- Email: [email protected]
[89] Phosphodiesterase from snake venom
[89]
PHOSPHODIESTERASE FROM SNAKE VENOM
561
utes) to remove insoluble material, and dialyzed overnight against 0.05 M NaHCO3. The extract is then made to a known volume. Such preparations are virtually free of specific glucose-6-phosphatase and may be used for assay of transphosphorylation activity with glucose as acceptor. Since acid phosphatases are readily extracted into solution in dilute salt solutions, the butanol extraction method has not been applied to these enzymes.
[89] Phosphodiesterase from Snake Venom R - - O - - P ( O 2 H ) - - O - - R ' + H20--, R--O--PO3H2 + R'OH
By G. C. BUTLER Assay Method Gulland and Jackson 1used diphenyl phosphate as substrate and measured the liberation of phenol. Sinsheimer and Koerner 2 measured the liberation of p-nitrophenol from bis(p-nitrophenyl)phosphate. The latter method is the most convenient procedure available at present for measuring phosphodiesterase activity. Neither of these methods will, however, detect the presence of 5'-nucleotidase, since this enzyme does not hydrolyze phenyl phosphates. The 5'-nucleotidase found in the venoms of Russell's viper, diamond rattlesnake, and water moccasin may be assayed by the liberation of inorganic phosphate from 5-AMP. The activities of both enzymes may be measured in a single digest by using as substrate the mixed deoxyoligonucleotides resulting from the action of deoxyribonuclease on DNA. The conversion of "uranium-insoluble" to "uranium-soluble ''~ phosphate is a measure of phosphodiesterase activity, and the liberation of inorganic phosphate indicates 5'-nucleotidase activity. This method of measuring diesterase activity is unreliable in the presence of appreciable amounts of 5'-nucleotidase because the latter enzyme removes the product of action of the former and may thus effect an activation of the diesterase. If the diesterase is to be used for the hydrolysis of polynucleotides, it is advisable to test its activity on the oligonucleotides.
Reagents Magnesium oligonucleotide (MgON) solution (0.5%). Dissolve 500 mg. of the dry powder, prepared by the method of Little 1 j. M. Gulland and E. M. Jackson, Biochem. J. 82, 590 (1938). 2 R. L. Sinsheimer and J. F. Koerner, J. Biol. Chem. 198, 293 (1952). R. O. Hurst, J. A. Little, and G. C. Butler, J. Biol. Chem. 188, 705 (1951).
562
ENZYMES IN PHOSPHATE METABOLISM
[89]
and Butler, 4 in 100 ml. of 0.05 M Na barbiturate-Na2C03 (Veronal-carbonate) buffer, pH 9.25. ~ Uranyl acetate-trichloroacetic acid reagent. Dissolve 1.56 g. of uranyl acetate in 100 ml. of 10 % trichloroacetic acid. Enzyme. For assay, dilute the enzyme solutions so that the phosphodiesterase gives, under the test conditions prescribed below, less than 75 % hydrolysis in 1 hour.
Procedure. Since magnesium oligonucleotide contains some "uraniumsoluble" nucleotides, the extent of this solubility must be measured before the action of the enzyme can be tested. To 2 ml. of the uranyl acetate solution (above) add 2 ml. of the MgON solution, stir the mixture, allow it to stand for !0 minutes, and then centrifuge it. Filter the supernatant solution through Whatman No. 44 paper. Dilute 1 ml. of the filtrate to 10 ml. with water, and determine the total phosphorus in 1 ml. by King's method2 After the total phosphorus content of the MgON solution has been determined, the percentage solubility in the uranium reagent may be calculated and used as the blank value for enzymic digestion. To 5 ml. of the MgON solution add 1 ml. of enzyme solution, and after 1 or 2 hours at 37 ° measure the percentage of phosphate soluble in the uranium reagent as described above. This value is a measure of the phosphodiesterase action. In another sample of the diluted uranium filtrate determine the inorganic phosphate which gives a measure of the phosphomonoesterase action. For rough assessment of enzyme activity the uranium precipitate may be observed visually, a small amount of precipitate indicating extensive phosphodiesterase action. This method is often useful in selecting samples, and in judging the dilution they need, for more precise measurement of enzymic activity. Purification Procedure
Principle. The method to be described is based on the finding of Hurst and Butler 7 that both the phosphodiesterase and the 5'-nucleotidase of snake venoms may be adsorbed from aqueous solutions onto packed cellulose fibers. Dilute solutions of neutral salts elute both these enzymes, the diesterase somewhat more readily than the 5'-nucleotidase. The following directions describe how Mr. I. G. Walker has applied this 4j. A. Little and G. C. Butler, J. Biol. Chem. 188, 695 (1951); see Vol. I I I [107]. s E. J. King and G. E. Delory, Enzymologia 81 278 (1940). e E. J. King, Biochem. J. 26, 292 (1932). 7 R. O. Hurst and G. C. Butler, J. Biol. Chem. 195, 91 (1951).
[89]
PHOSPHODIESTERASE FROM SNAKE VENOM
563
principle to the separation of the phosphatases in the venom of the water moccasin (Agkistrodon piscivorus), s The previously published procedure ~ has been modified to make it more versatile. The modified method can be applied to any snake venom by making, when necessary, adjustments in the concentration of the NaC1 solution used for elution. In general it is advisable to elute the phosphodiesterase with the weakest possible NaC1 solution. Preparation of the Cellulose Column. Tear up fifteen sheets of Whatman No. 5 filter paper 12.5 cm. in diameter into 0.5-inch squares, and disperse them in 700 ml. of water by 1 minute's agitation in a blendor. Pour about one-third of the resulting slurry into a glass tube 35 mm. in diameter and 50 cm. long, closed at the bottom with a coarse sintered glass disk 20 ram. in diameter. Remove air bubbles by stirring the contents of the tube with a long wire, adding water from time to time as required to dilute the suspension. When all the air bubbles are removed, fill the column with water and apply air pressure at the top. Permit water to pass out the bottom of the column until more slurry can be added, then repeat the whole process. After all the filter paper has been added in this way, pack the column of cellulose by forcing water through under air pressure until the rate of packing becomes very slow. Then cover the cellulose column with a fiat disk of filter paper, and continue packing with a weighted piston (a rubber stopper on the end of a glass rod with 500 g. applied at the top) until the column of cellulose is 15 cm. long. During this final packing keep the vessel full of water. When the piston is removed, siphon off the excess water. Preparation of Phosphodiesterase. Pour a solution of 150 mg. of lyophilized moccasin venom in 5 ml. of water onto the cellulose column, and as soon as the solution has soaked into the filter paper begin washing with 0.03 % NaC1 solution. Pass this NaC1 solution through the column (with gravity) at a rate of 0.5 to 1.0 ml./min., and collect the filtrate as successive 15-ml. fractions. Measure the optical densities of the fractions at 280 mu. Test all fractions having an optical density greater than 0.1 for enzymic activity. Pool those samples displaying both a strong phosphodiesterase and a weak 5'-nucleotidase action. Although some workers have reported verbally that they are unable to obtain the results reported by Hurst and B u t l e r / a number of others, personally instructed in the separation procedure, have used it with success. The most probable causes of failure are inadequate packing of the cellulose, use of too little filter paper per milligram of venom, and too rapid passage of the salt solution through the column. 8 The dried venom is obtained from Ross Allen's Reptile Institute, Silver Springs, Florida.
564
ENZYMES IN PHOSPHATE METABOLISM
[89]
Properties
Stability. Solutions prepared in the manner described above retain their diesterase activity during several months' storage at 5 ° if mold and bacterial growths are prevented. Merthiolate at a concentration of 0.01% is a suitable preservative, since it does not inhibit the enzyme. Merthiolate will, however, interfere with measurements of ultraviolet absorption. Activity. The phosphodiesterase from 300 mg. of lyophilized moccasin venom hydrolyzed completely 5 g. of MgON to mononucleotides in 5 hours with the liberation of only 2.4 % of inorganic phosphate. Specificity. The purified enzyme acts on polynucleotides (both ribose and deoxyribose types) to produce 5'-mononucleotides and on phenol diesters of phosphoric acid to liberate phenol. No inorganic phosphate is liberated from either of these two types of substrate. Effect of pH. The phosphodiesterase from Russell's viper venom, acting on a 0.3% solution of MgON in Veronal-carbonate buffer, shows maximum activity at p H 9.3. The activity falls off sharply below p H 9.1 and above p H 9.6. Inhibitors. The phosphodiesterase from rattlesnake venom is not inhibited b y sodium arsenate (0.002 M) or b y beryllium sulfate (0.001 M). The diesterase from Russell's viper venom is not inhibited b y sodium citrate (0.001 M), but its activity is completely suppressed b y cysteine hydrochloride (0.15 M). TABLE I MONOESTERASE AND DIESTERASE ACTIVITIES FROM MOCCASIN VENOM
Conversion of MgON, % Optical density, Time, U-insol. Total P to Concentration 280 m~ hr. to U-sol. inorganic P
Sample Whole venom Filtrate fraction no. 7 (15-ml. fractions) 8 9 10 11 12 13 Combined fractions 9-12
0.7 mg./ml. \ I From 150 mg. ~ of venom 1 From 150 mg. of venom
-0.20 0.54 1.72 1.88 0.62 0.22 0.12
1 1 1 1 1 1 1 1 1 2 3 24
35 0 14 67 92 53 31 21 72 92 100 --
17 0 0.27 0.64 0.27 0.32 0.14 0 --0.52 2.3
[90]
SPLEEN AND INTESTINAL PHOSPHODIESTERASES
565
TABLE II PHOSPHODIESTERASE ACTIVITY OF DIFFERENT VENOMS
Four samples of lyophilized venom were assayed for phosphodiesterase activity by the method of Sinsheimer and Koerner. 2 Ten milligrams of each sample was dissolved in 10 ml. of water; the resulting solutions gave the following relative phosphodiesterase activities after both 1 hour and 2 hours of incubation.
Venom Russell's viper Rattlesnake Water moccasin 1952• Water moccasin 1953~
Supplier
Relative phosphodiesterase activity
Haitkine Inst. Ross Allen Ross Allen Ross Allen
1.0 0.84 0.55 0.56
It was not possible to separate the phosphodiesterase satisfactorily from the 1952 sample on a cellulose column, although exactly the same procedure gave excellent results with the 1953 sample.
Pyrophosphatase Activity. C. W. Helleiner has found t h a t phosphodiesterase fractions eluted from a cellulose column containing w a t e r moccasin v e n o m h a v e parallel activities in hydrolyzing sym-diphenylpyrop h o s p h a t e to monophenylphosphate, and m a g n e s i u m oligonucleotide to mononucleotides.
[90] Spleen and Intestinal Phosphodiesterases By
LEON A. HEPPEL and R. J. HILMOE
I. Spleen Spleen contains several diesterase fractions which differ in heat stability, substrate specificity, and other properties. Only one fraction has been extensively purified, and it is described here.
Assay Method
Principle. The assay is a modification of the procedure introduced b y M a c F a d y e n . I As polynucleotide is hydrolyzed b y the nuclease, an increasing fraction becomes soluble in uranium acetate-perchloric reagent. The test involves incubating the diesterase with R N A or other polynucleotide, adding the reagent, centrifuging the precipitate, and measuring the optical density of the s u p e r n a t a n t solution at 260 mtz. 1 D. A. MacFadyen, J. Biol. Chem. 107, 299 (1934).
PHOSPHODIESTERASE FROM SNAKE VENOM
561
utes) to remove insoluble material, and dialyzed overnight against 0.05 M NaHCO3. The extract is then made to a known volume. Such preparations are virtually free of specific glucose-6-phosphatase and may be used for assay of transphosphorylation activity with glucose as acceptor. Since acid phosphatases are readily extracted into solution in dilute salt solutions, the butanol extraction method has not been applied to these enzymes.
[89] Phosphodiesterase from Snake Venom R - - O - - P ( O 2 H ) - - O - - R ' + H20--, R--O--PO3H2 + R'OH
By G. C. BUTLER Assay Method Gulland and Jackson 1used diphenyl phosphate as substrate and measured the liberation of phenol. Sinsheimer and Koerner 2 measured the liberation of p-nitrophenol from bis(p-nitrophenyl)phosphate. The latter method is the most convenient procedure available at present for measuring phosphodiesterase activity. Neither of these methods will, however, detect the presence of 5'-nucleotidase, since this enzyme does not hydrolyze phenyl phosphates. The 5'-nucleotidase found in the venoms of Russell's viper, diamond rattlesnake, and water moccasin may be assayed by the liberation of inorganic phosphate from 5-AMP. The activities of both enzymes may be measured in a single digest by using as substrate the mixed deoxyoligonucleotides resulting from the action of deoxyribonuclease on DNA. The conversion of "uranium-insoluble" to "uranium-soluble ''~ phosphate is a measure of phosphodiesterase activity, and the liberation of inorganic phosphate indicates 5'-nucleotidase activity. This method of measuring diesterase activity is unreliable in the presence of appreciable amounts of 5'-nucleotidase because the latter enzyme removes the product of action of the former and may thus effect an activation of the diesterase. If the diesterase is to be used for the hydrolysis of polynucleotides, it is advisable to test its activity on the oligonucleotides.
Reagents Magnesium oligonucleotide (MgON) solution (0.5%). Dissolve 500 mg. of the dry powder, prepared by the method of Little 1 j. M. Gulland and E. M. Jackson, Biochem. J. 82, 590 (1938). 2 R. L. Sinsheimer and J. F. Koerner, J. Biol. Chem. 198, 293 (1952). R. O. Hurst, J. A. Little, and G. C. Butler, J. Biol. Chem. 188, 705 (1951).
562
ENZYMES IN PHOSPHATE METABOLISM
[89]
and Butler, 4 in 100 ml. of 0.05 M Na barbiturate-Na2C03 (Veronal-carbonate) buffer, pH 9.25. ~ Uranyl acetate-trichloroacetic acid reagent. Dissolve 1.56 g. of uranyl acetate in 100 ml. of 10 % trichloroacetic acid. Enzyme. For assay, dilute the enzyme solutions so that the phosphodiesterase gives, under the test conditions prescribed below, less than 75 % hydrolysis in 1 hour.
Procedure. Since magnesium oligonucleotide contains some "uraniumsoluble" nucleotides, the extent of this solubility must be measured before the action of the enzyme can be tested. To 2 ml. of the uranyl acetate solution (above) add 2 ml. of the MgON solution, stir the mixture, allow it to stand for !0 minutes, and then centrifuge it. Filter the supernatant solution through Whatman No. 44 paper. Dilute 1 ml. of the filtrate to 10 ml. with water, and determine the total phosphorus in 1 ml. by King's method2 After the total phosphorus content of the MgON solution has been determined, the percentage solubility in the uranium reagent may be calculated and used as the blank value for enzymic digestion. To 5 ml. of the MgON solution add 1 ml. of enzyme solution, and after 1 or 2 hours at 37 ° measure the percentage of phosphate soluble in the uranium reagent as described above. This value is a measure of the phosphodiesterase action. In another sample of the diluted uranium filtrate determine the inorganic phosphate which gives a measure of the phosphomonoesterase action. For rough assessment of enzyme activity the uranium precipitate may be observed visually, a small amount of precipitate indicating extensive phosphodiesterase action. This method is often useful in selecting samples, and in judging the dilution they need, for more precise measurement of enzymic activity. Purification Procedure
Principle. The method to be described is based on the finding of Hurst and Butler 7 that both the phosphodiesterase and the 5'-nucleotidase of snake venoms may be adsorbed from aqueous solutions onto packed cellulose fibers. Dilute solutions of neutral salts elute both these enzymes, the diesterase somewhat more readily than the 5'-nucleotidase. The following directions describe how Mr. I. G. Walker has applied this 4j. A. Little and G. C. Butler, J. Biol. Chem. 188, 695 (1951); see Vol. I I I [107]. s E. J. King and G. E. Delory, Enzymologia 81 278 (1940). e E. J. King, Biochem. J. 26, 292 (1932). 7 R. O. Hurst and G. C. Butler, J. Biol. Chem. 195, 91 (1951).
[89]
PHOSPHODIESTERASE FROM SNAKE VENOM
563
principle to the separation of the phosphatases in the venom of the water moccasin (Agkistrodon piscivorus), s The previously published procedure ~ has been modified to make it more versatile. The modified method can be applied to any snake venom by making, when necessary, adjustments in the concentration of the NaC1 solution used for elution. In general it is advisable to elute the phosphodiesterase with the weakest possible NaC1 solution. Preparation of the Cellulose Column. Tear up fifteen sheets of Whatman No. 5 filter paper 12.5 cm. in diameter into 0.5-inch squares, and disperse them in 700 ml. of water by 1 minute's agitation in a blendor. Pour about one-third of the resulting slurry into a glass tube 35 mm. in diameter and 50 cm. long, closed at the bottom with a coarse sintered glass disk 20 ram. in diameter. Remove air bubbles by stirring the contents of the tube with a long wire, adding water from time to time as required to dilute the suspension. When all the air bubbles are removed, fill the column with water and apply air pressure at the top. Permit water to pass out the bottom of the column until more slurry can be added, then repeat the whole process. After all the filter paper has been added in this way, pack the column of cellulose by forcing water through under air pressure until the rate of packing becomes very slow. Then cover the cellulose column with a fiat disk of filter paper, and continue packing with a weighted piston (a rubber stopper on the end of a glass rod with 500 g. applied at the top) until the column of cellulose is 15 cm. long. During this final packing keep the vessel full of water. When the piston is removed, siphon off the excess water. Preparation of Phosphodiesterase. Pour a solution of 150 mg. of lyophilized moccasin venom in 5 ml. of water onto the cellulose column, and as soon as the solution has soaked into the filter paper begin washing with 0.03 % NaC1 solution. Pass this NaC1 solution through the column (with gravity) at a rate of 0.5 to 1.0 ml./min., and collect the filtrate as successive 15-ml. fractions. Measure the optical densities of the fractions at 280 mu. Test all fractions having an optical density greater than 0.1 for enzymic activity. Pool those samples displaying both a strong phosphodiesterase and a weak 5'-nucleotidase action. Although some workers have reported verbally that they are unable to obtain the results reported by Hurst and B u t l e r / a number of others, personally instructed in the separation procedure, have used it with success. The most probable causes of failure are inadequate packing of the cellulose, use of too little filter paper per milligram of venom, and too rapid passage of the salt solution through the column. 8 The dried venom is obtained from Ross Allen's Reptile Institute, Silver Springs, Florida.
564
ENZYMES IN PHOSPHATE METABOLISM
[89]
Properties
Stability. Solutions prepared in the manner described above retain their diesterase activity during several months' storage at 5 ° if mold and bacterial growths are prevented. Merthiolate at a concentration of 0.01% is a suitable preservative, since it does not inhibit the enzyme. Merthiolate will, however, interfere with measurements of ultraviolet absorption. Activity. The phosphodiesterase from 300 mg. of lyophilized moccasin venom hydrolyzed completely 5 g. of MgON to mononucleotides in 5 hours with the liberation of only 2.4 % of inorganic phosphate. Specificity. The purified enzyme acts on polynucleotides (both ribose and deoxyribose types) to produce 5'-mononucleotides and on phenol diesters of phosphoric acid to liberate phenol. No inorganic phosphate is liberated from either of these two types of substrate. Effect of pH. The phosphodiesterase from Russell's viper venom, acting on a 0.3% solution of MgON in Veronal-carbonate buffer, shows maximum activity at p H 9.3. The activity falls off sharply below p H 9.1 and above p H 9.6. Inhibitors. The phosphodiesterase from rattlesnake venom is not inhibited b y sodium arsenate (0.002 M) or b y beryllium sulfate (0.001 M). The diesterase from Russell's viper venom is not inhibited b y sodium citrate (0.001 M), but its activity is completely suppressed b y cysteine hydrochloride (0.15 M). TABLE I MONOESTERASE AND DIESTERASE ACTIVITIES FROM MOCCASIN VENOM
Conversion of MgON, % Optical density, Time, U-insol. Total P to Concentration 280 m~ hr. to U-sol. inorganic P
Sample Whole venom Filtrate fraction no. 7 (15-ml. fractions) 8 9 10 11 12 13 Combined fractions 9-12
0.7 mg./ml. \ I From 150 mg. ~ of venom 1 From 150 mg. of venom
-0.20 0.54 1.72 1.88 0.62 0.22 0.12
1 1 1 1 1 1 1 1 1 2 3 24
35 0 14 67 92 53 31 21 72 92 100 --
17 0 0.27 0.64 0.27 0.32 0.14 0 --0.52 2.3
[90]
SPLEEN AND INTESTINAL PHOSPHODIESTERASES
565
TABLE II PHOSPHODIESTERASE ACTIVITY OF DIFFERENT VENOMS
Four samples of lyophilized venom were assayed for phosphodiesterase activity by the method of Sinsheimer and Koerner. 2 Ten milligrams of each sample was dissolved in 10 ml. of water; the resulting solutions gave the following relative phosphodiesterase activities after both 1 hour and 2 hours of incubation.
Venom Russell's viper Rattlesnake Water moccasin 1952• Water moccasin 1953~
Supplier
Relative phosphodiesterase activity
Haitkine Inst. Ross Allen Ross Allen Ross Allen
1.0 0.84 0.55 0.56
It was not possible to separate the phosphodiesterase satisfactorily from the 1952 sample on a cellulose column, although exactly the same procedure gave excellent results with the 1953 sample.
Pyrophosphatase Activity. C. W. Helleiner has found t h a t phosphodiesterase fractions eluted from a cellulose column containing w a t e r moccasin v e n o m h a v e parallel activities in hydrolyzing sym-diphenylpyrop h o s p h a t e to monophenylphosphate, and m a g n e s i u m oligonucleotide to mononucleotides.
[90] Spleen and Intestinal Phosphodiesterases By
LEON A. HEPPEL and R. J. HILMOE
I. Spleen Spleen contains several diesterase fractions which differ in heat stability, substrate specificity, and other properties. Only one fraction has been extensively purified, and it is described here.
Assay Method
Principle. The assay is a modification of the procedure introduced b y M a c F a d y e n . I As polynucleotide is hydrolyzed b y the nuclease, an increasing fraction becomes soluble in uranium acetate-perchloric reagent. The test involves incubating the diesterase with R N A or other polynucleotide, adding the reagent, centrifuging the precipitate, and measuring the optical density of the s u p e r n a t a n t solution at 260 mtz. 1 D. A. MacFadyen, J. Biol. Chem. 107, 299 (1934).