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A coupled optical enzyme assay for phosphopentomutase
ANALYTICAL
BIOCHEMISTRY
A Coupled
123, 265-269
(1982)
Optical Enzyme Assay for Phosphopentomutase
MARIA GRAZIA TOZZI, ROBERTA CATALANI, Institute
of Biochemistry,
PIER LUIGI
Biophysics and Genetics, Via A. Volta 4, 56100
IPATA,
Faculty of Science, Piss. Italy
AND UMBERTO University
MURA
of Pisa,
Received December 4, 1981 Published assays for phosphopentomutase activity are based on acid lability differences between ribose l-phosphate and ribose S-phosphate. The present work describes a new method in which the isomerization of ribose 5-phosphate to ribose l-phosphate is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: ribose l-phosphate + adenine S phosphate + adenosine (adenosine phosphorylase); adenosine + HZ0 -) inosine + NH3 (adenosine deaminase). The method has been used to show some properties of Escherichia coli phosphopentomutase.
In the course of experiments on induction and repression of enzymes involved in exogenous purine compounds utilization in Bacillus cereus ( 1,2), it became obvious that a better method to measure the phosphopentomutase catalyzed interconversion of ribose 1-phosphate and ribose Sphosphate would be valuable. Because ribose l-phosphate is mainly produced by phosphorolytic cleavage of the A’-glycosidic bond of nucleosides, the mutase may be considered as a linkage between the carbohydrate and nucleoside metabolism, allowing the ribose moiety of the nucleosides to enter the sugar phosphate shunt. To our knowledge, no simple continuous assay has been reported for phosphoribomutase activity; sampling assays based on chemical differences between the two phosphopentoses have so far been used, in which
the formation of ribose l-phosphate from ribose Sphosphate is measured, by taking advantage of the acid lability of ribose lphosphate compared to ribose Sphosphate (3-6). On the other hand, phosphodeoxyribomutase activity has been measured spectrophotometrically, either by coupling it with deoxyribose-Sphosphate aldolase (7) or by following the formation of a hydrazone of deoxyribose 5-phosphate with phenylhydrazine (8). In this paper a coupled optical assay for phosphoribomutase is described and used to show some properties of the enzyme of Escherichia coli. The ribose l-phosphate formed from ribose-5-phosphate is determined spectrophotometrically, from the rate of its conversion to inosine, in the presence of adenine and excess adenosine phosphorylase and adenosine deaminase:
phosphoribomutase
ribose 5-phosphate t ribose l-phosphate
. ribose 1-phosphate
adenosine
phosphorylase
adenosine
deaminase
+ adenine .
adenosine + H,O
’ adenosine + Pi
) inosine + NH3 265
[II [21 [31
0003-2697/82/100265-05.$02.00/0 Copyright Q 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
266
TO221
MATERIALS
AND METHODS
Ribose 5-phosphate, glucose 1,6-diphosphate, deoxyribose 5-phosphate, adenine and other fine chemicals were purchased from Sigma Chemical Company (St. Louis, MO.). All other chemicals used were of reagent grade. Adenosine deaminase (EC 3.5.4.4) was obtained from Boehringer und Soehne and was diluted with water to give a concentration of 80 pg protein/ml before use. The contamination by inosine phosphoryIase of the diluted commercial enzyme preparation accounted for less than 0.02 phosphorylase units/ml. Adenosine phosphorylase was prepared from Bacillus cert~us, strain NCIB8 122, according to the procedure used to partially purify the same enzyme activity in Bacillus su~ti~is (9). B. cereus was grown in a standard medium supplemented with 20 mM glucose, which significantly repressed both adenosine deaminase and inosine phosphorylase ( 1). The final adenosine phosphorylase preparation was free of any detectable adenosine deaminase as well as inosine phosphorylase and contained 0.86 mg protein/ ml, with a specific activity of 1080 nmol adenosine formed/min/mg of protein. Phosphopentomutase was prepared from E. coli W essentially according to HammerJespersen and Munch-Petersen ( 10): 2 g of cells grown in glucose medium were harvested in stationary phase by centrifugation at 10,OOOg. The pellet, washed and suspended by 50 ml 0.1 M Tris-HCl, pH 7.4, containing 2 mM EDTA,’ was disrupted by sonic treatment. The supernatant at 10,OOOg of the homogenate was dialyzed overnight against 10 mM Tris-HCl, pH 7.4, 50 pM EDTA, concentrated by polyethylene glycol (PEG) to 3.5 ml, and ~hromatographed on Sephadex G-100 using 50 mM Tris-HCl, pH 7.4, 1 mM EDTA as eluant. Fractions containing ’ Abbreviations used: EDTA, ethylenediaminetetraacetic acid; PEG, polyethylene glycol.
ET AL.
most of the phosphopentomutase activity were pooled, concentrated by PEG to 11 ml, and dialyzed overnight against 10 mM TrisHCl, pH 7.4,50 FM EDTA. The final preparation contained 0.96 mg protein/ml, with a specific activity of 12 nmol of ribose lphosphate formed/min/mg of protein. Phosphopentomutase assay. The detailed standard procedure for the coupled optical enzyme assay used to measure the phosphopentomutase activity is the following: 1.7 ml of 0.1 M Tris-HCl buffer, pH 8.1, was pipetted into a cuvette, followed by 25 ~1 of 6.04 IIIM adenine, 75 ~1 of the adenosine phosphorylase preparations 50 ,cll of diluted adenosine deaminase, 20 ~1 of 1.35 mM glucose 1,6-diphosphate, phosphopentomutase preparation, and enough water to bring the volume to 2.1 ml. After a 3-min preincubation at 37”C, the reaction was started by addition of 100 ~1 of 80 mM ribose 5-phosphate. The decrease in absorbance at 265 nm, measured at 37°C against a reference cuvette containing the assay buffer, was monitored by a recording double-beam ACTA C III Beckman spectrophotometer. In the standard conditions, the assay is linear up to 15 min incubation or up to about 40% of adenine transformation. In these conditions no hypoxanthine is detectable in the assay mixture. RESULTS AND DISCUSSION
The coupled optical enzyme assay proposed to measure the phosphopentomutase activity has been used to show some properties of E. coli enzyme. A typical time course of the reaction is shown in Fig. 1. It can be seen that in the absence of the substrate (ribose 5-phosphate) the absorbance at 265 nm remained constant, showing the absence of adenase activity both in the auxiliary enzymes and in the mutase preparation. On the other hand, the auxiliary enzymes do not contain phospho~ntomutase activity, since the absorbance at 265 nm remained equally constant when the reaction
PHOSPHOPENTOMUTASE
was started with the mutase preparation instead of ribose Sphosphate (data not shown). A small increase in absorbance was constantly observed after ribose Sphosphate addition. This is to be ascribed to the presence of an unknown contaminant (which is not adenine) of commercial ribose Sphosphate, rather than to adenosine (an intermediate product in the assay) accumulation. In fact, the increase in absorbance, proportional to the ribose Sphosphate concentration, was observed even in the absence of both adenine and the auxiliary system. On the other hand, no effect on the initial absorbance increase was observed when a further strong excess of the auxiliary system in the assay condition was used. Finally, the small increase was not observed when deoxyribose Sphosphate was used as substrate. The rate of ribose l-phosphate formation as a function of increasing protein concentration is linear at least up to 0.3 mg of protein in the assay (Fig. 2). Moreover, the co-
257
ASSAY
pn
protein
Of
FIG. 2. Rate of ribose l-phosphate formation as a function of increasing protein concentration. The standard procedure described under Materials and Methods was used, with the indicated amounts of proteins of the mutase preparation. a, Fresh enzyme preparation; A, preparation stored 20 days at 4°C.
0.8. A
/;::111’.
0
1
z
3 ‘/[St
0 0
1
.B .-; 5E0.6 . c 0.6. P
2 Ribose
1
time (min) FIG. I. Time course of the change in absorbance at 265 nm during a coupled optical test for phosphopentomutase at 37’C. In a 3-ml cuvette containing 1.7 ml of 0.1 M Tris-Cl buffer, pH 8.1, the following additions were performed at the times indicated by the arrows: (a) 2.5 ~1 of 6.04 mM adenine; (b) 275 ~1 of a mixture containing 0.73 mM MnCl*, 98 pM glucose 1,6-diphosphate, 3.1 mU of phosphopentomutase, 730 mU of diIuted adenosine deaminase, and 80 mU of adenosine phosphorylase; and (c) 100 pl of 80 mM ribose Sphosphate.
n1 0.2. 0
0
4 I mM I
0 3 I/v 2
E & 0.4. m. 4
3 5-phosphate
1
/rli-.
0 1
2 deoxyribosa
1
2 3 I-phosphate
3
‘/I s1 4 ImM)
FIG. 3. Phosphopentomutase activity as a function of substrate concentration. The initial rates vs (A) ribose 5-phosphate and (B) deoxyribose 5-phosphate concentrations were measured in the standard assay conditions. Double reciprocal plots are shown in the insets.
TOZZI
ET AL.
10
PH
FIG. 4. Phospho~ntomutase activity as a function of pH. The standard assayconditions with 0.1 M Tris-HC1 buffer at the indicated pH values were used. Measurements were performed either with 1.8 mU (0) or 2.7 mU (0) of enzyme. The apparent catalytic activity of the auxitiary enzyme system (reported as percent of the maximum (A)) has been measured in the standard conditions in the absence of enzyme preparation; the reaction was started by addition of 100 aI of 2.3 mM ribose I-phosphate. Both mutase and auxiliary system activities have been calculated at different pH values by using the appropriate differences in extinction coefficients (I&,,,,,) between adenine and inosine (*).
incidence between the activity of a fresh enzyme preparation and the activity of the same enzyme preparation stored 20 days at
MnC$
OJW
FIG. 5. Enzyme activity as a function of MnC12 concentration. The standard procedure was used with the indicated final concentration of MnCl2.
20 Glucose
30 ‘7 l&3-diphosphate
(phII
FIG. 6. Enzyme activity as a function of glucose 1,6diphosphate concentration. The standard procedure was used with the indicated final ~ncentrat~on of glucose 1,6-diphosphate.
4°C furnishes evidence of a good stability of the mutase. Since both ribose l-phosphate and its 2deoxyderivative, deoxyribose 5-phosphate, are substrates for adenosine phosphorylase and both adenosine and deoxyadenosine are substrates for the calf intestinal mucose adenosine deaminase, ribose 5-phosphate as well as the deoxyribose 5-phosphate may be used as substrates. The effect of both substrates concentrations on the initial rates of the isomerization reaction catalyzed by E. coli enzyme is reported in Fig. 3. From double reciprocal plots a K, value of 0.87 mM was observed for the ribose 5-phosphate and of 1.33 mM for the deoxyribose 5-phosphate. The profile activity as a function of pH is shown in Fig. 4, In the same figure the differences in extinction coefficients at 265 nm between adenine and inosine, which have been used for enzymatic unit calculations at different pH values, are reported. The results obtained in the assay in which a higher amount of mutase preparation was used (Fig. 4) show the reliability of the method even in the pH region in which the catalytic activity of the auxiliary enzyme system is apparently reduced.
PHOSPHOPENTOMUTASE
The absolute requirements of metal ions and phosphorylated cofactors (i.e., either ribose 1,Sdiphosphate or glucose 1,6-diphosphate) for E. coli phosphopentomutase activity (10) have been tested. The enzyme activity dependence on MnCl, and glucose 1,6-diphosphate concentrations are reported in Figs. 5 and 6, respectively. The half-saturation concentration for MnCI, and glucose 1,Gdiphosphate calculated from double reciprocal plots are 20 and 2.2 PM, respectively.
2. Tozzi, M. G., Sgarrella, F., and Ipata, P. L. ( 198 1) Ital.
ACKNOWLEDGMENTS
REFERENCES 1. Tozzi, M. G., Sgarrella, F., and Ipata, P. L. (1981) Biochim.
Biophys.
Acta 678,460-466.
J. Biochem.
30, 172.
3. Klenow, H. (19.55) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 361-363. 4. Leloir, L. F., Cardini, C. E. (I 957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Voi. 3, 840-849. 5. Barth, P. T., Beacham, I. R., Ahmad, S. I., and Pritchard, R. H. (1968) Biochim. Biophys. Acta 161, X4-557.
6. Kammen, H. O., and Koo, R. ( 1969) 1. Biol. Chem. 244, 4888-4893. 7.
This paper was supported by the Italian CNR and by the Italian Board of Education.
269
ASSAY
Hoffee, P. A., and Robertson, B. C. ( 1969) f. Butteriol. 97, 1386-l 396.
8. Beacham, I. R. (1969) Biochim. Biophys. Acta 191, 158-161. 9. Senesi, S., Falcone, G., Mura, U., Sgarrella, F., and Ipata, P. L. (1976) FEBS Left. 64. 353-357. IO. Hammer-Jespersen, K., and Munch-Petersen, A. (1970)
Eur.
J. Biochem.
17, 397-407.
BIOCHEMISTRY
A Coupled
123, 265-269
(1982)
Optical Enzyme Assay for Phosphopentomutase
MARIA GRAZIA TOZZI, ROBERTA CATALANI, Institute
of Biochemistry,
PIER LUIGI
Biophysics and Genetics, Via A. Volta 4, 56100
IPATA,
Faculty of Science, Piss. Italy
AND UMBERTO University
MURA
of Pisa,
Received December 4, 1981 Published assays for phosphopentomutase activity are based on acid lability differences between ribose l-phosphate and ribose S-phosphate. The present work describes a new method in which the isomerization of ribose 5-phosphate to ribose l-phosphate is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: ribose l-phosphate + adenine S phosphate + adenosine (adenosine phosphorylase); adenosine + HZ0 -) inosine + NH3 (adenosine deaminase). The method has been used to show some properties of Escherichia coli phosphopentomutase.
In the course of experiments on induction and repression of enzymes involved in exogenous purine compounds utilization in Bacillus cereus ( 1,2), it became obvious that a better method to measure the phosphopentomutase catalyzed interconversion of ribose 1-phosphate and ribose Sphosphate would be valuable. Because ribose l-phosphate is mainly produced by phosphorolytic cleavage of the A’-glycosidic bond of nucleosides, the mutase may be considered as a linkage between the carbohydrate and nucleoside metabolism, allowing the ribose moiety of the nucleosides to enter the sugar phosphate shunt. To our knowledge, no simple continuous assay has been reported for phosphoribomutase activity; sampling assays based on chemical differences between the two phosphopentoses have so far been used, in which
the formation of ribose l-phosphate from ribose Sphosphate is measured, by taking advantage of the acid lability of ribose lphosphate compared to ribose Sphosphate (3-6). On the other hand, phosphodeoxyribomutase activity has been measured spectrophotometrically, either by coupling it with deoxyribose-Sphosphate aldolase (7) or by following the formation of a hydrazone of deoxyribose 5-phosphate with phenylhydrazine (8). In this paper a coupled optical assay for phosphoribomutase is described and used to show some properties of the enzyme of Escherichia coli. The ribose l-phosphate formed from ribose-5-phosphate is determined spectrophotometrically, from the rate of its conversion to inosine, in the presence of adenine and excess adenosine phosphorylase and adenosine deaminase:
phosphoribomutase
ribose 5-phosphate t ribose l-phosphate
. ribose 1-phosphate
adenosine
phosphorylase
adenosine
deaminase
+ adenine .
adenosine + H,O
’ adenosine + Pi
) inosine + NH3 265
[II [21 [31
0003-2697/82/100265-05.$02.00/0 Copyright Q 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
266
TO221
MATERIALS
AND METHODS
Ribose 5-phosphate, glucose 1,6-diphosphate, deoxyribose 5-phosphate, adenine and other fine chemicals were purchased from Sigma Chemical Company (St. Louis, MO.). All other chemicals used were of reagent grade. Adenosine deaminase (EC 3.5.4.4) was obtained from Boehringer und Soehne and was diluted with water to give a concentration of 80 pg protein/ml before use. The contamination by inosine phosphoryIase of the diluted commercial enzyme preparation accounted for less than 0.02 phosphorylase units/ml. Adenosine phosphorylase was prepared from Bacillus cert~us, strain NCIB8 122, according to the procedure used to partially purify the same enzyme activity in Bacillus su~ti~is (9). B. cereus was grown in a standard medium supplemented with 20 mM glucose, which significantly repressed both adenosine deaminase and inosine phosphorylase ( 1). The final adenosine phosphorylase preparation was free of any detectable adenosine deaminase as well as inosine phosphorylase and contained 0.86 mg protein/ ml, with a specific activity of 1080 nmol adenosine formed/min/mg of protein. Phosphopentomutase was prepared from E. coli W essentially according to HammerJespersen and Munch-Petersen ( 10): 2 g of cells grown in glucose medium were harvested in stationary phase by centrifugation at 10,OOOg. The pellet, washed and suspended by 50 ml 0.1 M Tris-HCl, pH 7.4, containing 2 mM EDTA,’ was disrupted by sonic treatment. The supernatant at 10,OOOg of the homogenate was dialyzed overnight against 10 mM Tris-HCl, pH 7.4, 50 pM EDTA, concentrated by polyethylene glycol (PEG) to 3.5 ml, and ~hromatographed on Sephadex G-100 using 50 mM Tris-HCl, pH 7.4, 1 mM EDTA as eluant. Fractions containing ’ Abbreviations used: EDTA, ethylenediaminetetraacetic acid; PEG, polyethylene glycol.
ET AL.
most of the phosphopentomutase activity were pooled, concentrated by PEG to 11 ml, and dialyzed overnight against 10 mM TrisHCl, pH 7.4,50 FM EDTA. The final preparation contained 0.96 mg protein/ml, with a specific activity of 12 nmol of ribose lphosphate formed/min/mg of protein. Phosphopentomutase assay. The detailed standard procedure for the coupled optical enzyme assay used to measure the phosphopentomutase activity is the following: 1.7 ml of 0.1 M Tris-HCl buffer, pH 8.1, was pipetted into a cuvette, followed by 25 ~1 of 6.04 IIIM adenine, 75 ~1 of the adenosine phosphorylase preparations 50 ,cll of diluted adenosine deaminase, 20 ~1 of 1.35 mM glucose 1,6-diphosphate, phosphopentomutase preparation, and enough water to bring the volume to 2.1 ml. After a 3-min preincubation at 37”C, the reaction was started by addition of 100 ~1 of 80 mM ribose 5-phosphate. The decrease in absorbance at 265 nm, measured at 37°C against a reference cuvette containing the assay buffer, was monitored by a recording double-beam ACTA C III Beckman spectrophotometer. In the standard conditions, the assay is linear up to 15 min incubation or up to about 40% of adenine transformation. In these conditions no hypoxanthine is detectable in the assay mixture. RESULTS AND DISCUSSION
The coupled optical enzyme assay proposed to measure the phosphopentomutase activity has been used to show some properties of E. coli enzyme. A typical time course of the reaction is shown in Fig. 1. It can be seen that in the absence of the substrate (ribose 5-phosphate) the absorbance at 265 nm remained constant, showing the absence of adenase activity both in the auxiliary enzymes and in the mutase preparation. On the other hand, the auxiliary enzymes do not contain phospho~ntomutase activity, since the absorbance at 265 nm remained equally constant when the reaction
PHOSPHOPENTOMUTASE
was started with the mutase preparation instead of ribose Sphosphate (data not shown). A small increase in absorbance was constantly observed after ribose Sphosphate addition. This is to be ascribed to the presence of an unknown contaminant (which is not adenine) of commercial ribose Sphosphate, rather than to adenosine (an intermediate product in the assay) accumulation. In fact, the increase in absorbance, proportional to the ribose Sphosphate concentration, was observed even in the absence of both adenine and the auxiliary system. On the other hand, no effect on the initial absorbance increase was observed when a further strong excess of the auxiliary system in the assay condition was used. Finally, the small increase was not observed when deoxyribose Sphosphate was used as substrate. The rate of ribose l-phosphate formation as a function of increasing protein concentration is linear at least up to 0.3 mg of protein in the assay (Fig. 2). Moreover, the co-
257
ASSAY
pn
protein
Of
FIG. 2. Rate of ribose l-phosphate formation as a function of increasing protein concentration. The standard procedure described under Materials and Methods was used, with the indicated amounts of proteins of the mutase preparation. a, Fresh enzyme preparation; A, preparation stored 20 days at 4°C.
0.8. A
/;::111’.
0
1
z
3 ‘/[St
0 0
1
.B .-; 5E0.6 . c 0.6. P
2 Ribose
1
time (min) FIG. I. Time course of the change in absorbance at 265 nm during a coupled optical test for phosphopentomutase at 37’C. In a 3-ml cuvette containing 1.7 ml of 0.1 M Tris-Cl buffer, pH 8.1, the following additions were performed at the times indicated by the arrows: (a) 2.5 ~1 of 6.04 mM adenine; (b) 275 ~1 of a mixture containing 0.73 mM MnCl*, 98 pM glucose 1,6-diphosphate, 3.1 mU of phosphopentomutase, 730 mU of diIuted adenosine deaminase, and 80 mU of adenosine phosphorylase; and (c) 100 pl of 80 mM ribose Sphosphate.
n1 0.2. 0
0
4 I mM I
0 3 I/v 2
E & 0.4. m. 4
3 5-phosphate
1
/rli-.
0 1
2 deoxyribosa
1
2 3 I-phosphate
3
‘/I s1 4 ImM)
FIG. 3. Phosphopentomutase activity as a function of substrate concentration. The initial rates vs (A) ribose 5-phosphate and (B) deoxyribose 5-phosphate concentrations were measured in the standard assay conditions. Double reciprocal plots are shown in the insets.
TOZZI
ET AL.
10
PH
FIG. 4. Phospho~ntomutase activity as a function of pH. The standard assayconditions with 0.1 M Tris-HC1 buffer at the indicated pH values were used. Measurements were performed either with 1.8 mU (0) or 2.7 mU (0) of enzyme. The apparent catalytic activity of the auxitiary enzyme system (reported as percent of the maximum (A)) has been measured in the standard conditions in the absence of enzyme preparation; the reaction was started by addition of 100 aI of 2.3 mM ribose I-phosphate. Both mutase and auxiliary system activities have been calculated at different pH values by using the appropriate differences in extinction coefficients (I&,,,,,) between adenine and inosine (*).
incidence between the activity of a fresh enzyme preparation and the activity of the same enzyme preparation stored 20 days at
MnC$
OJW
FIG. 5. Enzyme activity as a function of MnC12 concentration. The standard procedure was used with the indicated final concentration of MnCl2.
20 Glucose
30 ‘7 l&3-diphosphate
(phII
FIG. 6. Enzyme activity as a function of glucose 1,6diphosphate concentration. The standard procedure was used with the indicated final ~ncentrat~on of glucose 1,6-diphosphate.
4°C furnishes evidence of a good stability of the mutase. Since both ribose l-phosphate and its 2deoxyderivative, deoxyribose 5-phosphate, are substrates for adenosine phosphorylase and both adenosine and deoxyadenosine are substrates for the calf intestinal mucose adenosine deaminase, ribose 5-phosphate as well as the deoxyribose 5-phosphate may be used as substrates. The effect of both substrates concentrations on the initial rates of the isomerization reaction catalyzed by E. coli enzyme is reported in Fig. 3. From double reciprocal plots a K, value of 0.87 mM was observed for the ribose 5-phosphate and of 1.33 mM for the deoxyribose 5-phosphate. The profile activity as a function of pH is shown in Fig. 4, In the same figure the differences in extinction coefficients at 265 nm between adenine and inosine, which have been used for enzymatic unit calculations at different pH values, are reported. The results obtained in the assay in which a higher amount of mutase preparation was used (Fig. 4) show the reliability of the method even in the pH region in which the catalytic activity of the auxiliary enzyme system is apparently reduced.
PHOSPHOPENTOMUTASE
The absolute requirements of metal ions and phosphorylated cofactors (i.e., either ribose 1,Sdiphosphate or glucose 1,6-diphosphate) for E. coli phosphopentomutase activity (10) have been tested. The enzyme activity dependence on MnCl, and glucose 1,6-diphosphate concentrations are reported in Figs. 5 and 6, respectively. The half-saturation concentration for MnCI, and glucose 1,Gdiphosphate calculated from double reciprocal plots are 20 and 2.2 PM, respectively.
2. Tozzi, M. G., Sgarrella, F., and Ipata, P. L. ( 198 1) Ital.
ACKNOWLEDGMENTS
REFERENCES 1. Tozzi, M. G., Sgarrella, F., and Ipata, P. L. (1981) Biochim.
Biophys.
Acta 678,460-466.
J. Biochem.
30, 172.
3. Klenow, H. (19.55) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 361-363. 4. Leloir, L. F., Cardini, C. E. (I 957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Voi. 3, 840-849. 5. Barth, P. T., Beacham, I. R., Ahmad, S. I., and Pritchard, R. H. (1968) Biochim. Biophys. Acta 161, X4-557.
6. Kammen, H. O., and Koo, R. ( 1969) 1. Biol. Chem. 244, 4888-4893. 7.
This paper was supported by the Italian CNR and by the Italian Board of Education.
269
ASSAY
Hoffee, P. A., and Robertson, B. C. ( 1969) f. Butteriol. 97, 1386-l 396.
8. Beacham, I. R. (1969) Biochim. Biophys. Acta 191, 158-161. 9. Senesi, S., Falcone, G., Mura, U., Sgarrella, F., and Ipata, P. L. (1976) FEBS Left. 64. 353-357. IO. Hammer-Jespersen, K., and Munch-Petersen, A. (1970)
Eur.
J. Biochem.
17, 397-407.