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Enzymes hydrolyzing ribo- and desoxyribonucleic acids
NOTES FROM THE BIOCHEMICAL RESEARCH FOUNDATION.
Enzymes Hydrolyzing Ribo- and Desoxyribonucleic Acids.--CHARLES A. ZITTLE. Enzymes hydrolyzing ribonucleic acid and desoxyribonucleic acid are present in calf intestinal mucosa (I). For the present studies this material was partly purified (ME) by the procedure of Schmidt and Thannhauser (2) for alkaline phosphatase, without the aluminum hydroxide treatment. These preparations contained 12,5oo units of alkaline phosphatase * per gram assayed by the method of Huggins and Talalay (3). The preparation of Schmidt and Thannhauser (2) (ribonucleinase was absent) hydrolyzed ribonucleic acid. ME hydrolyzed ribonucleic acid completely, measured by its solubility in uranium reagent (4), in which nucleic acid components larger than mononucleotides are insoluble, and by the appearance of inorganic phosphate. This hydrolysis took place w i t h the liberation of acid, measured by the CO2 liberated from a NaHCO3 medium, pH 7.5. Mononucleotides are intermediates in the hydrolysis of desoxyribonucleic acid by intestinal mucosa enzyme (5) and presumably the same is true of the hydrolysis of ribonucleic acid and the secondary phosphoric acid groups are being measured. The striking difference (see Fig. I) between M E and ribonucleinase, which also liberates acid under these conditions (6), should serve to distinguish these enzymes since only the former is active with low concentrations of ribonucleic acid. The calf intestinal mucosa enzyme hydrolyzes desoxyribonucleic acid (I) only after the latter has been depolymerized by chemical or enzymatic means (7, 8). For the present studies desoxyribonucleie acid prepared by the method of Hammarsten (9) was depolymerized with an enzyme prepared from beef pancreas by the initial steps in McCarty's procedure (IO), which apparently gives tetranucleotides (II). It was confirmed (IO, 8) that the product was soluble in HC1; with uranium reagent (4) the amount of precipitate was the same before and after depolymerization. After M E had acted on the desoxyribonucleic acid-tetranucleotides a precipitate was no longer obtained with this reagent. With the desoxyribonucleic acid-tetranucleotides the liberation of acid by M E is about the same as with ribonucleic acid (Fig. I) in respect to rate and influence of concentration of substrate. In the fractionation of the calf intestinal mucosa performed so far phosphodiesterase activity (acid liberated from the nucleic acids) and the phosphomonoesterase activity (alkaline phosphatase) have run * T h e p h o s p h a t a s e d e t e r m i n a t i o n s were performed by Dr. F r a n c i s E. R e i n h a r t in ('(lilnection with a n o t h e r p r o g r a m . 379
3~0
~foc'+;;.'..vfc.'.,\*,. 7-~-:Sr.:A~'4C;~ .,+:OL'N,')A'7";ON XOTT~.S.
[}~. F. :.
parallel. Activity of the lg:tter has been great(,r than the former but this is in line with the observations of Schmidt and T h a n n h a u s e r (2) on diphenyl- and monophenylphosphates. These authors beliew.'d one enzyme hydrolyzed both esters. The present studies and those of others suggest t h a t the enzyme hydrolyzing ribonueleic acid and desoxyribonucleic acid-tetranucleotides, and alkaline phosphatase of animal origin are identical (this enzyme is of broader specificity than ribonucleinase which hydrolyzes only about a third of ribonucleic acid to mononucleotides). The reported isolation of phosphomonoesterase
od
Z
I
so O
4o
I.d 3o 13_ 2-.0
:+
+~+~L-.----+.--:
~-[i; /
+
x~
x
o 0
U
x.
I0
2_,0
30
NUCLEIC
40.
50
ACID,
60
70
80
90
MG./3.5 CC.
I00
FIG. I. Activity of ribonucleinase and intestinal mucosa enzyme with various concentrations of ribonucleic acid. Ribonucleinase ( X ) : Io 3' of e n z y m e with i.o cc. of o.I M NaHCOa, an a t m o s p h e r e of 5 per cent CO2 - 95 per cent N2, p H 7.5, total volume 3.5 cc. Intestinal mucosa enzyme ( + ) : same conditions with 0. 4 cc. of a solution of M E (about I per cent. solid m a t t e r ) . T h e o p t i m u m activity of M E lies above p H 7.5.
free of phosphodiesterase (I2) is at variance with this hypothesis but m a y have another explanation, for example, the activity of-alkaline phosphatase with monoesters is over IOO times greater than with diesters (2). Only the isolation of the pure enzyme can definitely decide this question. It appears, however, t h a t with only two enzymes (desoxyribonucleic acid-depolymerase and alkaline phosphatase) ribonucleic acid and desoxyribonucleic acid m a y be hydrolyzed to nucleotides and nucleosides. REFERENCES.
(I) KLEIN, W., Z. physiol. Chem., 207, I25 (I932). (2) SCHMIDT, G., AND THaNNHaUSER, S. J., J. Biol. Chem., z49, 369 (I943). (3) HUGGINS, C., AND TALALAY, P., J. Biol. Chem., x59, 399 (I945)-
May, 1946.1
I~IOCtiEMICAL [\I,,bI,~AR(~I[ |¢OUNI)ATION .N,'O'I'ES. )
,,
~
,
~I
(4) MACFAt)'/EN, l). A., J. Biol. Chem., lO7, 297 (1934). (5) (6) (7) (8)
(9) (1o) ([ I)
(12)
KLEIn, W., Z. physiol. Chem., 218, I64 (I933). BAIN, J. A., AN~ Ruscri, H. P., J. Biol. Chem., 153, 65!) (1944). SCHMIDT, G., PICKELS, E. G., AND LEVENE, I). A., J. Biol. Chem., 127, 25l (1939). LASKOWSKI, M., AND SEIDEL, M. 1~., Arch. Biochem., 7, 465 (1945). HAMMARSTEN,E., Biochem. Z., 144, 385 (1924). McCARTY, M., J. Gen. Physiol., 29, I23 (I946). FISCHER, F. G., BOETTGER, I., AND LEHMANN-ECHTERNACHT,[t., Z. physiol. Chem., 27 I, 246 (I941). GULLAND,J. MASSON, AND JACKSON,E. M., Biochem. J., 32, 590 (1938).
Enzymes Hydrolyzing Ribo- and Desoxyribonucleic Acids.--CHARLES A. ZITTLE. Enzymes hydrolyzing ribonucleic acid and desoxyribonucleic acid are present in calf intestinal mucosa (I). For the present studies this material was partly purified (ME) by the procedure of Schmidt and Thannhauser (2) for alkaline phosphatase, without the aluminum hydroxide treatment. These preparations contained 12,5oo units of alkaline phosphatase * per gram assayed by the method of Huggins and Talalay (3). The preparation of Schmidt and Thannhauser (2) (ribonucleinase was absent) hydrolyzed ribonucleic acid. ME hydrolyzed ribonucleic acid completely, measured by its solubility in uranium reagent (4), in which nucleic acid components larger than mononucleotides are insoluble, and by the appearance of inorganic phosphate. This hydrolysis took place w i t h the liberation of acid, measured by the CO2 liberated from a NaHCO3 medium, pH 7.5. Mononucleotides are intermediates in the hydrolysis of desoxyribonucleic acid by intestinal mucosa enzyme (5) and presumably the same is true of the hydrolysis of ribonucleic acid and the secondary phosphoric acid groups are being measured. The striking difference (see Fig. I) between M E and ribonucleinase, which also liberates acid under these conditions (6), should serve to distinguish these enzymes since only the former is active with low concentrations of ribonucleic acid. The calf intestinal mucosa enzyme hydrolyzes desoxyribonucleic acid (I) only after the latter has been depolymerized by chemical or enzymatic means (7, 8). For the present studies desoxyribonucleie acid prepared by the method of Hammarsten (9) was depolymerized with an enzyme prepared from beef pancreas by the initial steps in McCarty's procedure (IO), which apparently gives tetranucleotides (II). It was confirmed (IO, 8) that the product was soluble in HC1; with uranium reagent (4) the amount of precipitate was the same before and after depolymerization. After M E had acted on the desoxyribonucleic acid-tetranucleotides a precipitate was no longer obtained with this reagent. With the desoxyribonucleic acid-tetranucleotides the liberation of acid by M E is about the same as with ribonucleic acid (Fig. I) in respect to rate and influence of concentration of substrate. In the fractionation of the calf intestinal mucosa performed so far phosphodiesterase activity (acid liberated from the nucleic acids) and the phosphomonoesterase activity (alkaline phosphatase) have run * T h e p h o s p h a t a s e d e t e r m i n a t i o n s were performed by Dr. F r a n c i s E. R e i n h a r t in ('(lilnection with a n o t h e r p r o g r a m . 379
3~0
~foc'+;;.'..vfc.'.,\*,. 7-~-:Sr.:A~'4C;~ .,+:OL'N,')A'7";ON XOTT~.S.
[}~. F. :.
parallel. Activity of the lg:tter has been great(,r than the former but this is in line with the observations of Schmidt and T h a n n h a u s e r (2) on diphenyl- and monophenylphosphates. These authors beliew.'d one enzyme hydrolyzed both esters. The present studies and those of others suggest t h a t the enzyme hydrolyzing ribonueleic acid and desoxyribonucleic acid-tetranucleotides, and alkaline phosphatase of animal origin are identical (this enzyme is of broader specificity than ribonucleinase which hydrolyzes only about a third of ribonucleic acid to mononucleotides). The reported isolation of phosphomonoesterase
od
Z
I
so O
4o
I.d 3o 13_ 2-.0
:+
+~+~L-.----+.--:
~-[i; /
+
x~
x
o 0
U
x.
I0
2_,0
30
NUCLEIC
40.
50
ACID,
60
70
80
90
MG./3.5 CC.
I00
FIG. I. Activity of ribonucleinase and intestinal mucosa enzyme with various concentrations of ribonucleic acid. Ribonucleinase ( X ) : Io 3' of e n z y m e with i.o cc. of o.I M NaHCOa, an a t m o s p h e r e of 5 per cent CO2 - 95 per cent N2, p H 7.5, total volume 3.5 cc. Intestinal mucosa enzyme ( + ) : same conditions with 0. 4 cc. of a solution of M E (about I per cent. solid m a t t e r ) . T h e o p t i m u m activity of M E lies above p H 7.5.
free of phosphodiesterase (I2) is at variance with this hypothesis but m a y have another explanation, for example, the activity of-alkaline phosphatase with monoesters is over IOO times greater than with diesters (2). Only the isolation of the pure enzyme can definitely decide this question. It appears, however, t h a t with only two enzymes (desoxyribonucleic acid-depolymerase and alkaline phosphatase) ribonucleic acid and desoxyribonucleic acid m a y be hydrolyzed to nucleotides and nucleosides. REFERENCES.
(I) KLEIN, W., Z. physiol. Chem., 207, I25 (I932). (2) SCHMIDT, G., AND THaNNHaUSER, S. J., J. Biol. Chem., z49, 369 (I943). (3) HUGGINS, C., AND TALALAY, P., J. Biol. Chem., x59, 399 (I945)-
May, 1946.1
I~IOCtiEMICAL [\I,,bI,~AR(~I[ |¢OUNI)ATION .N,'O'I'ES. )
,,
~
,
~I
(4) MACFAt)'/EN, l). A., J. Biol. Chem., lO7, 297 (1934). (5) (6) (7) (8)
(9) (1o) ([ I)
(12)
KLEIn, W., Z. physiol. Chem., 218, I64 (I933). BAIN, J. A., AN~ Ruscri, H. P., J. Biol. Chem., 153, 65!) (1944). SCHMIDT, G., PICKELS, E. G., AND LEVENE, I). A., J. Biol. Chem., 127, 25l (1939). LASKOWSKI, M., AND SEIDEL, M. 1~., Arch. Biochem., 7, 465 (1945). HAMMARSTEN,E., Biochem. Z., 144, 385 (1924). McCARTY, M., J. Gen. Physiol., 29, I23 (I946). FISCHER, F. G., BOETTGER, I., AND LEHMANN-ECHTERNACHT,[t., Z. physiol. Chem., 27 I, 246 (I941). GULLAND,J. MASSON, AND JACKSON,E. M., Biochem. J., 32, 590 (1938).