APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1979, p. 159-161 0099-2240/79/07-0159/03 $02.00/0
Vol. 38, No. 1
NOTES Purification of an Endogenous Polynucleotide Phosphorylase from Brevibacterium JM98A HUEI-HSIUNG YANG,t D. W. THAYER,* AND S. P. YANG Departments of Biological Sciences and Food and Nutrition, Texas Tech University, Lubbock, Texas 79409 Received for publication 22 June 1978
Polynucleotide phosphorylase was purified from Brevibacterium JM98A (ATCC 29895). Homopolynucleotides were arsenolysed in the order polyadenylate > polycytidylic acid > polyuridylic acid >> polyguanylate. The products were ribonucleoside 5'-monophosphates. A polynucleotide phosphorylase was identified in the reduction of endogenous nucleic acid in Brevibacterium JM98A ATCC 29895 incubated at 550C and pH 10.5 (12). The present work was undertaken to purify and partially characterize this enzyme. Brevibacterium JM98A (9, 10) was used in all the experiments. Cultures were maintained on tryptic soy agar (Difco) at 40C and transferred monthly. Cells were grown on 500 ml of tryptic soy broth (Difco) in 2,800-ml baffled Erlenmeyer flasks. The cultures were incubated at 350C and agitated at 250 rpm on a gyratory shaker. Protein was determined with the Folin-Ciocalteu reagent as described by Herbert et al. (4). The quantity of 5'-nucleotide-phosphate was assayed with 5'-nucleotidase (3). Inorganic phosphate in the reaction mixture was estimated by the method of Chen et al. (1). Identification of the nucleotides in the supernatant and the cold HC104 fraction was carried out by ion-exchange chromatography (7). Polynucleotide phosphorylase activity was determined by the liberation of inorganic Pi from ADP (6). One unit of enzyme was defined as the amount of enzyme that liberated 1 umol of inorganic phosphate in 20 min. Arsenolysis of synthetic polynucleotides was carried out by the method of Singer and O'Brien (8). The cells were suspended in tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 7.4, 0.01 M) and disrupted by sonification. The cellular constituents were fractionated by differential centrifugation into various sedimentation fractions described by Imada et al. (5). The S-1 fraction was separated from the debris t Present address: U. S. Department of Agriculture, Forest Service, Forest Products Laboratory, Madison, WI 53705.
by centrifugation at 26,500 x g for 45 min. The S-2 (soluble) fraction and the R-4 (ribosomal) fraction were removed from the S-2 fraction by centrifugation at 105,000 x g for 90 min. The enzyme was purified from the soluble fraction by precipitation with streptomycin, ammonium sulfate fractionation (6), zinc chloride precipitation, and diethylaminoethyl (DEAE)cellulose separation (8). The purification of the polynucleotide phosphorylase activity is summarized in Table 1. Fraction III, which was the supernatant solution following ZnCl2 precipitation, arsenolysed polyadenylic acid [poly(A)] at a greater rate than polycytidylic acid [poly(C)] or polyuridylic acid [poly(U)] (Fig. 1). Polyguanylate [poly(G)] was not attacked by the enzyme. The addition of poly(U) to poly(A) inhibited the rate of arsenolysis (Fig. 2). The polymerization of ADP by enzyme fractions III and IV was studied. The Km for synthesis of poly(A) from ADP by the ZnCl2 supernatant was 2.5 x 10-2 M, but it was 4.2 x 10' M by the DEAE-cellulose fraction TABLE 1. Purification ofpolynucleotide phosphorylase from Brevibacterium JM98A Fraction
I S-2 II Streptomycin precipitation and ammonium sulfate fractionation III ZnCl2 precipitation IV DEAE-cellulose peak fraction a EU, Enzyme units.
159
Total Vol-
unme
Pr-Total
tai (mg/
ac-
Sp ct
(EU/
MDtivity m) (EU)m) (Ml)ml
160 2.98 33.6 0.07 50 4.70 35.7 0.15
30 1.18 10 0.12
6.0 0.20 2.7 2.20
160
APPL. ENVIRON. MICROBIOL.
NOTES
4
(Fig. 3 and 4). Poly(G) was not attacked by Brevibacterium JM98A polynucleotide phosphorylase. Enzymes from Bacillus stearothermophilus and Thermus aquaticus showed a strong preference for poly(A) in the phosphorolysis reaction and did not attack poly(G) (11). But the phosphorolysis of poly(U) by the enzymes from those two
0 x
31
-i
21 I_
,' 20
I
E
1 16
%.v
I
0I la O 12 S 0
I
C
0
I
I
I
I
II
-I 0 I 2 3 4 5 6 7
8
c
(ADP)
.I' 4
0
5 5 4 2 Time, hr FIG. 1. Arsenolysis of polynucleotides. The reaction mixture contained 3.0 ml of 0.1 M Tris-buffer, pH 8.4, containing 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, 0.05 M Na2HAsO4, I mol of polynucleotide P, and 0.2 ml of enzyme solution. A control lacking polynucleotides was also prepared. Absorbancy was measured at 257, 280, 256, or 262 nm for poly(A) (0), poly(C) (5), poly(G) (A), orpoly(U) (x), 1
FIG. 3. Lineweaver-Burk plots of Pi release by fraction III enzyme as a function of ADP concentration.
12.0 z i
w
-J Cl)
respectively. The following molar absorptivity at Am.. was used to estimate the concentration of mononucleotides: 5'-AMP = 15.1 x 103; 5'-CMP = 13.0; 5'GMP = 12.2 x 103; and 5'- UMP = 10.0 x 103.
0J 1 1.0 I.
_ el
0.3
-3
c 0 a
.0
0.2
U C
3 5 (ADP) MX 1-2
7
FIG. 4. Lineweaver-Burk plots of Pi release by fraction IV enzyme as a function of ADP concentra-
EC
8
-I 0
tion.
0.1
sources was faster than that of poly(C); the opposite result was obtained in the present study. The rate of arsenolysis of a poly(A-U) mixture was strikingly dependent upon their relative 0 concentrations in the incubation mixture. When 2 3 4 5 6 1 Time, hr poly(A) and poly(U) are present, a helical strucFIG. 2. Arsenolysis of various mixttures ofpoly(A) ture is formed (2), and the rate of depolymeriand poly(U). The reaction mixtures ar.e as described zation is lower than that of the individual singlein Fig. 1. Symbols: (0) poly(A); (x) 3 poly(A) plus 1 stranded polymer. The Km value for the partially purified polypoly(U); (O), poly(A) plus I poly(L 9; and (A), 1 poly(A) plus 3 poly(U). nucleotide phosphorylase of Brevibacterium 4
1
NOTES
VOL. 38, 1979
JM98A was 4.2 mM and decreased during purification. The Km of polynucleotide phosphorylase of Brevibacterium JM98A was larger than the Km of 2.5 mM for the polynucleotide phosphorylase of B. stearothermophilus.
6. 7.
We are grateful for the helpful suggestions of R. C. Albin, J. S. Sevall, and C. L. Baugh. This research was supported by funds from The Dodge Jones Foundation and the State of Texas.
LITERATURE CITED 1. Chen, P. S., T. Y. Toribara, and H. Warner. 1956. Microdetermination of phosphorus. Anal. Chem. 28: 1756-1758. 2. Grunberg-Manago, M. 1959. Phosphorolyse et configuration macromoleculaire des polyribonucleotides biosynthetiques et des acides ribonucleiques. J. Mol. Biol. 1:240-259. 3. Heppel, L A., and R. J. Hilmoe. 1955. "5" Nucleotidases 5-AMP + H20 Adenosine + P. L. "5" Nucleotidase of seminal plasma. Methods Enzymol. 2:546-548. 4. Herbert, D., P. J. Phipps, and R. E. Strange. 1971. Chemical analysis of microbial cells, p. 209-344. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 5B. Academic Press, Inc., New York. 5. Imada, A., Y. Nakao, and K. Ogata. 1962. Excretion of
8.
9.
10.
11.
12.
161
5'-nucleotides by bacteria. Part III. Degradation of ribonucleic acid in a Bacillus by its own polynucleotide phosphorylase. Agric. Biol. Chem. 26:611-623. Kimbi, Y., and U. S. Littauer. 1968. Polynucleotide phosphorylase from Escherichia coli. Methods Enzymol. 12B:513-519. Ogata, K., A. Imada, and Y. Nakao. 1962. Excretion of 5'-nucleotides by bacteria. Part I. Accumulation of 5'nucleotides in the culture fluid of a BaciUus during growth. Agric. Biol. Chem. 26:586-595. Singer, M. F., and B. M. O'Brien. 1963. Polynucleotide phosphorylase ofMicrococcus lysodeikticus. II. Further purification of the enzyme and the arsenolysis of polyribonucleotides. J. Biol. Chem. 238:328-335. Thayer, D. W., and J. 0. Murray. 1977. Physiological, biochemical and morphological characteristics of mesquite wood-digesting bacteria. J. Gen. Microbiol. 101: 71-77. Thayer, D. W., S. P. Yang, A. B. Key, H. H. Yang, and J. W. Barker. 1975. Production of cattle feed by the growth of bacteria on mesquite wood. Dev. Ind. Microbiol. 16:465-474. Wood, J. N., and D. W. Hutchinson. 1976. Thermostable polynucleotide phosphorylases from Bacillus stearothermophilus and Thermus aquaticus. Nucleic Acids Res. 3:219-229. Yang, H.-H., D. W. Thayer, and S. P. Yang. 1979. Reduction of endogenous nucleic acid in a single-cell protein. Appl. Environ. Microbiol. 38:143-147.
Vol. 38, No. 1
NOTES Purification of an Endogenous Polynucleotide Phosphorylase from Brevibacterium JM98A HUEI-HSIUNG YANG,t D. W. THAYER,* AND S. P. YANG Departments of Biological Sciences and Food and Nutrition, Texas Tech University, Lubbock, Texas 79409 Received for publication 22 June 1978
Polynucleotide phosphorylase was purified from Brevibacterium JM98A (ATCC 29895). Homopolynucleotides were arsenolysed in the order polyadenylate > polycytidylic acid > polyuridylic acid >> polyguanylate. The products were ribonucleoside 5'-monophosphates. A polynucleotide phosphorylase was identified in the reduction of endogenous nucleic acid in Brevibacterium JM98A ATCC 29895 incubated at 550C and pH 10.5 (12). The present work was undertaken to purify and partially characterize this enzyme. Brevibacterium JM98A (9, 10) was used in all the experiments. Cultures were maintained on tryptic soy agar (Difco) at 40C and transferred monthly. Cells were grown on 500 ml of tryptic soy broth (Difco) in 2,800-ml baffled Erlenmeyer flasks. The cultures were incubated at 350C and agitated at 250 rpm on a gyratory shaker. Protein was determined with the Folin-Ciocalteu reagent as described by Herbert et al. (4). The quantity of 5'-nucleotide-phosphate was assayed with 5'-nucleotidase (3). Inorganic phosphate in the reaction mixture was estimated by the method of Chen et al. (1). Identification of the nucleotides in the supernatant and the cold HC104 fraction was carried out by ion-exchange chromatography (7). Polynucleotide phosphorylase activity was determined by the liberation of inorganic Pi from ADP (6). One unit of enzyme was defined as the amount of enzyme that liberated 1 umol of inorganic phosphate in 20 min. Arsenolysis of synthetic polynucleotides was carried out by the method of Singer and O'Brien (8). The cells were suspended in tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 7.4, 0.01 M) and disrupted by sonification. The cellular constituents were fractionated by differential centrifugation into various sedimentation fractions described by Imada et al. (5). The S-1 fraction was separated from the debris t Present address: U. S. Department of Agriculture, Forest Service, Forest Products Laboratory, Madison, WI 53705.
by centrifugation at 26,500 x g for 45 min. The S-2 (soluble) fraction and the R-4 (ribosomal) fraction were removed from the S-2 fraction by centrifugation at 105,000 x g for 90 min. The enzyme was purified from the soluble fraction by precipitation with streptomycin, ammonium sulfate fractionation (6), zinc chloride precipitation, and diethylaminoethyl (DEAE)cellulose separation (8). The purification of the polynucleotide phosphorylase activity is summarized in Table 1. Fraction III, which was the supernatant solution following ZnCl2 precipitation, arsenolysed polyadenylic acid [poly(A)] at a greater rate than polycytidylic acid [poly(C)] or polyuridylic acid [poly(U)] (Fig. 1). Polyguanylate [poly(G)] was not attacked by the enzyme. The addition of poly(U) to poly(A) inhibited the rate of arsenolysis (Fig. 2). The polymerization of ADP by enzyme fractions III and IV was studied. The Km for synthesis of poly(A) from ADP by the ZnCl2 supernatant was 2.5 x 10-2 M, but it was 4.2 x 10' M by the DEAE-cellulose fraction TABLE 1. Purification ofpolynucleotide phosphorylase from Brevibacterium JM98A Fraction
I S-2 II Streptomycin precipitation and ammonium sulfate fractionation III ZnCl2 precipitation IV DEAE-cellulose peak fraction a EU, Enzyme units.
159
Total Vol-
unme
Pr-Total
tai (mg/
ac-
Sp ct
(EU/
MDtivity m) (EU)m) (Ml)ml
160 2.98 33.6 0.07 50 4.70 35.7 0.15
30 1.18 10 0.12
6.0 0.20 2.7 2.20
160
APPL. ENVIRON. MICROBIOL.
NOTES
4
(Fig. 3 and 4). Poly(G) was not attacked by Brevibacterium JM98A polynucleotide phosphorylase. Enzymes from Bacillus stearothermophilus and Thermus aquaticus showed a strong preference for poly(A) in the phosphorolysis reaction and did not attack poly(G) (11). But the phosphorolysis of poly(U) by the enzymes from those two
0 x
31
-i
21 I_
,' 20
I
E
1 16
%.v
I
0I la O 12 S 0
I
C
0
I
I
I
I
II
-I 0 I 2 3 4 5 6 7
8
c
(ADP)
.I' 4
0
5 5 4 2 Time, hr FIG. 1. Arsenolysis of polynucleotides. The reaction mixture contained 3.0 ml of 0.1 M Tris-buffer, pH 8.4, containing 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, 0.05 M Na2HAsO4, I mol of polynucleotide P, and 0.2 ml of enzyme solution. A control lacking polynucleotides was also prepared. Absorbancy was measured at 257, 280, 256, or 262 nm for poly(A) (0), poly(C) (5), poly(G) (A), orpoly(U) (x), 1
FIG. 3. Lineweaver-Burk plots of Pi release by fraction III enzyme as a function of ADP concentration.
12.0 z i
w
-J Cl)
respectively. The following molar absorptivity at Am.. was used to estimate the concentration of mononucleotides: 5'-AMP = 15.1 x 103; 5'-CMP = 13.0; 5'GMP = 12.2 x 103; and 5'- UMP = 10.0 x 103.
0J 1 1.0 I.
_ el
0.3
-3
c 0 a
.0
0.2
U C
3 5 (ADP) MX 1-2
7
FIG. 4. Lineweaver-Burk plots of Pi release by fraction IV enzyme as a function of ADP concentra-
EC
8
-I 0
tion.
0.1
sources was faster than that of poly(C); the opposite result was obtained in the present study. The rate of arsenolysis of a poly(A-U) mixture was strikingly dependent upon their relative 0 concentrations in the incubation mixture. When 2 3 4 5 6 1 Time, hr poly(A) and poly(U) are present, a helical strucFIG. 2. Arsenolysis of various mixttures ofpoly(A) ture is formed (2), and the rate of depolymeriand poly(U). The reaction mixtures ar.e as described zation is lower than that of the individual singlein Fig. 1. Symbols: (0) poly(A); (x) 3 poly(A) plus 1 stranded polymer. The Km value for the partially purified polypoly(U); (O), poly(A) plus I poly(L 9; and (A), 1 poly(A) plus 3 poly(U). nucleotide phosphorylase of Brevibacterium 4
1
NOTES
VOL. 38, 1979
JM98A was 4.2 mM and decreased during purification. The Km of polynucleotide phosphorylase of Brevibacterium JM98A was larger than the Km of 2.5 mM for the polynucleotide phosphorylase of B. stearothermophilus.
6. 7.
We are grateful for the helpful suggestions of R. C. Albin, J. S. Sevall, and C. L. Baugh. This research was supported by funds from The Dodge Jones Foundation and the State of Texas.
LITERATURE CITED 1. Chen, P. S., T. Y. Toribara, and H. Warner. 1956. Microdetermination of phosphorus. Anal. Chem. 28: 1756-1758. 2. Grunberg-Manago, M. 1959. Phosphorolyse et configuration macromoleculaire des polyribonucleotides biosynthetiques et des acides ribonucleiques. J. Mol. Biol. 1:240-259. 3. Heppel, L A., and R. J. Hilmoe. 1955. "5" Nucleotidases 5-AMP + H20 Adenosine + P. L. "5" Nucleotidase of seminal plasma. Methods Enzymol. 2:546-548. 4. Herbert, D., P. J. Phipps, and R. E. Strange. 1971. Chemical analysis of microbial cells, p. 209-344. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 5B. Academic Press, Inc., New York. 5. Imada, A., Y. Nakao, and K. Ogata. 1962. Excretion of
8.
9.
10.
11.
12.
161
5'-nucleotides by bacteria. Part III. Degradation of ribonucleic acid in a Bacillus by its own polynucleotide phosphorylase. Agric. Biol. Chem. 26:611-623. Kimbi, Y., and U. S. Littauer. 1968. Polynucleotide phosphorylase from Escherichia coli. Methods Enzymol. 12B:513-519. Ogata, K., A. Imada, and Y. Nakao. 1962. Excretion of 5'-nucleotides by bacteria. Part I. Accumulation of 5'nucleotides in the culture fluid of a BaciUus during growth. Agric. Biol. Chem. 26:586-595. Singer, M. F., and B. M. O'Brien. 1963. Polynucleotide phosphorylase ofMicrococcus lysodeikticus. II. Further purification of the enzyme and the arsenolysis of polyribonucleotides. J. Biol. Chem. 238:328-335. Thayer, D. W., and J. 0. Murray. 1977. Physiological, biochemical and morphological characteristics of mesquite wood-digesting bacteria. J. Gen. Microbiol. 101: 71-77. Thayer, D. W., S. P. Yang, A. B. Key, H. H. Yang, and J. W. Barker. 1975. Production of cattle feed by the growth of bacteria on mesquite wood. Dev. Ind. Microbiol. 16:465-474. Wood, J. N., and D. W. Hutchinson. 1976. Thermostable polynucleotide phosphorylases from Bacillus stearothermophilus and Thermus aquaticus. Nucleic Acids Res. 3:219-229. Yang, H.-H., D. W. Thayer, and S. P. Yang. 1979. Reduction of endogenous nucleic acid in a single-cell protein. Appl. Environ. Microbiol. 38:143-147.
