion of ribose 5-phosphate to ribulose 5-phosphate gave a value of 72. 2 Ribulose 5-phosphate and xylulose 5-phosphate differ only in their configuration about carbon-3, and addition of epimerase does not change the absorbance of pure ribulose 5-phosphate. Consequently, the same absorption coefficient is used in calculating the activity of both enzymes.
A
290
EPIMERASE ISOMERASE
0.2
0
5
10
15
TIME (min)
FIG. 1. A s s a y of D-ribulose-5-phosphate 3-epimerase.
[14]
PENTOSE-PHOSPHATE
I S O M E R A S E AND E P I M E R A S E
65
If y = initial velocity in absorbance units per minute, then for a 2.00-ml volume: Rate in mieromoles/min = y X 2.00/0.072 Notes
1. The commercially available sodium salt can give an absorbance less than 0.04 at 290 nm. Ketopentose phosphates in the ribose 5-phosphate may be destroyed by treatment with alkali if desired2 2. The commercial spinach enzyme (75 units/mg) is very suitable. Once prepared, the solution may be stored at --20 ° and thawed when required. Repeated freezing and thawing does not appear to affect the enzyme and may be beneficial by inactivating traces of contaminating ribulose-5-phosphate 3-epimerase. The yeast enzyme (230 units/mg) contains less epimerase impurity but is inactivated by repeated freezing and thawing. 3. The chromophore has its absorption peak at 278-280 nm. A wavelength of 290 nm is used to reduce interference from proteins and nucleic acids. The exact structure of the absorbing chromophore is not certain, but the absorption coefficient is in the range of values for simple straightchain ketose phosphates. ~ After approximately 65 min at 37 ° in 50 mM triethanolamine.HC1 buffer, pH 7.4, the absorbance of the isomerase product begins to increase spontaneously and nearly doubles over the next 4 hr. This increase occurs even after ultrafiltration and is independent of the presence of enzyme2 For this reason the addition of epimerase after equilibrium has been reached should not be long delayed. In 50 mM sodium phosphate buffer the absorbance began to increase spontaneously after only 20 min, and it is recommended that the use of this buffer be avoided, although it is employed in the isomerase assay at 280 nm described by Knowles et al2 4. The validity of the isomerase assay has been checked against the phloroglucinol2 and cysteine-carbazole~ colorimetric methods and that of the assay for epimerase against the spectrophotometric method at 340 nm. 2 5. A number of substances such as EDTA, EGTA, 8-hydroxyquinoline, dithiothreitol, and mercaptoethanol, interfere by reacting with the product of the isgmerase reaction to give chromophores of greater absorb3F. Dickens and D. H. Williamson,Biochem. J. 64, 567 (1956). G. R. Gray and R. Barker, Biochemistry 9, 2454 (1970). T. Wood, unpublished results, 1972. ~F. C. Knowles, M. K. Pon, and N. G. Pon, Anal. Biochem. 29, 40 (1969).
66
ENZYME ASSAY PROCEDURES
[15]
ance at 290 nm. Thus, 10 t~l of 1 mM E D T A can mimic the reaction of 0.15 unit of epimerase. Consequently, the absence of artifacts should be checked by testing, in the assay, the buffers used to dissolve the enzymes. 6. The method for isomerase gave values close to those obtained by the 340 nm assay with undialyzed extracts of rat muscle, heart, kidney, intestine, brain, lung, spleen, and uterus, but spuriously high values were obtained with extracts of rat liver and rat blood hemolysates2
Applications The isomerase assay has been used to follow the purification of the enzyme from spinach 7 and other sources, 8 in the determination of Michaelis constants, 7,s and for the assay of isomerase activity in animal tissues, s,9 The epimerase assay has been used to follow the purification of the enzyme and determine its Michaelis constant, s 7 F. C. Knowles and N. G. Pon, Anal. Biochem. 24, 305 (1968). 8 M. E. Kiely, A. L. Stuart, and T. Wood, Biochim. Biophys. Acta 293, 534 (1973). F. C. Kauffman, J. Neurochem. 19, 1 (1972).
[15] F r u c t o s e - d i p h o s p h a t e A l d o l a s e , P y r u v a t e K i n a s e , a n d Pyridine Nucleotide-Linked Activities after Electrophoresis
By
WALTER A. SUSOR, EDWARD PENHOET, a n d WILLIAM J. RUTTER
A number of methods have been used to localize specific enzyme activities after electrophoresis. The widely used method of choice of most enzymes that can be coupled to the reduction of NAD or NADP is the coupled reduction of a tetrazolium dye to form an insoluble pigment. This method as applied to the detection of fructose-diphosphate aldolase 1 is described in detail below. There are in addition several enzymes which are more readily coupled to the oxidation of NADH or NADPH. These can be assayed by the disappearance of fluorescence or UV absorbance of the reduced nucleotides. The second method presented here was designed for the detection of pyruvate kinase, ~ an enzyme that can be coupled to the oxidation of 1 E. Penhoet, T. l~ajkumar, and W. J. Rutter, Proc. Nat. Acad. Sci. U.S. 56, 1275 (1966). 2 W. A. Susor and W. J. Rutter, Anal. Biochem. 43, 147 (1971).