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Changes in ribonuclease activity in rat liver following hepatectomy
BIOCHIMICA ET BIOPHYSICA ACTA
027
SHORT COMMUNICATIONS BBA 93313
Chonges in ribonucleose octivity in rot liver following hepotectomy There appears to be no simple correlation between the level of ribonuclease activity in cells and tissues and their metabolic activity z-4. Exposure of mice and rats to X-rays leads to a rapid increase in ribonuclease activity of lymphoid organs 5. In part at least this was an indirect effect of radiation, as it could be induced b y irradiation of the head only with the thymus shielded e, a procedure that also caused a t e m p o r a r y rise in mitotic activity in the thymus ~. To test whether ribonuclease activity also increases after other procedures that cause a t e m p o r a r y rise in mitotic activity, measurements were made on regenerating liver at different times after hepatectomy. The experimental procedures were designed to measure the total amount of alkaline fibonuclease present and do not reflect alterations in specific enzyme inhibitors or in intracellular localisation. Hepatectomy. Surgical removal under ether anaesthetic was carried out as described b y HIGGINS AND ANDERSON8 on male Wistar rats aged between 21 and 42 days. 65-75 % of the total liver was removed, leaving within the peritoneum the right lateral lobe and the small caudate lobe. Analysis o[ the liver. At intervals between 3 h and 8 days after hepatectomy the animals were injected intraperitoneally with I #C/g body weight of uniformly 3H-labelled thymidine with a specific activity of 8.8 C/mmole. Absorption from the peritoneum was complete in less than IO rain and the rats were killed with ether 15 min after injection when the livers were removed and immediately weighed. The tissue was cut up and homogenized in 5 ml of ice-cold distilled water at o ° using a glass homogenizer fitted with a Teflon plunger, rotated at about 20o0 rev./min for I min and then immersed in a salt-ice mixture at --lO% The frozen homogenate was cooled to --7 °o and thawed at o ° three times, after which particle-bound ribonuclease appeared to be completely solubilised and the ribonuclease activity of the supernatant was not increased further even when thirty cycles of freezing and thawing were carried out. DNA was extracted from the homogenate according to the procedure of CRADDOCK9 and counted b y scintillation in a Tri-carb Packard Counter. The D N A content of the homogenate was determined colourmetrically b y the DlSCHE z° method. Removal o] ribonuclease inhibitors. Heating at an acid p H precipitates the inhibitory proteins but has no effect on the activity of alkaline ribonuclease 5. To I ml of the repeatedly frozen and thawed tissue homogenate was added o.I ml of o.i M acetate buffer at p H 3.4 and the whole heated to 65 ° for 15 min and then centrifuged for IO min at 5000 rev./min. The effectiveness of the removal of ribonuclease inhibitors was checked b y adding known concentrations of crystalline pancreatic ribonuclease to the tissue homogenate treated in this way. The increase in ribonuclease activity corresponded to the amount of pancreatic ribonuclease added. The addition to the heated homogenate of p-chloromercuribenzoate, which inactivates ribonuclease inhibitor s, did not alter the ribonuclease activity of the homogenate. Biochim. Biophys. Acta, 157 (1968) 627-629
?
"2
-,q
I
5 5 5 5 5 4
Number o[ animals
ACTIVITY
AND
55i5.o8 5022 6026. 4 50±6 51±4. 9 5625.2
INCORPORATION
2.8 ± 0 . 2 8 0.8220.09 i.i ~o.i 5 0.9 ~ o . I5 1. 7 ± 0 . 2 3 1.9 ~ o . 2 8
Wet wt. (g)
INTO
5.6 ± 0 . 7 3 o.952o.17 1.43±o.41 2.2320.66 2.6220.35 2.2620.24
D N A (rag)
Per whole liver*
OF THYMIDINE
Body wt. (g)
RATE
2.9 ± 0 . 8 0.20520.03 3.42 2 1.o 7.38 2 1 . 6 6.8 ± i . o 2.47 ~ o . 2 6
**
TIMES
AFTER
4306028700 5464025200 56o5oi IIoo 6175026250 8112029000 342oo~28oo
Radioactivity (counts~rain)
AT DIFFERENT
Ribonuclease (mt~g)
LIVER
* ± after the values are t h e s t a n d a r d deviations w i t h i n the g r o u p of animals. activity/rag D N A after h e p a t e c t o m y . E x p r e s s e d as activity/rag D N A in control
Control 20 3° 54 72 192
Time alter hepatectomy (h)
RIBONUCLEANE
TABLE
i.o 0.4 4.4 6.2 4.8 2.0
Ribonuclease
i.o 7.3 4.9 3.6 3.9 1. 9
Thymidine incorporation
Change in activity** relative to control
HEPATECTOMY
c/?
z
>
o
H
©
m
b~ GO
SHORT COMMUNICATIONS
029
Estimation o/ribonuclease activity. A modification of the acid-soluble nucleotide assay 11 was used. A I °/o solution of purified yeast RNA (Worthington) was added to the supernatant of the tissue homogenate after removal of inhibitor and incubated for 30 min at 25 ° in p H 8 Tris buffer. The reaction was stopped and the undigested RNA precipitated b y adding an ice cooled mixture of perchloric acid and ethanol. The absorbance of the supernatant was measured at 260 m# and the increase in absorption over that of the homogenate without RNA was a direct measure of the ribonuclease activity of the homogenate. The ribonuclease activity was expressed as the equivalent in #g of crystalline pancreatic ribonuclease (Worthington). Table I shows that there is a sharp rise in ribonuclease activity of the liver homogenate following hepatectomy but that this increase occurs after the peak of DNA synthesis. While enzymatic activities are known to v a r y at different stages in the mitotic cycle, the increased ribonuclease activity cannot be explained in this way because it occurs too late and persists for too long. The total time occupied b y the "G 2 phase" and mitosis in regenerating liver extends only over 3-4 h following the period of DNA synthesis TM, after which the majority of the cells are not in a division cycle. The increase in ribonuclease synthesis coincides with the return of the hepatocytes to differentiated function. Following the stimulus for division by hepatectomy the pattern of protein synthesis must undergo a drastic change. From being predominantly engaged in making proteins that are excreted, such as albumin, the cells synthesise material needed for their own reproduction. Following regeneration the reverse process must occur and the rise in ribonuclease synthesis at this time m a y be associated with the replacement of some of the messenger RNA, coding for proteins needed for mitosis, b y messenger RNA coding for proteins that are made by non-dividing liver cells. The material will be submitted b y D. M. as part of a Ph.D. Thesis of the Hebrew University, Jerusalem. Rogo// Institute, Beilinson Hospital, Petah Tiqva (Israel) Chester Beatty Research Institute, Sutton, Surrey (Great Britain)
D. MAOR P. ALEXANDER
L. LEDOUX, S. BRANDLI AND C. DELAEPE, Nature, 181 (1958) 913 . S. BRONXz, Biochim. Biophys. Acta, 24 (1957) 5 °2. A. K. CHAKRAVORTY AND H. BUSCH, Biochem. Pharmacol., 16 (1967) 367. E. BRESNICK, J. SAGE AND K. LANCLOS, Bioehim. Biophys. Acta, 114 (1966) 631. P. P. WEYMOUTH, Radiation Res., 8 (1958) 307 . D. MAOR AND P. ALEXANDER, Intern. J. Radiation Biol., 6 (I963) 93. D. MAOR AND P. ALEXANDER, Nature, 205 (1965) 4 o. C. M. HIGGINS AND R. M. ANDERSON, Arch. Pathol., 12 (1931) 186. C. G. CRADDOCK, J. Clin. Invest. 41 (1962) 360. Z. DlSCHE, i n E. CHARGAFF AND J. N. DAVIDSON, The Nucleic Acids, Vol. 2, A c a d e m i c Press, N e w Y o r k , 1955, p. 283. i i C. B. ANFINSEN, J. Biol. Chem., 207 (1954) 2Ol. 12 J. I. FABRIKANT, Radiation Res., 31 (1967) 304 . I 2 3 4 5 6 7 8 9 lO
Received February 28th, 1968 Bioehim. Biophys. Acta, 157 (1968) 6 2 7 - 6 2 9
027
SHORT COMMUNICATIONS BBA 93313
Chonges in ribonucleose octivity in rot liver following hepotectomy There appears to be no simple correlation between the level of ribonuclease activity in cells and tissues and their metabolic activity z-4. Exposure of mice and rats to X-rays leads to a rapid increase in ribonuclease activity of lymphoid organs 5. In part at least this was an indirect effect of radiation, as it could be induced b y irradiation of the head only with the thymus shielded e, a procedure that also caused a t e m p o r a r y rise in mitotic activity in the thymus ~. To test whether ribonuclease activity also increases after other procedures that cause a t e m p o r a r y rise in mitotic activity, measurements were made on regenerating liver at different times after hepatectomy. The experimental procedures were designed to measure the total amount of alkaline fibonuclease present and do not reflect alterations in specific enzyme inhibitors or in intracellular localisation. Hepatectomy. Surgical removal under ether anaesthetic was carried out as described b y HIGGINS AND ANDERSON8 on male Wistar rats aged between 21 and 42 days. 65-75 % of the total liver was removed, leaving within the peritoneum the right lateral lobe and the small caudate lobe. Analysis o[ the liver. At intervals between 3 h and 8 days after hepatectomy the animals were injected intraperitoneally with I #C/g body weight of uniformly 3H-labelled thymidine with a specific activity of 8.8 C/mmole. Absorption from the peritoneum was complete in less than IO rain and the rats were killed with ether 15 min after injection when the livers were removed and immediately weighed. The tissue was cut up and homogenized in 5 ml of ice-cold distilled water at o ° using a glass homogenizer fitted with a Teflon plunger, rotated at about 20o0 rev./min for I min and then immersed in a salt-ice mixture at --lO% The frozen homogenate was cooled to --7 °o and thawed at o ° three times, after which particle-bound ribonuclease appeared to be completely solubilised and the ribonuclease activity of the supernatant was not increased further even when thirty cycles of freezing and thawing were carried out. DNA was extracted from the homogenate according to the procedure of CRADDOCK9 and counted b y scintillation in a Tri-carb Packard Counter. The D N A content of the homogenate was determined colourmetrically b y the DlSCHE z° method. Removal o] ribonuclease inhibitors. Heating at an acid p H precipitates the inhibitory proteins but has no effect on the activity of alkaline ribonuclease 5. To I ml of the repeatedly frozen and thawed tissue homogenate was added o.I ml of o.i M acetate buffer at p H 3.4 and the whole heated to 65 ° for 15 min and then centrifuged for IO min at 5000 rev./min. The effectiveness of the removal of ribonuclease inhibitors was checked b y adding known concentrations of crystalline pancreatic ribonuclease to the tissue homogenate treated in this way. The increase in ribonuclease activity corresponded to the amount of pancreatic ribonuclease added. The addition to the heated homogenate of p-chloromercuribenzoate, which inactivates ribonuclease inhibitor s, did not alter the ribonuclease activity of the homogenate. Biochim. Biophys. Acta, 157 (1968) 627-629
?
"2
-,q
I
5 5 5 5 5 4
Number o[ animals
ACTIVITY
AND
55i5.o8 5022 6026. 4 50±6 51±4. 9 5625.2
INCORPORATION
2.8 ± 0 . 2 8 0.8220.09 i.i ~o.i 5 0.9 ~ o . I5 1. 7 ± 0 . 2 3 1.9 ~ o . 2 8
Wet wt. (g)
INTO
5.6 ± 0 . 7 3 o.952o.17 1.43±o.41 2.2320.66 2.6220.35 2.2620.24
D N A (rag)
Per whole liver*
OF THYMIDINE
Body wt. (g)
RATE
2.9 ± 0 . 8 0.20520.03 3.42 2 1.o 7.38 2 1 . 6 6.8 ± i . o 2.47 ~ o . 2 6
**
TIMES
AFTER
4306028700 5464025200 56o5oi IIoo 6175026250 8112029000 342oo~28oo
Radioactivity (counts~rain)
AT DIFFERENT
Ribonuclease (mt~g)
LIVER
* ± after the values are t h e s t a n d a r d deviations w i t h i n the g r o u p of animals. activity/rag D N A after h e p a t e c t o m y . E x p r e s s e d as activity/rag D N A in control
Control 20 3° 54 72 192
Time alter hepatectomy (h)
RIBONUCLEANE
TABLE
i.o 0.4 4.4 6.2 4.8 2.0
Ribonuclease
i.o 7.3 4.9 3.6 3.9 1. 9
Thymidine incorporation
Change in activity** relative to control
HEPATECTOMY
c/?
z
>
o
H
©
m
b~ GO
SHORT COMMUNICATIONS
029
Estimation o/ribonuclease activity. A modification of the acid-soluble nucleotide assay 11 was used. A I °/o solution of purified yeast RNA (Worthington) was added to the supernatant of the tissue homogenate after removal of inhibitor and incubated for 30 min at 25 ° in p H 8 Tris buffer. The reaction was stopped and the undigested RNA precipitated b y adding an ice cooled mixture of perchloric acid and ethanol. The absorbance of the supernatant was measured at 260 m# and the increase in absorption over that of the homogenate without RNA was a direct measure of the ribonuclease activity of the homogenate. The ribonuclease activity was expressed as the equivalent in #g of crystalline pancreatic ribonuclease (Worthington). Table I shows that there is a sharp rise in ribonuclease activity of the liver homogenate following hepatectomy but that this increase occurs after the peak of DNA synthesis. While enzymatic activities are known to v a r y at different stages in the mitotic cycle, the increased ribonuclease activity cannot be explained in this way because it occurs too late and persists for too long. The total time occupied b y the "G 2 phase" and mitosis in regenerating liver extends only over 3-4 h following the period of DNA synthesis TM, after which the majority of the cells are not in a division cycle. The increase in ribonuclease synthesis coincides with the return of the hepatocytes to differentiated function. Following the stimulus for division by hepatectomy the pattern of protein synthesis must undergo a drastic change. From being predominantly engaged in making proteins that are excreted, such as albumin, the cells synthesise material needed for their own reproduction. Following regeneration the reverse process must occur and the rise in ribonuclease synthesis at this time m a y be associated with the replacement of some of the messenger RNA, coding for proteins needed for mitosis, b y messenger RNA coding for proteins that are made by non-dividing liver cells. The material will be submitted b y D. M. as part of a Ph.D. Thesis of the Hebrew University, Jerusalem. Rogo// Institute, Beilinson Hospital, Petah Tiqva (Israel) Chester Beatty Research Institute, Sutton, Surrey (Great Britain)
D. MAOR P. ALEXANDER
L. LEDOUX, S. BRANDLI AND C. DELAEPE, Nature, 181 (1958) 913 . S. BRONXz, Biochim. Biophys. Acta, 24 (1957) 5 °2. A. K. CHAKRAVORTY AND H. BUSCH, Biochem. Pharmacol., 16 (1967) 367. E. BRESNICK, J. SAGE AND K. LANCLOS, Bioehim. Biophys. Acta, 114 (1966) 631. P. P. WEYMOUTH, Radiation Res., 8 (1958) 307 . D. MAOR AND P. ALEXANDER, Intern. J. Radiation Biol., 6 (I963) 93. D. MAOR AND P. ALEXANDER, Nature, 205 (1965) 4 o. C. M. HIGGINS AND R. M. ANDERSON, Arch. Pathol., 12 (1931) 186. C. G. CRADDOCK, J. Clin. Invest. 41 (1962) 360. Z. DlSCHE, i n E. CHARGAFF AND J. N. DAVIDSON, The Nucleic Acids, Vol. 2, A c a d e m i c Press, N e w Y o r k , 1955, p. 283. i i C. B. ANFINSEN, J. Biol. Chem., 207 (1954) 2Ol. 12 J. I. FABRIKANT, Radiation Res., 31 (1967) 304 . I 2 3 4 5 6 7 8 9 lO
Received February 28th, 1968 Bioehim. Biophys. Acta, 157 (1968) 6 2 7 - 6 2 9