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[72] d -Ribulose-1,5-diphosphate oxygenase
484
CARBOXYL&SES ANn DECARBOXYLASES
[72]
HCO3- or 3-phosphoglycerate (competitive inhibitor with C021) might give even better preservation of enzyme activity during preparation than either alone.
Summary The extraction, purification, and assay procedures described herein allow the preparation of mg amounts of highly pure RuDPCase from up to 6 samples within 24 hr. The purification occurs under mild conditions, where minimal alteration or contamination of the native enzyme should be expected. The procedure is suited for a variety of applications where large amounts of purified protein are not required, especially where estimates of the specific activity of the enzyme in several tissue samples are required.
[72] D-Ribulose-l,5-diphosphate Oxygenase By G. H. LORIMER,T. J. ANDREWS,and N. E. TOLBERT D-Ribulose 1,5-diphosphate + O5
Mg2+
, 2-phosphoglycolate+ 3-phosphoglycerate- + 2H+
Assay Method Principle. Ribulose-diphosphate oxygenase catalyzes the irreversible oxidation of D-ribulose 1,5-diphosphate to 2-phosphoglycolate and 3-phosphoglycerate with the stoichiometry indicated by the above equation. 1-3 The activity of ribulose-diphosphate oxygenase is associated with that of ribulose-diphosphate carboxylase, and the oxygenase activity has been detected in the leaves of a variety of higher plants. The separation of ribulose-diphosphate oxygenase and ribulose-diphosphate carboxylase has not been achieved, and circumstantial evidence indicates that the same protein catalyzes both reactions. 2 Assay methods based upon the disappearance of ribulose diphosphate or the appearance of 3-phosphoglycerate are considered inadvisable, since carboxylation of the substrate would seriously interfere if traces of C02 were present. The assay methods employed therefore follow oxygen consumption either 1 G. Bowes, W. L. Ogren, and R. H. Hageman, Biochem. Biophys. Res. Commun. 45, 716 (1971). 2 T. J. Andrews, G. H. Lorimer, and N. E. Tolbert, Biochemistry 12, 11 (1973). 3G. H. Lorimer, T. J. Andrews, and N. E. Tolbert, Biochemistry 12, 18 (1973).
[72]
D-RIBULOSE-1,5-DIPHOSPHATE OXYGENASE
485
manometrically2 or by the use of an oxygen electrode. Parallel assays may be run for the ribulose diphosphate carboxylase activity. 4
Reagents Ammediol (2-amino-2-methyl-l,3-propanediol)-HC1, 0.2 M, pH 9.3 MgC12, 0.1 M EDTA, 0.1 M Dithiothreitol, 8 mM Ribulose diphosphate, Na~ (Sigma), 25 mM
Procedure. MANOMETRIC METHOD. Sensitivity is maximized by using small flasks (total volume about 3 ml). The reaction mixture contains 100 ~moles 2-amino-2-methyl-l,3-propanediol (ammediol)-HC1 at pH 9.3; 10 ~moles MgCl_,; 1 ~mole EDTA; 0.4 ~moles dithiothreitol, and 0.5-2 mg of enzyme protein in a volume of 0.92 ml. The side arm contains 2 ~moles of ribulose diphosphate in 0.08 ml. Prior to use, the enzyme is passed through a small column of Sephadex G-25 equilibrated with a solution of 25 mM glycylglycine-KOH at pH 7.7, 1 mM EDTA, and 1 mM dithiothreitol. The flasks are attached to the manometer (bath temperature, 25 °) and shaken (150 oscillations min-1) for 3 min with pure oxygen passing through and are equilibrated for a further 9 rain while closed. The reaction is started by tipping in the ribulose diphosphate. Readings are taken every 90 sec. A short lag is usually observed, followed by a linear reaction which continues until the ribulose diphosphate becomes limiting. Data are expressed as micromoles 02 uptake per milligram of protein per unit of time. A control in which ribulose diphosphate is omitted is run with each batch of assays. A control with boiled enzyme has a negligible rate. An all-glass submarine-type Warburg respirometer is greatly preferred, for other models lose 02 from tubing connectors. OXYGEN ELECTRODE METHOD. An electrode having a minimal "dead volume" behind the membrane is required. Before use, the enzyme is passed through Sephadex G-25 as described above. The dithiothreitol concentration is adjusted at this stage so that the amount carried over into the reaction mixture with the enzyme solution will result in a final dithiothreitol concentration in the range 0.1-0.4 mM. The assay is carried out at 25% Two milliliters of air-saturated buffer solution, containing 0.125 M ammediol-HC1 at pH 9.3 and 12.5 mM MgC12, is pipetted into the electrode chamber, followed by enzyme solution and water to give a total volume of 2.4 ml. A blank rate of 02 consumption, due largely to the oxidation of dithiothreitol, is observed at this stage. At dithiothreitol concentrations greater than 0.4 raM, this rate becomes unacceptably 4M. Wishnick and M. D. Lane, this series, Vol. 23, p. 570.
486
CARBOXYLASES AND DECARBOXYLASES
[72]
large. The oxygenase reaction is initiated by the addition of 2 tLmoles of ribulose diphosphate in 0.1 ml. At 02 concentrations in the region of air saturation (approximately 0.23 mM), the enzymic rate shows very nearly first-order dependence on 02 concentration. This is to be expected since the Km of ribulose-diphosphate oxygenase for O2 is 0.75 n~l~r (equivalent to an atmosphere containing about 65% 02).2 A knowledge of the O2 concentration prevailing at the time of the rate measurement is therefore of critical importance. Oxygenase activity is conveniently expressed as the first-order rate constant with respect to 02, calculated thus: (Enzymic rate - blank rate)/O~ concentration min-1 If the rates are expressed in chart divisions min -1 and the 02 concentration in chart divisions, the need to calibrate the electrode in absolute terms is avoided. Purification Procedure
Since the ribulose-diphosphate oxygenase reaction appears to be catalyzed by the same protein as ribulose-diphosphate carboxylase, the reader is referred to previously published procedures for the purification of ribulose diphosphate carboxylase. 4 k preparation greatly enriched with ribulose-diphosphate carboxylase and oxygenase activities, although not completely pure, may be obtained by sucrose density gradient centrifugation of leaf extracts from which the membranous components have been removed. This may be done on a small scale in swinging buckets ~ or on a larger scale by the use of a zonal rotor. 2 The following procedure is similar to that of Goldthwaite and Bogorad 5 but adapted to a zonal rotor. A 600-ml linear (by volume) density gradient from 15 to 30% (w/w) sucrose in 25 mM glycylglycine-KOH buffer at pH 7.7 is loaded into a B-30 (International Equipment Co.) zonal rotor from the rim, followed by sufficient 35% (w/w) sucrose to fill the rotor. Thirty milliliters of the enzyme solution is then pumped into the rotor core followed by a 50-ml overlay of the buffer. Centrifugation is then conducted for 4.5 hr at 50,000 rpm, after which the gradient is displaced by pumping 35% (w/w) sucrose into the rim of the rotor. The protein concentration is monitored by passage of the eluate through a recording UV monitor. A large peak of fraction 1 protein, which contains both the oxygenase and carboxylase activities, is well separated from less rapidly sedimenting contaminants. The apparent asymmetry of the fraction 1 protein peak is due to dilution incurred by the radial nature of the migration. 5j. j. Goldthwaite and L. Bogorad, Anal. Biochem. 41, 57 (1971).
[72]
D-RIBULOSE-1,5-DIPHOSPHATE OXYGENASE
487
Properties The oxygenase reaction is specific for ribulose diphosphate. No reaction can be detected with fructose 1,6-diphosphate, fructose 6-phosphate, 3-phosphoglycerate, ribulose 5-phosphate, or ribulose. 2 The Km (ribulose diphosphate), measured under a gas phase of 100% oxygen, which is not saturating, is approximately 0.2 mM. 2 The Km (05) is approximately 0.75 mMY The pH optimum of the ribulose-diphosphate oxygenase-catalyzed reaction is approximately 9.3.5 The presence of MgC12 and a sulfhydrylreducing reagent, such as dithiothreitol, is required for full activity. The enzyme should be preincubated with the MgC12 for an hour before assaying. Upon storage in the cold at 4 ° or at --18 °, the enzyme from some sources (i.e., spinach and tobacco leaves) may lose 25 to 75% of its activity in a few days, but full activity can be restored by heating to 55 ° for 10 min in the presence of 50 mM MgC12 and a sulfhydryl reagent.
CARBOXYL&SES ANn DECARBOXYLASES
[72]
HCO3- or 3-phosphoglycerate (competitive inhibitor with C021) might give even better preservation of enzyme activity during preparation than either alone.
Summary The extraction, purification, and assay procedures described herein allow the preparation of mg amounts of highly pure RuDPCase from up to 6 samples within 24 hr. The purification occurs under mild conditions, where minimal alteration or contamination of the native enzyme should be expected. The procedure is suited for a variety of applications where large amounts of purified protein are not required, especially where estimates of the specific activity of the enzyme in several tissue samples are required.
[72] D-Ribulose-l,5-diphosphate Oxygenase By G. H. LORIMER,T. J. ANDREWS,and N. E. TOLBERT D-Ribulose 1,5-diphosphate + O5
Mg2+
, 2-phosphoglycolate+ 3-phosphoglycerate- + 2H+
Assay Method Principle. Ribulose-diphosphate oxygenase catalyzes the irreversible oxidation of D-ribulose 1,5-diphosphate to 2-phosphoglycolate and 3-phosphoglycerate with the stoichiometry indicated by the above equation. 1-3 The activity of ribulose-diphosphate oxygenase is associated with that of ribulose-diphosphate carboxylase, and the oxygenase activity has been detected in the leaves of a variety of higher plants. The separation of ribulose-diphosphate oxygenase and ribulose-diphosphate carboxylase has not been achieved, and circumstantial evidence indicates that the same protein catalyzes both reactions. 2 Assay methods based upon the disappearance of ribulose diphosphate or the appearance of 3-phosphoglycerate are considered inadvisable, since carboxylation of the substrate would seriously interfere if traces of C02 were present. The assay methods employed therefore follow oxygen consumption either 1 G. Bowes, W. L. Ogren, and R. H. Hageman, Biochem. Biophys. Res. Commun. 45, 716 (1971). 2 T. J. Andrews, G. H. Lorimer, and N. E. Tolbert, Biochemistry 12, 11 (1973). 3G. H. Lorimer, T. J. Andrews, and N. E. Tolbert, Biochemistry 12, 18 (1973).
[72]
D-RIBULOSE-1,5-DIPHOSPHATE OXYGENASE
485
manometrically2 or by the use of an oxygen electrode. Parallel assays may be run for the ribulose diphosphate carboxylase activity. 4
Reagents Ammediol (2-amino-2-methyl-l,3-propanediol)-HC1, 0.2 M, pH 9.3 MgC12, 0.1 M EDTA, 0.1 M Dithiothreitol, 8 mM Ribulose diphosphate, Na~ (Sigma), 25 mM
Procedure. MANOMETRIC METHOD. Sensitivity is maximized by using small flasks (total volume about 3 ml). The reaction mixture contains 100 ~moles 2-amino-2-methyl-l,3-propanediol (ammediol)-HC1 at pH 9.3; 10 ~moles MgCl_,; 1 ~mole EDTA; 0.4 ~moles dithiothreitol, and 0.5-2 mg of enzyme protein in a volume of 0.92 ml. The side arm contains 2 ~moles of ribulose diphosphate in 0.08 ml. Prior to use, the enzyme is passed through a small column of Sephadex G-25 equilibrated with a solution of 25 mM glycylglycine-KOH at pH 7.7, 1 mM EDTA, and 1 mM dithiothreitol. The flasks are attached to the manometer (bath temperature, 25 °) and shaken (150 oscillations min-1) for 3 min with pure oxygen passing through and are equilibrated for a further 9 rain while closed. The reaction is started by tipping in the ribulose diphosphate. Readings are taken every 90 sec. A short lag is usually observed, followed by a linear reaction which continues until the ribulose diphosphate becomes limiting. Data are expressed as micromoles 02 uptake per milligram of protein per unit of time. A control in which ribulose diphosphate is omitted is run with each batch of assays. A control with boiled enzyme has a negligible rate. An all-glass submarine-type Warburg respirometer is greatly preferred, for other models lose 02 from tubing connectors. OXYGEN ELECTRODE METHOD. An electrode having a minimal "dead volume" behind the membrane is required. Before use, the enzyme is passed through Sephadex G-25 as described above. The dithiothreitol concentration is adjusted at this stage so that the amount carried over into the reaction mixture with the enzyme solution will result in a final dithiothreitol concentration in the range 0.1-0.4 mM. The assay is carried out at 25% Two milliliters of air-saturated buffer solution, containing 0.125 M ammediol-HC1 at pH 9.3 and 12.5 mM MgC12, is pipetted into the electrode chamber, followed by enzyme solution and water to give a total volume of 2.4 ml. A blank rate of 02 consumption, due largely to the oxidation of dithiothreitol, is observed at this stage. At dithiothreitol concentrations greater than 0.4 raM, this rate becomes unacceptably 4M. Wishnick and M. D. Lane, this series, Vol. 23, p. 570.
486
CARBOXYLASES AND DECARBOXYLASES
[72]
large. The oxygenase reaction is initiated by the addition of 2 tLmoles of ribulose diphosphate in 0.1 ml. At 02 concentrations in the region of air saturation (approximately 0.23 mM), the enzymic rate shows very nearly first-order dependence on 02 concentration. This is to be expected since the Km of ribulose-diphosphate oxygenase for O2 is 0.75 n~l~r (equivalent to an atmosphere containing about 65% 02).2 A knowledge of the O2 concentration prevailing at the time of the rate measurement is therefore of critical importance. Oxygenase activity is conveniently expressed as the first-order rate constant with respect to 02, calculated thus: (Enzymic rate - blank rate)/O~ concentration min-1 If the rates are expressed in chart divisions min -1 and the 02 concentration in chart divisions, the need to calibrate the electrode in absolute terms is avoided. Purification Procedure
Since the ribulose-diphosphate oxygenase reaction appears to be catalyzed by the same protein as ribulose-diphosphate carboxylase, the reader is referred to previously published procedures for the purification of ribulose diphosphate carboxylase. 4 k preparation greatly enriched with ribulose-diphosphate carboxylase and oxygenase activities, although not completely pure, may be obtained by sucrose density gradient centrifugation of leaf extracts from which the membranous components have been removed. This may be done on a small scale in swinging buckets ~ or on a larger scale by the use of a zonal rotor. 2 The following procedure is similar to that of Goldthwaite and Bogorad 5 but adapted to a zonal rotor. A 600-ml linear (by volume) density gradient from 15 to 30% (w/w) sucrose in 25 mM glycylglycine-KOH buffer at pH 7.7 is loaded into a B-30 (International Equipment Co.) zonal rotor from the rim, followed by sufficient 35% (w/w) sucrose to fill the rotor. Thirty milliliters of the enzyme solution is then pumped into the rotor core followed by a 50-ml overlay of the buffer. Centrifugation is then conducted for 4.5 hr at 50,000 rpm, after which the gradient is displaced by pumping 35% (w/w) sucrose into the rim of the rotor. The protein concentration is monitored by passage of the eluate through a recording UV monitor. A large peak of fraction 1 protein, which contains both the oxygenase and carboxylase activities, is well separated from less rapidly sedimenting contaminants. The apparent asymmetry of the fraction 1 protein peak is due to dilution incurred by the radial nature of the migration. 5j. j. Goldthwaite and L. Bogorad, Anal. Biochem. 41, 57 (1971).
[72]
D-RIBULOSE-1,5-DIPHOSPHATE OXYGENASE
487
Properties The oxygenase reaction is specific for ribulose diphosphate. No reaction can be detected with fructose 1,6-diphosphate, fructose 6-phosphate, 3-phosphoglycerate, ribulose 5-phosphate, or ribulose. 2 The Km (ribulose diphosphate), measured under a gas phase of 100% oxygen, which is not saturating, is approximately 0.2 mM. 2 The Km (05) is approximately 0.75 mMY The pH optimum of the ribulose-diphosphate oxygenase-catalyzed reaction is approximately 9.3.5 The presence of MgC12 and a sulfhydrylreducing reagent, such as dithiothreitol, is required for full activity. The enzyme should be preincubated with the MgC12 for an hour before assaying. Upon storage in the cold at 4 ° or at --18 °, the enzyme from some sources (i.e., spinach and tobacco leaves) may lose 25 to 75% of its activity in a few days, but full activity can be restored by heating to 55 ° for 10 min in the presence of 50 mM MgC12 and a sulfhydryl reagent.