Volume 9, number 1
MOLECULAR• CELLULARBIOCHEMISTRY
October 31, 1975
THE KINETICS OF PANCREATIC RIBONUCLEASE REACTION WITH ALKALINE AND ACIDIC FORMS OF POLY A Zafar M. IQBAL*
Laboratory of Molecular Pharmacology, Division of Cancer Treatment, National Institutes of Health, National Cancer Institute, Bethesda, Maryland 20014 (Received November 11, 1974)
Summary
Materials and Methods
The RNase hydrolysis of random-coil (alkaline form) poly A follows biphasic kinetics at low salt concentrations. However, its resistance to RNase increases with the ionic strength. Helical (acidic form) poly A is also susceptible to RNase but its hydrolysis follows first-order kinetics, and its resistance increases as the pH is lowered. These conformation-dependent kinetics of poly A hydrolysis are similar to those obtained in the hydrolysis of cellular RNA and reovirus doublestranded RNA.
Polyadenylate-3H (18-51 mCi/mM of polyadenylate phosphorus; m. wt. approx. 2-4 × 105) and polyadenylate (m. wt. 1.16 x 106) were obtained from Schwarz BioResearch Inc., Orangeburg, N.J. Hexadecyltrimethylammoniumbromide (CTAB) was purchased from Eastman Kodak Co., Rochester, N.Y., and N-tri(hydroxymethyl) methyl-2-aminoethane sulfonic acid (TES) from CalBiochem., Los Angeles, Calif. Pancreatic ribonuclease (phosphate free) was purchased from Worthington Biochemical Corp., N.J.
Introduction Poly A, at low salt concentrations, is susceptible to pancreatic ribonuclease (p-RNase) 1, but acquires resistance with the increase in ionic strength. Poly A undergoes some pH-dependent conformation changes which can affect its reaction with the RNase 2-7. In this report, the kinetics of p-RNase hydrolysis of poly A at low salt concentrations are re-examined. Both the random coil alkaline poly A and helical acidic poly A forms are susceptible to p-RNase but produce different kinetics of hydrolysis. The RNase resistance in alkaline form increased with the salt concentration (up to 0.25 i), whereas in helical acidic form RNase resistance increased as the pH was lowered. * Present address: Departmentof Pharmacology,Schoolof Medicine,Case WesternReserveUniversity,Cleveland,Ohio 44106.
Ribonuclease assay The susceptibility of poly A-3H to p-RNase (20-25 #g/ml) was studied at 25 ° either in neutral pH buffer containing 0.02 M Na-TES, 2 × 10 -4 u EDTA, or in 0.05 M acetate buffer at acidic pH's. Undigested poly A was precipitated by CTAB in the presence of EDTA 8. Trichloroacetic acid precipitation of poly A was used to compare the efficiency of CTAB precipitation. Results Poly A-3H at neutral pH and low salt concentration (0.02 M NaC1, 25 °) is highly susceptible to p-RNase (Fig. 1). These results are in agreement with the previous report 1. RNase hydrolysis of poly A produced biphasic kinetics,
Dr. W. Junk b.v. Publishers - The Hague, The Netherlands
17
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F i g . 1. E f f i c i e n c y o f C T A B a n d T C A p r e c i p i t a t i o n o f p o l y A resistant to p-RNase
( p H 7, 25°). 0.02 M N a C I : © - - O
TCA;
A z~ CTAB0.1 MNaC1 • - - e TCA; A - - • CTAB. with an initial fast phase followed by a slower phase extending over 1 hour. The resistance to RNase was estimated by extrapolating the slow phase of the reaction to zero-time (Fig. 1). This zero-intercept gives a better estimate of the total resistance than the resistance measured at one particular time during the course of the reaction. The slow phase of the reaction appears to be relatively independent of salt concentrations (Figs. 1 & 2). The relative efficiency of CTAB and TCA precipitation of undigested poly A is illustrated in Figure 1. Under com10 ~
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i
.
i
t
i
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parable salt conditions CTAB precipitated a significantly higher fraction of undigested poly A than TCA, indicating that CTAB is capable of precipitating smaller oligonucleotides. The salt-dependence of RNase susceptibility of poly A at pH 7 is shown in Figure 2. The kinetics of hydrolysis are again essentially biphasic. However, the RNase-resistant fraction, as estimated by the zero-intercept of the second phase, increased with the salt concentration. At lower salt concentrations, the RNase attack appears to be quite random and extensive. However, at 0.25 M NaC1, the reaction proceeded at a near-zero rate with poly A achieving over 90 % resistance. By comparison, no such saltdependent increase was observed in the resistance of poly C-3H. Poly C-3H was completely digested by RNase, even at 1 M NaC1. It is also reported that in the presence of magnesium ions poly A is protected from the RNase attack 1. As shown in Figure 1, only about 5 % poly A was resistant to RNase at 0.02 M NaC1 (CTAB precipitation), but in the presence of increasing magnesium concentration, higher fractions of poly A appeared resistant to the RNase attack (Fig. 3). With 10 m g Mg 2+, about Ioo
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F i g . 2. T h e s a l t - d e p e n d e n c e p-RNase.
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Mg 2+ (rnM) Fig. 3. The protection of random-coil poly A from p-RNase digestion (0.02 M NaC1) caused by magnesium.
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95 % of poly A was protected from RNase attack at 0.02 M NaC1. Such a low concentration of magnesium did not interfere with the RNase activity or the efficiency of CTAB precipitation, and the kinetics of hydrolysis remained biphasic. Since poly A is a homopolymer, it was expected that its hydrolysis would follow a constant rate. The gradual decrease in the rate of its reaction after an initial fast linear phase was not found to be due to the product-inhibition or progressive inactivation of the enzyme. There was no significant effect on the rate of the second phase of poly A-RNase reaction even when poly A-3H was isotopically diluted with cold poly A (10/tg/ml). The addition of fresh poly A-3H during the course of the reaction again produced biphasic kinetics. The enzyme in the reaction mixture was present in large excess of the substrate. Poly A is reported to assume a double helical conformation at acidic pH's 2'3'7. It is quite conceivable that this change in poly A conformation may have significant effect on its reaction with RNase. This reaction was carried out at 3 different pH values below 7, and the salt concentration was kept at a minimum
(0.02 ~). At pH 6 about 20 % poly A showed resistance to RNase, while at neutral pH only 5 % poly A was resistant (Fig. 4). The kinetics in both cases were biphasic and similar to those shown in Figure 1 - with an initial fast phase (first 20 min.) followed by a slower phase (20-65 min). However, L0
z
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Fig. 4. The kinetics of p-RNase hydrolysis of helical poly A at different pH values. • - • pH 7; , k - - A pH 6; © - - © pH 5.5, /X--A pH 5.
when the pH was lowered to 5.5 and 5, the RNase hydrolysis of poly A-3H followed first order kinetics. At these p H values, RNase was active and was able to completely digest cell RNA. These first-order kinetics are similar to those obtained with synthetic double-stranded copolymer poly A : poly U and natural helical reovirus RNA 9. The rate of hydrolysis of acidic helical poly A was reduced with the lowering of the pH, whereas, in the case of poly A : poly U, the rate of RNase hydrolysis was reduced with increasing salt concentrations.
Discussion
These experiments show that both the random coil (alkaline form) and helical (acidic form) poly A are susceptible to p-RNase. The apparent difference seems to be that the helical poly A follows a first-order kinetics of RNase digestion, and the random-coil poly A shows biphasic kinetics. Furthermore, the RNase resistance of random-coil poly A increases with the salt concentration, whereas the helical poly A becomes increasingly resistant to p-RNase as the pH is lowered. The first-order kinetics of helical poly A is consistent with those obtained with poly A : poly U and reovirus double-stranded RNA 9, and might be due to the exonuclease activity of p-RNase. The biphasic kinetics of alkaline poly A, however, presents an interesting situation. Since random-coil poly A has some short-range basestacking 4'5, it seems quite likely that the phosphodiester bonds of the bases in stack may not be as susceptible to RNase as the rest of the bonds in the random coil. RNase perhaps forms a complex with the stacked bases, thus protecting them while the rest of the random coil is hydrolyzed. It may then be speculated that, with increasing ionic strength, either the base-stackRNase complexes become more stable, or the number of base-residues per stack increases. This might explain the salt-dependence of alkaline poly A to p-RNase. It has also been suggested that p-RNase first attacks poly A randomly and then progressively attacks the oligomers produced 1. It may be that the RNase continues to attack the oligomer until the chain length reaches a level below which the 19
RNase cannot recognize it. Then, in the presence of salt, the polymer becomes less extended, and the minimum chain length recognized by RNase might actually possess more base residues than it did in the absence of salt. Furthermore, it is known that the p-RNase attack on RNA produces oligomers terminating in 2'-3' cyclic phosphates. Cyclic phosphate mononucleotides are then produced which are hydrolyzed to corresponding nucleotides 1°. It may be speculated that the biphasic kinetics of random-coil poly A is due to the increasing production of cyclized terminal nucleotides which perhaps gradually slow down the RNase activity. It seems likely, however, that a combination of the above-mentioned interpretations may be responsible for the biphasic kinetics of RNase hydrolysis of alkaline poly A. This report points out the fact that the differences in the kinetics of RNase hydrolysis can be helpful in understanding the conformation of the substrate molecules.
Acknowledgements The author thanks Dr. K. W. KOHN for many helpful discussions, and appreciates the technical assistance of Mrs. F. L. FRANCIS.
References 1. Beers, R. F. J. Biol. Chem. 235, 2393 2398 (1960). 2. Janik, B., Sommer, R. G. and Bobst, A. M. Biochem. Biophys. Acta 281,152 168 (1972). 3. Holcomb, D. N. and Tinoco, I. Bipolymers 3, 121-133 (1965). 4. Leng, M. and Felsenfeld, G. J. Mol. Biol. 15,455 466 (1966). 5. Eisenberg, H. and FeIsenfeld, G. J. Mol. Biol. 30, 17 37 (1967). 6. Steiner, R. and Millar, D. B. S. in Biological Polyelectrolytes (Veis, A., Ed.) Biological Macromolecules Series Vol. 3 pp. 65 129, M. Dekker, N.Y. (1970). 7. Brahms, J., Michelson, A. M. and van Holde, K. E. J. MoI. Biol. 15, 467-488 (1966). 8. Sibatani, A. Anal. Biochem. 33,279 285 (1970). 9. Iqbal, Z. M. and Kohn, K. W. Arch. Biochem. Biophys. in press (1974). 10. Roberts, G. C. K., Dennis, E. H., Meadows, D. H., Cohen, J. S. and Jardetzky, O. Proc. Nat'l. Acad. Sci., U.S. 62, 1151---1158 (1969).
20
MOLECULAR• CELLULARBIOCHEMISTRY
October 31, 1975
THE KINETICS OF PANCREATIC RIBONUCLEASE REACTION WITH ALKALINE AND ACIDIC FORMS OF POLY A Zafar M. IQBAL*
Laboratory of Molecular Pharmacology, Division of Cancer Treatment, National Institutes of Health, National Cancer Institute, Bethesda, Maryland 20014 (Received November 11, 1974)
Summary
Materials and Methods
The RNase hydrolysis of random-coil (alkaline form) poly A follows biphasic kinetics at low salt concentrations. However, its resistance to RNase increases with the ionic strength. Helical (acidic form) poly A is also susceptible to RNase but its hydrolysis follows first-order kinetics, and its resistance increases as the pH is lowered. These conformation-dependent kinetics of poly A hydrolysis are similar to those obtained in the hydrolysis of cellular RNA and reovirus doublestranded RNA.
Polyadenylate-3H (18-51 mCi/mM of polyadenylate phosphorus; m. wt. approx. 2-4 × 105) and polyadenylate (m. wt. 1.16 x 106) were obtained from Schwarz BioResearch Inc., Orangeburg, N.J. Hexadecyltrimethylammoniumbromide (CTAB) was purchased from Eastman Kodak Co., Rochester, N.Y., and N-tri(hydroxymethyl) methyl-2-aminoethane sulfonic acid (TES) from CalBiochem., Los Angeles, Calif. Pancreatic ribonuclease (phosphate free) was purchased from Worthington Biochemical Corp., N.J.
Introduction Poly A, at low salt concentrations, is susceptible to pancreatic ribonuclease (p-RNase) 1, but acquires resistance with the increase in ionic strength. Poly A undergoes some pH-dependent conformation changes which can affect its reaction with the RNase 2-7. In this report, the kinetics of p-RNase hydrolysis of poly A at low salt concentrations are re-examined. Both the random coil alkaline poly A and helical acidic poly A forms are susceptible to p-RNase but produce different kinetics of hydrolysis. The RNase resistance in alkaline form increased with the salt concentration (up to 0.25 i), whereas in helical acidic form RNase resistance increased as the pH was lowered. * Present address: Departmentof Pharmacology,Schoolof Medicine,Case WesternReserveUniversity,Cleveland,Ohio 44106.
Ribonuclease assay The susceptibility of poly A-3H to p-RNase (20-25 #g/ml) was studied at 25 ° either in neutral pH buffer containing 0.02 M Na-TES, 2 × 10 -4 u EDTA, or in 0.05 M acetate buffer at acidic pH's. Undigested poly A was precipitated by CTAB in the presence of EDTA 8. Trichloroacetic acid precipitation of poly A was used to compare the efficiency of CTAB precipitation. Results Poly A-3H at neutral pH and low salt concentration (0.02 M NaC1, 25 °) is highly susceptible to p-RNase (Fig. 1). These results are in agreement with the previous report 1. RNase hydrolysis of poly A produced biphasic kinetics,
Dr. W. Junk b.v. Publishers - The Hague, The Netherlands
17
1.0
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0.1
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F i g . 1. E f f i c i e n c y o f C T A B a n d T C A p r e c i p i t a t i o n o f p o l y A resistant to p-RNase
( p H 7, 25°). 0.02 M N a C I : © - - O
TCA;
A z~ CTAB0.1 MNaC1 • - - e TCA; A - - • CTAB. with an initial fast phase followed by a slower phase extending over 1 hour. The resistance to RNase was estimated by extrapolating the slow phase of the reaction to zero-time (Fig. 1). This zero-intercept gives a better estimate of the total resistance than the resistance measured at one particular time during the course of the reaction. The slow phase of the reaction appears to be relatively independent of salt concentrations (Figs. 1 & 2). The relative efficiency of CTAB and TCA precipitation of undigested poly A is illustrated in Figure 1. Under com10 ~
,
i
.
i
t
i
i
parable salt conditions CTAB precipitated a significantly higher fraction of undigested poly A than TCA, indicating that CTAB is capable of precipitating smaller oligonucleotides. The salt-dependence of RNase susceptibility of poly A at pH 7 is shown in Figure 2. The kinetics of hydrolysis are again essentially biphasic. However, the RNase-resistant fraction, as estimated by the zero-intercept of the second phase, increased with the salt concentration. At lower salt concentrations, the RNase attack appears to be quite random and extensive. However, at 0.25 M NaC1, the reaction proceeded at a near-zero rate with poly A achieving over 90 % resistance. By comparison, no such saltdependent increase was observed in the resistance of poly C-3H. Poly C-3H was completely digested by RNase, even at 1 M NaC1. It is also reported that in the presence of magnesium ions poly A is protected from the RNase attack 1. As shown in Figure 1, only about 5 % poly A was resistant to RNase at 0.02 M NaC1 (CTAB precipitation), but in the presence of increasing magnesium concentration, higher fractions of poly A appeared resistant to the RNase attack (Fig. 3). With 10 m g Mg 2+, about Ioo
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F i g . 2. T h e s a l t - d e p e n d e n c e p-RNase.
• 18
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of random-coil
I
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•0.25M.
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CTAB p r e c i p i t a t i o n . O - - © 0.02 M; A - - / X 0.05 M;
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Mg 2+ (rnM) Fig. 3. The protection of random-coil poly A from p-RNase digestion (0.02 M NaC1) caused by magnesium.
LO
95 % of poly A was protected from RNase attack at 0.02 M NaC1. Such a low concentration of magnesium did not interfere with the RNase activity or the efficiency of CTAB precipitation, and the kinetics of hydrolysis remained biphasic. Since poly A is a homopolymer, it was expected that its hydrolysis would follow a constant rate. The gradual decrease in the rate of its reaction after an initial fast linear phase was not found to be due to the product-inhibition or progressive inactivation of the enzyme. There was no significant effect on the rate of the second phase of poly A-RNase reaction even when poly A-3H was isotopically diluted with cold poly A (10/tg/ml). The addition of fresh poly A-3H during the course of the reaction again produced biphasic kinetics. The enzyme in the reaction mixture was present in large excess of the substrate. Poly A is reported to assume a double helical conformation at acidic pH's 2'3'7. It is quite conceivable that this change in poly A conformation may have significant effect on its reaction with RNase. This reaction was carried out at 3 different pH values below 7, and the salt concentration was kept at a minimum
(0.02 ~). At pH 6 about 20 % poly A showed resistance to RNase, while at neutral pH only 5 % poly A was resistant (Fig. 4). The kinetics in both cases were biphasic and similar to those shown in Figure 1 - with an initial fast phase (first 20 min.) followed by a slower phase (20-65 min). However, L0
z
i, ~- 0,1 z w n~
z 0.0t
20
40 TIME (rain)
60
Fig. 4. The kinetics of p-RNase hydrolysis of helical poly A at different pH values. • - • pH 7; , k - - A pH 6; © - - © pH 5.5, /X--A pH 5.
when the pH was lowered to 5.5 and 5, the RNase hydrolysis of poly A-3H followed first order kinetics. At these p H values, RNase was active and was able to completely digest cell RNA. These first-order kinetics are similar to those obtained with synthetic double-stranded copolymer poly A : poly U and natural helical reovirus RNA 9. The rate of hydrolysis of acidic helical poly A was reduced with the lowering of the pH, whereas, in the case of poly A : poly U, the rate of RNase hydrolysis was reduced with increasing salt concentrations.
Discussion
These experiments show that both the random coil (alkaline form) and helical (acidic form) poly A are susceptible to p-RNase. The apparent difference seems to be that the helical poly A follows a first-order kinetics of RNase digestion, and the random-coil poly A shows biphasic kinetics. Furthermore, the RNase resistance of random-coil poly A increases with the salt concentration, whereas the helical poly A becomes increasingly resistant to p-RNase as the pH is lowered. The first-order kinetics of helical poly A is consistent with those obtained with poly A : poly U and reovirus double-stranded RNA 9, and might be due to the exonuclease activity of p-RNase. The biphasic kinetics of alkaline poly A, however, presents an interesting situation. Since random-coil poly A has some short-range basestacking 4'5, it seems quite likely that the phosphodiester bonds of the bases in stack may not be as susceptible to RNase as the rest of the bonds in the random coil. RNase perhaps forms a complex with the stacked bases, thus protecting them while the rest of the random coil is hydrolyzed. It may then be speculated that, with increasing ionic strength, either the base-stackRNase complexes become more stable, or the number of base-residues per stack increases. This might explain the salt-dependence of alkaline poly A to p-RNase. It has also been suggested that p-RNase first attacks poly A randomly and then progressively attacks the oligomers produced 1. It may be that the RNase continues to attack the oligomer until the chain length reaches a level below which the 19
RNase cannot recognize it. Then, in the presence of salt, the polymer becomes less extended, and the minimum chain length recognized by RNase might actually possess more base residues than it did in the absence of salt. Furthermore, it is known that the p-RNase attack on RNA produces oligomers terminating in 2'-3' cyclic phosphates. Cyclic phosphate mononucleotides are then produced which are hydrolyzed to corresponding nucleotides 1°. It may be speculated that the biphasic kinetics of random-coil poly A is due to the increasing production of cyclized terminal nucleotides which perhaps gradually slow down the RNase activity. It seems likely, however, that a combination of the above-mentioned interpretations may be responsible for the biphasic kinetics of RNase hydrolysis of alkaline poly A. This report points out the fact that the differences in the kinetics of RNase hydrolysis can be helpful in understanding the conformation of the substrate molecules.
Acknowledgements The author thanks Dr. K. W. KOHN for many helpful discussions, and appreciates the technical assistance of Mrs. F. L. FRANCIS.
References 1. Beers, R. F. J. Biol. Chem. 235, 2393 2398 (1960). 2. Janik, B., Sommer, R. G. and Bobst, A. M. Biochem. Biophys. Acta 281,152 168 (1972). 3. Holcomb, D. N. and Tinoco, I. Bipolymers 3, 121-133 (1965). 4. Leng, M. and Felsenfeld, G. J. Mol. Biol. 15,455 466 (1966). 5. Eisenberg, H. and FeIsenfeld, G. J. Mol. Biol. 30, 17 37 (1967). 6. Steiner, R. and Millar, D. B. S. in Biological Polyelectrolytes (Veis, A., Ed.) Biological Macromolecules Series Vol. 3 pp. 65 129, M. Dekker, N.Y. (1970). 7. Brahms, J., Michelson, A. M. and van Holde, K. E. J. MoI. Biol. 15, 467-488 (1966). 8. Sibatani, A. Anal. Biochem. 33,279 285 (1970). 9. Iqbal, Z. M. and Kohn, K. W. Arch. Biochem. Biophys. in press (1974). 10. Roberts, G. C. K., Dennis, E. H., Meadows, D. H., Cohen, J. S. and Jardetzky, O. Proc. Nat'l. Acad. Sci., U.S. 62, 1151---1158 (1969).
20
