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Ribonuclease. IV. The effect of whole body x-irradiation on the ribonuclease system of rat liver
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
60,
7-13 (1956)
Ribonuclease. IV. The Effect of Whole Body X-Irradiation on the Ribonuclease System of Rat Liver’ Jay S. Roth From the William Goldman Isotope Laboratory, Division Hahnemnnn Medical College, Philadelphia,
Biological Pennsylvania
of
Chemistry,
Received July 25, 1955 INTRODUCTION
In spite of extensive research, the lethal and carcinogenic effects of x-radiation on higher animals still remain unexplained on a molecular or biochemical basis. Attempbs to unravel the complex sequence of events occurring after whole body x-irradiation have involved many aspects of the chemistry and physiology of cells, tissues, and the intact animal. Because of t,he probable close relationship of nucleic acid metabolism t,o cell division and growth, t,he effects of radiation on this metabolism are of interest. In this paper the action of 600 r. of whole body x-irradiation on the ribonuclease (RNase) system in various fractions of rat liver has been studied. In an earlier report (I), the effect of 600 r. of total body irradiation on the RNase activity of liver homogenates was investigated. Since this earlier report was published, it has been demonstrated that there are probably two RNases in rat liver (2, 3) as well as an inhibitor for at least one of them (3). It is obvious, therefore, that the changes in RNase acbivity observed in tfhe earlier study may have been due to alterations in any one, or more, of the components of this system. It seemed worth while to repeat the work, t,his time assaying separately for each component. In this way it may be possible to relate changes in ribonucleic acid (RNA) metabolism that occur aft’er irradiation (5-7) to the activities of the intracellular RNases and the inhibitor, and to gain some insight into the functions of these cell constituents. 1 This project was supported by a grant from the U. S. Atomic Energy Commission, Contract AT(30-1) 1069 and, in part, by a grant from the Damon Runyon Memorial Fund. 7
8
JAY S. R(YTH MATERIALS
AND METHODS
TWO experiments, I and II, were performed which will be described separately. In each experiment, 96 male rats of the Wistar strain were used, 48 controls and 48 x-irradiated. The method of irradiation has been described (1). The radiation factors, which were the same in both experiments, were as follows: 240 kv., 15 ma., filter 1.0 mm. Cu and 1.0 mm. Al, target distance 50 cm. A total of 600 r. was delivered at a rate of approximately 67 r./min. In Expt. I, the rats weighed between 180 and 220 g., and in Expt. II, the animals which were obtained from a different source, weighed between 150 and 175 g. The controls were sham-irradiated in Expt. II. All the animals were given ample water but food was withdrawn for 24 hr. after the experiments started. Thereafter, the controls were pair-fed against the irradiated animals for 7-8 days with a standard stock diet. By the end of this period, the food consumption of the irradiated animals was nearly normal, and ad Zibitum feeding was resumed. Four control and four irradiated animals were autopsied each day that assays were run. The rats were etherized and then exsanguinated by heart puncture. Livers were removed, washed with ice-cold 0.25 M sucrose (containing 1.8 X lo-* M CaCL), blotted dry, and weighed. Each liver, or a portion of it, was homogenized in a Ten Broeck homogenizer with 9 vol. of cold sucrose solution, and the nuclei and unbroken cells were removed by centrifuging at 500 X g in an International PR-2 refrigerated centrifuge. The supernatant from this was then centrifuged in a Spinco model L preparative ultracentrifuge at 5000 X g for 15 min. to remove mitochondria. The pink fluffy coat was added to the microsome fraction.After washing the mitochondria once with sucrose solution, they were recentrifuged at 5000 X g for an additional 15 min., and the resulting mitochondrial pellet was made up to 25 ml. in sucrose. The supernatant obtained from the first centrifugation of the mitochondria was spun at 59,800 X g (av.) for 1 hr., and the supernatant above the resulting microsome pellet was poured off and recentrifuged for an additional 30 min. at 59,800 X g. This final supernatant is referred to as the supernatant fraction. All determinations were performed in duplicate, except where noted, within 15 min. after preparation of the homogenate or fraction. RNase activity was assayed by the method of Roth and Milstein (8), and, in Expt. I, assays were carried out at pH’s 7.8 and 5.8 on mitochondria, and at pH 7.8 on the supernatant fraction. Assays were also made for inhibitor on the latter fraction. In Expt. II, RNase assays were performed at pH’s 7.8 and 5.8 on the whole homogenate and mitochondria, and inhibitor activity was determined in the supernatant fraction. The experimental details of the RNase assays are given in connection with the figures. Crystalline pancreatic RNase was supplied by the Worthington Biochemical Corp., Freehold, N. J. Stock solutions containing 50 fig. of enzyme/ml. were prepared in 0.1% gelatin solution and stored in the refrigerator. Dilute enzyme solutions, used in the inhibitor assay, were prepared daily from the stock solution using 0.1% gelatin solution as diluent. A complete description of the inhibitor and the method for its assay will be the subject of a paper to be published shortly. In Expt. I, animals were autopsied on the lst, 2nd, 3rd, and 5th days after irradiation; of those animals not autopsied (28), 17 died within 30 days after the start of the experiment. Animals were autopsied in Expt. II on the lst, 2nd, 3rd, 5th, 8th, lOth, and 12th days
!I
RIBONUCLEASE. IV
after irradiation, and of those animals not autopsied (20), 11 died by the end of 30 days. Two controls also died in this second experiment. Nikogen determinations were performed by the micro-Kjeldahl method. RESULTS
Mitochondria Experiment I. The RNase activities at the two pH values are plotted in Fig. 1. At pH 5.8, the activities in the irradiated livers tended t’o he lower (20-24 %) than the control values on days 1,2, and 3. This decrease is not considered to be particularly significant in view of the results of Expt. II (below) where no decrease from control values was observed. At pH 7.8, however, there was a significant drop in mitochondrial RNase activity of irradiated animals, the decrease reaching a maximum of 48 % on day 5. Experiment II. The results for mitochondrial RNase activity are
-
::
y;s Q ,oY -
-
8
0
0
OpJ X X
-21
- -.-. 8x X *c-------- p -7 -20
$6 d II:
PH 7.8
PH5.8
10-
x
c
v $-., x . ..\
-24 -
X
x
-25
-33
2-
“*-...sv X
2
3
5
123
x --.
x
x -45
I
0 x
x
-48
5
DAY FIG. 1. Experiment I: RNase activity in separated mitochondria from the livers of irradiated and control rats. X = irradiated animals, 0 = controls. Solid line is the average of the controls; dotted line is the average of the irradiated rats. The values below the experimental points are per cent differences of irradiated averages from control averages. The assay system consisted of 0.25 ml. of mitochondria, 1.0 ml. of 1% P32-labeled RNA, 1.0 ml. of VeronalLacetate buffer, pH 5.8 or 7.8, and 0.75 ml. of HzO. The mixture was incubated at 37°C. for 30 min. and unhydrolyzed RNA then precipitated with 3 ml. of acid-alcohol (8) and the radioactivity of an aliquot of the filtrate determined. Relative activit,v (the ordinate)
is counts/mg.
N X 10-a.
10
JAY
6.
II 123
5
8
IO
I2
ROTH
X -8-5-6 -3, -44 1111111,11111 123 5 8
-33
IO
-10 12
DAY FIG. 2. Experiment II: RNase activity in separated mitochondria from the livers of irradiated and control rats. The assay system was the same as that described in Fig. 1. Single determinations were made on each mitochondrial preparation. X = irradiated animals, 0 = controls. Solid line is the average of the controls; dotted line the average of the irradiated rats. The values below the experimental points are per cent differences of irradiated averages from control averages. Relative activity (the ordinate) is counts/mg. N X 10-a.
plotted in Fig. 2. At pH 5.8, the values for the irradiated animals did not differ significantly from the control values on any of the days assays were performed. Both the control and irradiated activities declined considerably on the second and third day but recovered on the fourth day. Considerable variability was noted in the individual values in both experiments, but this has been observed generally with mitochondrial RNase activities. At pH 7.8, the RNase activity of the mitochondria of irradiated rats did not fall significantly below the control level until the fifth day, when the decreasewas 31%. On this day, the four control values averaged somewhat lower than on most of the other days of measurement, and this may have contributed to a smaller decreaseon the fifth day than was observed in Expt. I. On the eighth day, the decrease in mitochondrial RNase activity was maximal (-44 %), and on the tenth and twelfth days, recovery appeared to set in, the values being 33 and 10% less than the controls, respectively. In view of the difference in the source and ages of the animals in the two experiments, these results may be considered to be fairly consistent. The different response of the RNase activity at pH’s 5.8 and 7.8 could be taken as evidence
RIBONUCLEASE.
IV
11
for the view that these activities represent two different enzymes; however, other interpretations are still possible. Since irradiation of certain tissues may result in a decrease in RnTA synthesis (5-7), it is tempting to speculate that the enzyme which is active at pH 7.8 may be related to t,his process. RNase Inhibitor The inhibitor, which occurs in the supernatant fraction, is a heatlabile, nondialyzable substance sensitive to certain -SW reagents. Large quantities of RNase are normally bound to it.2 This inhibitor is of interest, as it may furnish a clue as to the function of intracellular RNases and to the elucidation of their relationships to RNA and prot,ein synthesis. In Expts. I and II, a decrease of 12 and 41%, respectively, in inhibitor activit,y was noted in irradiated animals on the first day after irradiation. By the second day, activity had returned to control values in both experiments, and no further significant change was observed, although in Expt. I the values in the irradiated animals tended to be somewhat higher than the controls (3-15 %) on the second, t,hird, and fifth days. Initial changes in inhibitor activity may bc quite transient, and it would be of interest to measure activity at intervals less than 24 hr. after irradiation. Whole Homogenate In a previous paper (I), Wistar rats were subjected to 600 r. of whole body x-irradiation, and RNase activity of liver homogenates was assayed at’ pH F.O. Under these conditions, there was an increase in RNase activity of approximately 50% on days 2 and 3 postirradiation, followed by a decline t’o 25% of control activity by day 8. Thereafter, a rise to somewhat above control levels occurred by day 12. The changes recorded in RNase activity of whole homogenate, measured at pH 5.8 in Expt. II, were considerably less in the present report, although they varied approximately in the same direction as previously, being +2, +S, -8, -20, +13, -4, and $4 on the lst, 2nd, 3rd, 5th, 8th, and 12th day aft’er irradiation, respectively. The reason for the discrepancy is not, apparent. It is possible that the younger rats, used in the present work, are more resistant to changes in the particular system under study. In Expt. II, although variations in RNase activity, measured nD pH 7.8, of whole homogenate were noted, between the controls and 2 Roth, J. S., unpublished
results.
12
JAY S. ROTH
x-irradiated animals, these variations showed no particular trend or correlation with any of the above results. Also, no significant changes were observed in the RNase activity of the supernatant fractions in either experiment. DISCUSSION The relationship between inhibitor activity and RNase activity of the whole homogenate (measured at pH 7.8) could be either direct or inverse. Since a large quantity of RNase is normally bound by the inhibitor, inactivation of the latter by irradiation might release RNase activity. It is also possible that the inhibitor-enzyme complex, which apparently still has additional sites available for combination with added RNase,2 may have these active sites destroyed by radiation without breaking the enzyme-inhibitor bonds, and thus without release of RNase. Consideration of the data of Expt. II, day 10, favors the former view. On this day, whole homogenate RNase activity was +29%, isolated mitochondria RNase activity - 33 %, and inhibitor activity + 12 %, all measured at pH 7.8. Release of enzyme by the inhibitor might have been sufficient to overcome the lowered mitochondrial activity and give a net increase in homogenate activity. In vitro irradiation of the supernatant fraction is being carried out at present in an effort to provide more definite answers to the above questions. ACKNOWLEDGMENTS The author wishes to acknowledge the competent assistance of Miss Jeanne Boyd and Miss Malena Der Avedisian in carrying out the analyses.
SUMMARY 1. Rats were subjected to 600 r. of whole body x-irradiation, and RNase assays were carried out on liver homogenates and fractions, at intervals after irradiation. 2. A significant decrease was noted in the RNase activity, measured at pH 7.8, of separated mitochondria prepared from the livers of irradiated animals. 3. RNase
inhibitor
activity
in the supernatant
fractions
obtained
from the livers of irradiated rats, was depressed by 12 and 41%, in two experiments, 1 day after irradiation, when compared to control values. Recovery to control levels was observed by the second day after exposure.
RIBONUCLEASE.
IV
13
4. The RNase activity of whole liver homogenate, measured at pH 5.8, showed only small changes from control values, in irradiated animals, while activity at pH 7.8 in whole homogenate or supernatant fraction from irradiated animals did not show significant changes from the control levels. 5. Some implications of these results were discussed. REFERENCES 1. ROTH, J. S., EICHEL, H. J., WASE, A., ALPER, C., AND BOYD, M. J., Arch. Biothem. and Biophys. 44,95 (1953). 2. ROTH, J. S., J. Biol. Chem. 208, 181 (1954). 3. DELAMIRANDE,G.,ALLARD,C.,DACOSTA, H.C., AND CANTERO,A.,S~~~~~~ 119, 351 (1954). ~.‘RoTH, J. S., Federation Proc. 14,272 (1955). 5. ABRAMS, R., Arch. Biochem. 30,90 (1951). 6. THOMSON, J.F., TOURTELLOTTE, W. W., CARTTAR, M.S., AND STORER, J.B., Arch. Biochem. and Biophys. 42, 185 (1953). 7. ORD, M. G., AND STOCKEN,L. A., Physiol. Revs. 33,356 (1953). 8. ROTH, J. S., AND MILSTEIN, S. W., J. Biol. Chem. 196,489 (1952).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
60,
7-13 (1956)
Ribonuclease. IV. The Effect of Whole Body X-Irradiation on the Ribonuclease System of Rat Liver’ Jay S. Roth From the William Goldman Isotope Laboratory, Division Hahnemnnn Medical College, Philadelphia,
Biological Pennsylvania
of
Chemistry,
Received July 25, 1955 INTRODUCTION
In spite of extensive research, the lethal and carcinogenic effects of x-radiation on higher animals still remain unexplained on a molecular or biochemical basis. Attempbs to unravel the complex sequence of events occurring after whole body x-irradiation have involved many aspects of the chemistry and physiology of cells, tissues, and the intact animal. Because of t,he probable close relationship of nucleic acid metabolism t,o cell division and growth, t,he effects of radiation on this metabolism are of interest. In this paper the action of 600 r. of whole body x-irradiation on the ribonuclease (RNase) system in various fractions of rat liver has been studied. In an earlier report (I), the effect of 600 r. of total body irradiation on the RNase activity of liver homogenates was investigated. Since this earlier report was published, it has been demonstrated that there are probably two RNases in rat liver (2, 3) as well as an inhibitor for at least one of them (3). It is obvious, therefore, that the changes in RNase acbivity observed in tfhe earlier study may have been due to alterations in any one, or more, of the components of this system. It seemed worth while to repeat the work, t,his time assaying separately for each component. In this way it may be possible to relate changes in ribonucleic acid (RNA) metabolism that occur aft’er irradiation (5-7) to the activities of the intracellular RNases and the inhibitor, and to gain some insight into the functions of these cell constituents. 1 This project was supported by a grant from the U. S. Atomic Energy Commission, Contract AT(30-1) 1069 and, in part, by a grant from the Damon Runyon Memorial Fund. 7
8
JAY S. R(YTH MATERIALS
AND METHODS
TWO experiments, I and II, were performed which will be described separately. In each experiment, 96 male rats of the Wistar strain were used, 48 controls and 48 x-irradiated. The method of irradiation has been described (1). The radiation factors, which were the same in both experiments, were as follows: 240 kv., 15 ma., filter 1.0 mm. Cu and 1.0 mm. Al, target distance 50 cm. A total of 600 r. was delivered at a rate of approximately 67 r./min. In Expt. I, the rats weighed between 180 and 220 g., and in Expt. II, the animals which were obtained from a different source, weighed between 150 and 175 g. The controls were sham-irradiated in Expt. II. All the animals were given ample water but food was withdrawn for 24 hr. after the experiments started. Thereafter, the controls were pair-fed against the irradiated animals for 7-8 days with a standard stock diet. By the end of this period, the food consumption of the irradiated animals was nearly normal, and ad Zibitum feeding was resumed. Four control and four irradiated animals were autopsied each day that assays were run. The rats were etherized and then exsanguinated by heart puncture. Livers were removed, washed with ice-cold 0.25 M sucrose (containing 1.8 X lo-* M CaCL), blotted dry, and weighed. Each liver, or a portion of it, was homogenized in a Ten Broeck homogenizer with 9 vol. of cold sucrose solution, and the nuclei and unbroken cells were removed by centrifuging at 500 X g in an International PR-2 refrigerated centrifuge. The supernatant from this was then centrifuged in a Spinco model L preparative ultracentrifuge at 5000 X g for 15 min. to remove mitochondria. The pink fluffy coat was added to the microsome fraction.After washing the mitochondria once with sucrose solution, they were recentrifuged at 5000 X g for an additional 15 min., and the resulting mitochondrial pellet was made up to 25 ml. in sucrose. The supernatant obtained from the first centrifugation of the mitochondria was spun at 59,800 X g (av.) for 1 hr., and the supernatant above the resulting microsome pellet was poured off and recentrifuged for an additional 30 min. at 59,800 X g. This final supernatant is referred to as the supernatant fraction. All determinations were performed in duplicate, except where noted, within 15 min. after preparation of the homogenate or fraction. RNase activity was assayed by the method of Roth and Milstein (8), and, in Expt. I, assays were carried out at pH’s 7.8 and 5.8 on mitochondria, and at pH 7.8 on the supernatant fraction. Assays were also made for inhibitor on the latter fraction. In Expt. II, RNase assays were performed at pH’s 7.8 and 5.8 on the whole homogenate and mitochondria, and inhibitor activity was determined in the supernatant fraction. The experimental details of the RNase assays are given in connection with the figures. Crystalline pancreatic RNase was supplied by the Worthington Biochemical Corp., Freehold, N. J. Stock solutions containing 50 fig. of enzyme/ml. were prepared in 0.1% gelatin solution and stored in the refrigerator. Dilute enzyme solutions, used in the inhibitor assay, were prepared daily from the stock solution using 0.1% gelatin solution as diluent. A complete description of the inhibitor and the method for its assay will be the subject of a paper to be published shortly. In Expt. I, animals were autopsied on the lst, 2nd, 3rd, and 5th days after irradiation; of those animals not autopsied (28), 17 died within 30 days after the start of the experiment. Animals were autopsied in Expt. II on the lst, 2nd, 3rd, 5th, 8th, lOth, and 12th days
!I
RIBONUCLEASE. IV
after irradiation, and of those animals not autopsied (20), 11 died by the end of 30 days. Two controls also died in this second experiment. Nikogen determinations were performed by the micro-Kjeldahl method. RESULTS
Mitochondria Experiment I. The RNase activities at the two pH values are plotted in Fig. 1. At pH 5.8, the activities in the irradiated livers tended t’o he lower (20-24 %) than the control values on days 1,2, and 3. This decrease is not considered to be particularly significant in view of the results of Expt. II (below) where no decrease from control values was observed. At pH 7.8, however, there was a significant drop in mitochondrial RNase activity of irradiated animals, the decrease reaching a maximum of 48 % on day 5. Experiment II. The results for mitochondrial RNase activity are
-
::
y;s Q ,oY -
-
8
0
0
OpJ X X
-21
- -.-. 8x X *c-------- p -7 -20
$6 d II:
PH 7.8
PH5.8
10-
x
c
v $-., x . ..\
-24 -
X
x
-25
-33
2-
“*-...sv X
2
3
5
123
x --.
x
x -45
I
0 x
x
-48
5
DAY FIG. 1. Experiment I: RNase activity in separated mitochondria from the livers of irradiated and control rats. X = irradiated animals, 0 = controls. Solid line is the average of the controls; dotted line is the average of the irradiated rats. The values below the experimental points are per cent differences of irradiated averages from control averages. The assay system consisted of 0.25 ml. of mitochondria, 1.0 ml. of 1% P32-labeled RNA, 1.0 ml. of VeronalLacetate buffer, pH 5.8 or 7.8, and 0.75 ml. of HzO. The mixture was incubated at 37°C. for 30 min. and unhydrolyzed RNA then precipitated with 3 ml. of acid-alcohol (8) and the radioactivity of an aliquot of the filtrate determined. Relative activit,v (the ordinate)
is counts/mg.
N X 10-a.
10
JAY
6.
II 123
5
8
IO
I2
ROTH
X -8-5-6 -3, -44 1111111,11111 123 5 8
-33
IO
-10 12
DAY FIG. 2. Experiment II: RNase activity in separated mitochondria from the livers of irradiated and control rats. The assay system was the same as that described in Fig. 1. Single determinations were made on each mitochondrial preparation. X = irradiated animals, 0 = controls. Solid line is the average of the controls; dotted line the average of the irradiated rats. The values below the experimental points are per cent differences of irradiated averages from control averages. Relative activity (the ordinate) is counts/mg. N X 10-a.
plotted in Fig. 2. At pH 5.8, the values for the irradiated animals did not differ significantly from the control values on any of the days assays were performed. Both the control and irradiated activities declined considerably on the second and third day but recovered on the fourth day. Considerable variability was noted in the individual values in both experiments, but this has been observed generally with mitochondrial RNase activities. At pH 7.8, the RNase activity of the mitochondria of irradiated rats did not fall significantly below the control level until the fifth day, when the decreasewas 31%. On this day, the four control values averaged somewhat lower than on most of the other days of measurement, and this may have contributed to a smaller decreaseon the fifth day than was observed in Expt. I. On the eighth day, the decrease in mitochondrial RNase activity was maximal (-44 %), and on the tenth and twelfth days, recovery appeared to set in, the values being 33 and 10% less than the controls, respectively. In view of the difference in the source and ages of the animals in the two experiments, these results may be considered to be fairly consistent. The different response of the RNase activity at pH’s 5.8 and 7.8 could be taken as evidence
RIBONUCLEASE.
IV
11
for the view that these activities represent two different enzymes; however, other interpretations are still possible. Since irradiation of certain tissues may result in a decrease in RnTA synthesis (5-7), it is tempting to speculate that the enzyme which is active at pH 7.8 may be related to t,his process. RNase Inhibitor The inhibitor, which occurs in the supernatant fraction, is a heatlabile, nondialyzable substance sensitive to certain -SW reagents. Large quantities of RNase are normally bound to it.2 This inhibitor is of interest, as it may furnish a clue as to the function of intracellular RNases and to the elucidation of their relationships to RNA and prot,ein synthesis. In Expts. I and II, a decrease of 12 and 41%, respectively, in inhibitor activit,y was noted in irradiated animals on the first day after irradiation. By the second day, activity had returned to control values in both experiments, and no further significant change was observed, although in Expt. I the values in the irradiated animals tended to be somewhat higher than the controls (3-15 %) on the second, t,hird, and fifth days. Initial changes in inhibitor activity may bc quite transient, and it would be of interest to measure activity at intervals less than 24 hr. after irradiation. Whole Homogenate In a previous paper (I), Wistar rats were subjected to 600 r. of whole body x-irradiation, and RNase activity of liver homogenates was assayed at’ pH F.O. Under these conditions, there was an increase in RNase activity of approximately 50% on days 2 and 3 postirradiation, followed by a decline t’o 25% of control activity by day 8. Thereafter, a rise to somewhat above control levels occurred by day 12. The changes recorded in RNase activity of whole homogenate, measured at pH 5.8 in Expt. II, were considerably less in the present report, although they varied approximately in the same direction as previously, being +2, +S, -8, -20, +13, -4, and $4 on the lst, 2nd, 3rd, 5th, 8th, and 12th day aft’er irradiation, respectively. The reason for the discrepancy is not, apparent. It is possible that the younger rats, used in the present work, are more resistant to changes in the particular system under study. In Expt. II, although variations in RNase activity, measured nD pH 7.8, of whole homogenate were noted, between the controls and 2 Roth, J. S., unpublished
results.
12
JAY S. ROTH
x-irradiated animals, these variations showed no particular trend or correlation with any of the above results. Also, no significant changes were observed in the RNase activity of the supernatant fractions in either experiment. DISCUSSION The relationship between inhibitor activity and RNase activity of the whole homogenate (measured at pH 7.8) could be either direct or inverse. Since a large quantity of RNase is normally bound by the inhibitor, inactivation of the latter by irradiation might release RNase activity. It is also possible that the inhibitor-enzyme complex, which apparently still has additional sites available for combination with added RNase,2 may have these active sites destroyed by radiation without breaking the enzyme-inhibitor bonds, and thus without release of RNase. Consideration of the data of Expt. II, day 10, favors the former view. On this day, whole homogenate RNase activity was +29%, isolated mitochondria RNase activity - 33 %, and inhibitor activity + 12 %, all measured at pH 7.8. Release of enzyme by the inhibitor might have been sufficient to overcome the lowered mitochondrial activity and give a net increase in homogenate activity. In vitro irradiation of the supernatant fraction is being carried out at present in an effort to provide more definite answers to the above questions. ACKNOWLEDGMENTS The author wishes to acknowledge the competent assistance of Miss Jeanne Boyd and Miss Malena Der Avedisian in carrying out the analyses.
SUMMARY 1. Rats were subjected to 600 r. of whole body x-irradiation, and RNase assays were carried out on liver homogenates and fractions, at intervals after irradiation. 2. A significant decrease was noted in the RNase activity, measured at pH 7.8, of separated mitochondria prepared from the livers of irradiated animals. 3. RNase
inhibitor
activity
in the supernatant
fractions
obtained
from the livers of irradiated rats, was depressed by 12 and 41%, in two experiments, 1 day after irradiation, when compared to control values. Recovery to control levels was observed by the second day after exposure.
RIBONUCLEASE.
IV
13
4. The RNase activity of whole liver homogenate, measured at pH 5.8, showed only small changes from control values, in irradiated animals, while activity at pH 7.8 in whole homogenate or supernatant fraction from irradiated animals did not show significant changes from the control levels. 5. Some implications of these results were discussed. REFERENCES 1. ROTH, J. S., EICHEL, H. J., WASE, A., ALPER, C., AND BOYD, M. J., Arch. Biothem. and Biophys. 44,95 (1953). 2. ROTH, J. S., J. Biol. Chem. 208, 181 (1954). 3. DELAMIRANDE,G.,ALLARD,C.,DACOSTA, H.C., AND CANTERO,A.,S~~~~~~ 119, 351 (1954). ~.‘RoTH, J. S., Federation Proc. 14,272 (1955). 5. ABRAMS, R., Arch. Biochem. 30,90 (1951). 6. THOMSON, J.F., TOURTELLOTTE, W. W., CARTTAR, M.S., AND STORER, J.B., Arch. Biochem. and Biophys. 42, 185 (1953). 7. ORD, M. G., AND STOCKEN,L. A., Physiol. Revs. 33,356 (1953). 8. ROTH, J. S., AND MILSTEIN, S. W., J. Biol. Chem. 196,489 (1952).