ANALYTICAL
BIOCHEMISTRY
75,
660-663
(1976)
A Rapid Assay for Ribokinase A
rapid
method
capable
of
detecting
[@*P]ATP is converted to ribose onto charcoal. The assay is linear of at least 4 X IOF mg of enzyme/ml.
low
5-[32P]phosphate in enzyme
The first enzyme in the biosynthetic (EC 2.7.1.15), catalyzes the conversion
by Eq. [ll. Ribose + ATP-
Mg2+
levels
of
ribokinase
which concentration
is
is not
to
given.
absorbable a lower limit
utilization of ribose, ribokinase of ribose to ribose 5phosphate
Ribose 5phosphate
+ ADP.
ill
This enzyme was of interest to us as a means of obtaining radiolabeled ribose 5-phosphate from ribose of high specific activity. The previous assay for ribokinase (1) relied upon precipitation of ribose 5-phosphate with Ba*+ and determination of the ribose remaining in the supernatant solution by the orcinol test. We have found several shortcomings in this method and have therefore developed a rapid and sensitive assay utilizing the transfer of 32P from [Y-~~P]ATP to ribose. METHODS
AND MATERIALS
[Y-~~P]ATP was prepared by the method of Glynn and Chappell (2). Other chemicals were from usual commercial sources. Ribokinase was prepared as previously described (1). Protein concentrations was determined by the method of Lowry et al. (3) using bovine serum albumin as a standard. The ribokinase assay is similar to the procedure described by Switzer for 5phosphoribosylpyrophosphate synthetase (4). Assay was initiated by addition of a small aliquot of enzyme (2-20 pg) to 0.5 ml of 0.1 M potassium phosphate buffer, pH 7.5, at 37°C which contained 5 mM ribose, 2 mM MgCl,, and 3 mM [y-32P]ATP (60,000-100,000 cpm). The reaction was stopped by addition of 0.5 ml of cold 5% perchloric acid, and the solution was incubated at 0°C for 15 min. Acid-washed Norit activated charcoal (0.3 ml of 20%, v/v, prepared according to Zimmerman, Ref. (5) was added, and the mixture was incubated for 15 min at 0°C. Sodium pyrophosphate (0.2 ml of 50 mM), pH 7.0, containing 5 mg/ml of bovine serum albumin was added, the material was mixed vigorously and centrifuged, and 0.5 ml of the supernatant solution was placed in 10 ml of Aquasol (New England Nuclear) which was counted in a liquid scintillation spectrometer. Recovery of
660 CopyrIght 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved
661
SHORT COMMUNICATIONS 91
I
I
I
I
I
0.40mg/ml
6-
7-
:
6-
T zJ
5-
;. B 4.-s E g6 3" ae 2-
TIME
(min.)
FIG. 1. Percentage of conversion of [y-3ZP]ATP to nonadsorbable 32P versus time. Fifty microliters of ribokinase containing the indicated total protein concentrations resulted in a linear increase of nonadsorbable 3zP with time. Ten percent conversion of ATP to ribose 5-phosphate represents approximately 6000 cpm measured. Controls contained either no ribose, 5 mM ribose 5-phosphate in place of ribose, or ribokinase, or heat-inactivated ribokinase (0.4 mg/ml, 100°C for I5 min). All controls showed a negligible increase in counts.
ribose Sphosphate was tested by resubjecting an aliquot of charcoaltreated assay solution to the charcoal adsorption procedure. RESULTS
Figure 1 shows the time course for 32P transfer to ribose by different concentrations of ribokinase. A ratio of 5:3:1 for ribose:ATP:Mg2+ was present. All reactions were essentially linear. Figure 2 shows that the transfer reaction was directly proportional to concentration of enzyme. In the absence of either active ribokinase or ribose no increase in counts per minute above background levels was found for up to 20 min. Ribose Sphosphate also gave no increase in counts, indicating that the ribokinase preparation was not contaminated by Sphosphoribosylpyrophosphate synthetase, phosphoribokinase, or an ATPase. Essentially 100% recovery of synthesized ribose 5-r2P]phosphate was obtained. An aliquot of supernatant containing 4277 cpnxJO.5 ml after
662
SHORT COMMUNICATIONS
Protein
Concentration.
n-q/ml
FIG. 2. Rate of increase of nonadsorbable 32P versus protein slopes of Fig. 1 are plotted as a function of protein concentration.
concentration.
The
the first charcoal adsorption contained 1429 cpm/OS ml after a second charcoal adsorption, resulting in threefold dilution. Thus both [Y-~~P]ATP adsorption and ribose 5-[32P]phosphate recovery are quantitative. DISCUSSION
We have found the previously reported assay procedure for ribokinase activity difficult to use quantitatively. The method is subject to incomplete precipitation of ribose Sphosphate by ethanolic Ba2+, coprecipitation of ribose, and lowered color yield in the orcinol test for ribose due to the presence of ethanol. The method reported here is rapid and, since it does not require extensive sample manipulation, a precisely controlled analytical procedure is not required. The availability of commercial [Y-~~]ATP makes the assay particularly convenient. However, this assay would be of restricted use in preparations containing phosphoribosylpyrophosphate synthetase, phosphoribokinase, or an ATPase. The presence of the two former enzymes would result in too great an increase in nonadsorbable 32P while an ATPase would result in a spurious increase. The best control for background activity not due to ribokinase is to conduct the assay in the presence of preadded ribose Sphosphate instead of ribose. ACKNOWLEDGMENTS This work was supported by the U. S. Public Health Service (NIH-l-ROl-GM20835). We thank Dr. Daniel Purich for a gift of [yzP]ATP.
REFERENCES 1. Horecker, Chem.
B. L., Gibbs, M., Klenow, 207, 393-403.
H., and Smyrniotis,
P. Z. (1954) J. Biol.
663
SHORTCOMMUNICATIONS 2. Glynn, I. M., and Chappell, 3. Lowry, 0. H., Rosebrough, Chem. 193,265-275. 4. Switzer, R. L. (1%9)J. Biol. 5. Zimmerman, S. B. (1963) in N. O., eds.), pp. 258-262,
J. B. (1%4) Biochem. J. 90, 147-149. N. J.. Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem.
244, 2854-2863.
Methods in Enzymology, (Colowick, Academic Press, New York.
S. P., and Kaplan,
ROBERT K. GOITEIN STANLEY M. PARSONS Department of Chemistry University of California Santa Barbara, California 93106 Received February 9, 1976; accepted
May
20, 1976
BIOCHEMISTRY
75,
660-663
(1976)
A Rapid Assay for Ribokinase A
rapid
method
capable
of
detecting
[@*P]ATP is converted to ribose onto charcoal. The assay is linear of at least 4 X IOF mg of enzyme/ml.
low
5-[32P]phosphate in enzyme
The first enzyme in the biosynthetic (EC 2.7.1.15), catalyzes the conversion
by Eq. [ll. Ribose + ATP-
Mg2+
levels
of
ribokinase
which concentration
is
is not
to
given.
absorbable a lower limit
utilization of ribose, ribokinase of ribose to ribose 5phosphate
Ribose 5phosphate
+ ADP.
ill
This enzyme was of interest to us as a means of obtaining radiolabeled ribose 5-phosphate from ribose of high specific activity. The previous assay for ribokinase (1) relied upon precipitation of ribose 5-phosphate with Ba*+ and determination of the ribose remaining in the supernatant solution by the orcinol test. We have found several shortcomings in this method and have therefore developed a rapid and sensitive assay utilizing the transfer of 32P from [Y-~~P]ATP to ribose. METHODS
AND MATERIALS
[Y-~~P]ATP was prepared by the method of Glynn and Chappell (2). Other chemicals were from usual commercial sources. Ribokinase was prepared as previously described (1). Protein concentrations was determined by the method of Lowry et al. (3) using bovine serum albumin as a standard. The ribokinase assay is similar to the procedure described by Switzer for 5phosphoribosylpyrophosphate synthetase (4). Assay was initiated by addition of a small aliquot of enzyme (2-20 pg) to 0.5 ml of 0.1 M potassium phosphate buffer, pH 7.5, at 37°C which contained 5 mM ribose, 2 mM MgCl,, and 3 mM [y-32P]ATP (60,000-100,000 cpm). The reaction was stopped by addition of 0.5 ml of cold 5% perchloric acid, and the solution was incubated at 0°C for 15 min. Acid-washed Norit activated charcoal (0.3 ml of 20%, v/v, prepared according to Zimmerman, Ref. (5) was added, and the mixture was incubated for 15 min at 0°C. Sodium pyrophosphate (0.2 ml of 50 mM), pH 7.0, containing 5 mg/ml of bovine serum albumin was added, the material was mixed vigorously and centrifuged, and 0.5 ml of the supernatant solution was placed in 10 ml of Aquasol (New England Nuclear) which was counted in a liquid scintillation spectrometer. Recovery of
660 CopyrIght 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved
661
SHORT COMMUNICATIONS 91
I
I
I
I
I
0.40mg/ml
6-
7-
:
6-
T zJ
5-
;. B 4.-s E g6 3" ae 2-
TIME
(min.)
FIG. 1. Percentage of conversion of [y-3ZP]ATP to nonadsorbable 32P versus time. Fifty microliters of ribokinase containing the indicated total protein concentrations resulted in a linear increase of nonadsorbable 3zP with time. Ten percent conversion of ATP to ribose 5-phosphate represents approximately 6000 cpm measured. Controls contained either no ribose, 5 mM ribose 5-phosphate in place of ribose, or ribokinase, or heat-inactivated ribokinase (0.4 mg/ml, 100°C for I5 min). All controls showed a negligible increase in counts.
ribose Sphosphate was tested by resubjecting an aliquot of charcoaltreated assay solution to the charcoal adsorption procedure. RESULTS
Figure 1 shows the time course for 32P transfer to ribose by different concentrations of ribokinase. A ratio of 5:3:1 for ribose:ATP:Mg2+ was present. All reactions were essentially linear. Figure 2 shows that the transfer reaction was directly proportional to concentration of enzyme. In the absence of either active ribokinase or ribose no increase in counts per minute above background levels was found for up to 20 min. Ribose Sphosphate also gave no increase in counts, indicating that the ribokinase preparation was not contaminated by Sphosphoribosylpyrophosphate synthetase, phosphoribokinase, or an ATPase. Essentially 100% recovery of synthesized ribose 5-r2P]phosphate was obtained. An aliquot of supernatant containing 4277 cpnxJO.5 ml after
662
SHORT COMMUNICATIONS
Protein
Concentration.
n-q/ml
FIG. 2. Rate of increase of nonadsorbable 32P versus protein slopes of Fig. 1 are plotted as a function of protein concentration.
concentration.
The
the first charcoal adsorption contained 1429 cpm/OS ml after a second charcoal adsorption, resulting in threefold dilution. Thus both [Y-~~P]ATP adsorption and ribose 5-[32P]phosphate recovery are quantitative. DISCUSSION
We have found the previously reported assay procedure for ribokinase activity difficult to use quantitatively. The method is subject to incomplete precipitation of ribose Sphosphate by ethanolic Ba2+, coprecipitation of ribose, and lowered color yield in the orcinol test for ribose due to the presence of ethanol. The method reported here is rapid and, since it does not require extensive sample manipulation, a precisely controlled analytical procedure is not required. The availability of commercial [Y-~~]ATP makes the assay particularly convenient. However, this assay would be of restricted use in preparations containing phosphoribosylpyrophosphate synthetase, phosphoribokinase, or an ATPase. The presence of the two former enzymes would result in too great an increase in nonadsorbable 32P while an ATPase would result in a spurious increase. The best control for background activity not due to ribokinase is to conduct the assay in the presence of preadded ribose Sphosphate instead of ribose. ACKNOWLEDGMENTS This work was supported by the U. S. Public Health Service (NIH-l-ROl-GM20835). We thank Dr. Daniel Purich for a gift of [yzP]ATP.
REFERENCES 1. Horecker, Chem.
B. L., Gibbs, M., Klenow, 207, 393-403.
H., and Smyrniotis,
P. Z. (1954) J. Biol.
663
SHORTCOMMUNICATIONS 2. Glynn, I. M., and Chappell, 3. Lowry, 0. H., Rosebrough, Chem. 193,265-275. 4. Switzer, R. L. (1%9)J. Biol. 5. Zimmerman, S. B. (1963) in N. O., eds.), pp. 258-262,
J. B. (1%4) Biochem. J. 90, 147-149. N. J.. Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem.
244, 2854-2863.
Methods in Enzymology, (Colowick, Academic Press, New York.
S. P., and Kaplan,
ROBERT K. GOITEIN STANLEY M. PARSONS Department of Chemistry University of California Santa Barbara, California 93106 Received February 9, 1976; accepted
May
20, 1976
