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Lysine catabolism by liver after partial hepatectomy
346
PRELIMINARY NOTES
small as o.5/~g per g body wt. Control experiments with extracts from other mouse tissues (spleen, liver, kidney, lung), injected at a concentration up to IO times higher, were completely negative. The granulocytosis promoting activity of the partially parified extract was retained after prolonged dialysis against distilled water, suggesting that the granulocytosis factor is associated with a large molecule. The biological activity was completely destroyed by heating for IO min at 9 o°. Experiments are under way in order to gain more information on the chemical characteristics of the granulocytosis factor as well as on the nature of the induced leukocytosis. It remains to be ascertained whether the salivary factor itself is responsible for the discharge of granulocytes from the bone marrow, or whether it acts by stimulating the production of other substances (e.g. Leukocytosis-promoting factor 7, Leukopoietin-G s, L I P , or others) and, in the former case, whether the granulocytosis factor exerts a granulopoietic effect on the bone marrow. This work was supported b y C.N.R. and by Merck-Sharp and Dohme.
Chemistry Department, School of Medicine, Universit~ Cattolica S. Cuore, Rome (Italy)
P. U. ANGELETTI M. L. SALVI F. CAPANI L. FRATI
I R. LEvI-MONTALClNI AND P. U. ANGELETTI, Proc. Intern. Conf. on Salivary Glands and their Secretions, Washington, 1962, P e r g a m o n , 1964, p. 129. 2 R. LEvI-MONTALClNI, Science, 143 (1964) lO 5. 3 R. LEvI-MONTALClNI AND S. COHEN, Ann. N . Y . Acad. Sci., 85 (196o) 324 . 4 S. COHEN, J. Biol. Chem., 237 (1962) 1555. 5 P- U. ANGELETTI, M. L. SALVI, R. L. CHESANOW AND S. COHEN, Experientia, 20 (19641 146. 6 0 . LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 7 V. MENKIN, Ann. N . Y . Acad. Sci., 59 (I955) 956. 8 H. R. BIERMAI% Ann. N . Y . Acad. Sci., 113 (1964) 753. 9 A. S. GORDON, E. S. HANDLER, C. D. SIEGEL, B. S. DORNFEST AND J. Lo BuE, Ann. N . Y . Acad. Sci., 113 (1964) 766.
Received August 23rd, 1965 Biochim. Biophys. Acta,
III
(1965) 344-346
PN 2 1 1 1 0
Lysine catabolism by liver after partial hepatectomy Removal ot two-thirds of the liver of the rat leads to an increase in the pool size of hepatic free lysine l& The increase appears to begin immediately after the operation and a peak (about a three-fold rise) is achieved 6 h later. At this time, the pool begins to decline and by 12 h after the operation it has almost returned to the preoperative level 2. Unpublished studies (M. KOGA AND I. LIEBERMAN) have provided evidence that the increase in liver lysine results from the inability of the one-third portion of the Biochim. Biophys. Acta, i i i (1965) 346-348
347
P R E L I M I N A R Y NOTES
liver to degrade the amounts of lysine that are now available to it. The mechanism of the reduction of the pool to the preoperative level (before an increase in liver mass occurs) has, however, not been studied. The most likely possibility would seem to be an enhancement in the ability of the liver remnant to degrade lysine. The recent demonstration of lysine breakdown by cell-free (mitochondrial) preparations from liver 3 and the identification of saccharopine (e-N-(L-glutaryl-2)-L-lysine) 4,5 as an intermediate of lysine catabolism 3 allowed this possibility to be tested. Comparisons have now been made of the rate of lysine breakdown by rat liver mitochondria as a Iunction of the time after partial hepatectomy (Table I). As the table shows, partial hepatectomy caused a rise in the ability of the mitochondria to produce saccharopine and CO s from lysine. Under the conditions used, the formation of these products was linear with time and proportional to the amount of mitochondrial protein. TABLE
I
RATE OF LYSINE CATABOLISM BY LIVER MITOCHONDRIA FROM SHAM AND PARTIALLY HEPATECTOMIZED RATS
L i v e r m i t o c h o n d r i a ( 2 - 4 m g of protein) w e r e i n c u b a t e d in e v a c u a t e d T h u n b e r g t u b e s in r e a c t i o n m i x t u r e s c o n t a i n i n g L14C]lysine as p r e v i o u s l y described s. A f t e r 3 ° min, o. 5 m l of o.2 5 M HC1Oa w a s a d d e d t h r o u g h t h e sidearms, t h e released CO 2 w a s t r a p p e d in t h e K O H p r e s e n t in t h e stoppers, a n d t h e acidified r e a c t i o n m i x t u r e s w e r e n e u t r a l i z e d w i t h I
Hepateetomy
Time
Saccharopine
CO S
after operation
(counts/min per mg protein)
(counts/rain per m e protein)
(h) Sham
Partial
o
I I ooo
IISO
6
12 6 o o
IIOO
12
IO 500
II30
O
12 500
1 3 6
1730 27 40O
9 12
1060 1050 297 ° 3180
21 7OO
363 °
To gain some insight into the mechanism o~ the enhancement, attempts were made to prepare soluble enzyme preparations from the mitochondria. It has not yet been possible to prepare in soluble form the activity that converts lysine to saccharopine. Soluble preparations that actively degraded saccharopine to ~-aminoadipic, ~-ketoadipic, and glutaric acids (small amounts of =-ketoglutaric acid and CO s were also formed) have, however, been obtained by sonic oscillation. With these preparations, no increase in the rate of saccharopine breakdown was found between o and Biochim. Biophys. Acla, i i i (1965) 3 4 6 - 3 4 8
348
PRELIMINARY NOTES
6 h after partial hepatectomy. It would seem, therefore, that the enhanced activity of the liver mitochondria either reflects a rise only in the rate of conversion of lysine to saccharopine or a change unrelated to the levels of the mitochondrial enzymes.
Department of Microbiology, University of Pittsburgh, School of Medicine, Pittsburgh, Pa. (U.S.A.) I 2 3 4 5
KAZUYA IRVING
HIGASHINO* LIEBERMAN
V. FERRARI AND 1~. D. HARKNESS, J. Physiol., 124 (1954) 443M. FujlOI~A, M. KOGA AND I. LIEB~RMAIq, J. Biol. Chem., 238 (I963) 34Ol. K. HIGASHINO AND I. LIEBERMAN, Biochem. Biophys. Res. Commun., 2o (I965) 285. S. DARLING AND P. O. LARSEN, Acta Chem. Scan&, 15 (1961) 743. A. KJAER AND P. O. LARSEN, A a a Chem. Scan&, 15 (I961) 75 o.
Received August 2oth, 1965 * Present address: Third Department of Internal Medicine, Osaka University Medical School, Osaka, Japan. Biochim. Biophys. Acta, i i i (1965) 346-348
PRELIMINARY NOTES
small as o.5/~g per g body wt. Control experiments with extracts from other mouse tissues (spleen, liver, kidney, lung), injected at a concentration up to IO times higher, were completely negative. The granulocytosis promoting activity of the partially parified extract was retained after prolonged dialysis against distilled water, suggesting that the granulocytosis factor is associated with a large molecule. The biological activity was completely destroyed by heating for IO min at 9 o°. Experiments are under way in order to gain more information on the chemical characteristics of the granulocytosis factor as well as on the nature of the induced leukocytosis. It remains to be ascertained whether the salivary factor itself is responsible for the discharge of granulocytes from the bone marrow, or whether it acts by stimulating the production of other substances (e.g. Leukocytosis-promoting factor 7, Leukopoietin-G s, L I P , or others) and, in the former case, whether the granulocytosis factor exerts a granulopoietic effect on the bone marrow. This work was supported b y C.N.R. and by Merck-Sharp and Dohme.
Chemistry Department, School of Medicine, Universit~ Cattolica S. Cuore, Rome (Italy)
P. U. ANGELETTI M. L. SALVI F. CAPANI L. FRATI
I R. LEvI-MONTALClNI AND P. U. ANGELETTI, Proc. Intern. Conf. on Salivary Glands and their Secretions, Washington, 1962, P e r g a m o n , 1964, p. 129. 2 R. LEvI-MONTALClNI, Science, 143 (1964) lO 5. 3 R. LEvI-MONTALClNI AND S. COHEN, Ann. N . Y . Acad. Sci., 85 (196o) 324 . 4 S. COHEN, J. Biol. Chem., 237 (1962) 1555. 5 P- U. ANGELETTI, M. L. SALVI, R. L. CHESANOW AND S. COHEN, Experientia, 20 (19641 146. 6 0 . LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 7 V. MENKIN, Ann. N . Y . Acad. Sci., 59 (I955) 956. 8 H. R. BIERMAI% Ann. N . Y . Acad. Sci., 113 (1964) 753. 9 A. S. GORDON, E. S. HANDLER, C. D. SIEGEL, B. S. DORNFEST AND J. Lo BuE, Ann. N . Y . Acad. Sci., 113 (1964) 766.
Received August 23rd, 1965 Biochim. Biophys. Acta,
III
(1965) 344-346
PN 2 1 1 1 0
Lysine catabolism by liver after partial hepatectomy Removal ot two-thirds of the liver of the rat leads to an increase in the pool size of hepatic free lysine l& The increase appears to begin immediately after the operation and a peak (about a three-fold rise) is achieved 6 h later. At this time, the pool begins to decline and by 12 h after the operation it has almost returned to the preoperative level 2. Unpublished studies (M. KOGA AND I. LIEBERMAN) have provided evidence that the increase in liver lysine results from the inability of the one-third portion of the Biochim. Biophys. Acta, i i i (1965) 346-348
347
P R E L I M I N A R Y NOTES
liver to degrade the amounts of lysine that are now available to it. The mechanism of the reduction of the pool to the preoperative level (before an increase in liver mass occurs) has, however, not been studied. The most likely possibility would seem to be an enhancement in the ability of the liver remnant to degrade lysine. The recent demonstration of lysine breakdown by cell-free (mitochondrial) preparations from liver 3 and the identification of saccharopine (e-N-(L-glutaryl-2)-L-lysine) 4,5 as an intermediate of lysine catabolism 3 allowed this possibility to be tested. Comparisons have now been made of the rate of lysine breakdown by rat liver mitochondria as a Iunction of the time after partial hepatectomy (Table I). As the table shows, partial hepatectomy caused a rise in the ability of the mitochondria to produce saccharopine and CO s from lysine. Under the conditions used, the formation of these products was linear with time and proportional to the amount of mitochondrial protein. TABLE
I
RATE OF LYSINE CATABOLISM BY LIVER MITOCHONDRIA FROM SHAM AND PARTIALLY HEPATECTOMIZED RATS
L i v e r m i t o c h o n d r i a ( 2 - 4 m g of protein) w e r e i n c u b a t e d in e v a c u a t e d T h u n b e r g t u b e s in r e a c t i o n m i x t u r e s c o n t a i n i n g L14C]lysine as p r e v i o u s l y described s. A f t e r 3 ° min, o. 5 m l of o.2 5 M HC1Oa w a s a d d e d t h r o u g h t h e sidearms, t h e released CO 2 w a s t r a p p e d in t h e K O H p r e s e n t in t h e stoppers, a n d t h e acidified r e a c t i o n m i x t u r e s w e r e n e u t r a l i z e d w i t h I
Hepateetomy
Time
Saccharopine
CO S
after operation
(counts/min per mg protein)
(counts/rain per m e protein)
(h) Sham
Partial
o
I I ooo
IISO
6
12 6 o o
IIOO
12
IO 500
II30
O
12 500
1 3 6
1730 27 40O
9 12
1060 1050 297 ° 3180
21 7OO
363 °
To gain some insight into the mechanism o~ the enhancement, attempts were made to prepare soluble enzyme preparations from the mitochondria. It has not yet been possible to prepare in soluble form the activity that converts lysine to saccharopine. Soluble preparations that actively degraded saccharopine to ~-aminoadipic, ~-ketoadipic, and glutaric acids (small amounts of =-ketoglutaric acid and CO s were also formed) have, however, been obtained by sonic oscillation. With these preparations, no increase in the rate of saccharopine breakdown was found between o and Biochim. Biophys. Acla, i i i (1965) 3 4 6 - 3 4 8
348
PRELIMINARY NOTES
6 h after partial hepatectomy. It would seem, therefore, that the enhanced activity of the liver mitochondria either reflects a rise only in the rate of conversion of lysine to saccharopine or a change unrelated to the levels of the mitochondrial enzymes.
Department of Microbiology, University of Pittsburgh, School of Medicine, Pittsburgh, Pa. (U.S.A.) I 2 3 4 5
KAZUYA IRVING
HIGASHINO* LIEBERMAN
V. FERRARI AND 1~. D. HARKNESS, J. Physiol., 124 (1954) 443M. FujlOI~A, M. KOGA AND I. LIEB~RMAIq, J. Biol. Chem., 238 (I963) 34Ol. K. HIGASHINO AND I. LIEBERMAN, Biochem. Biophys. Res. Commun., 2o (I965) 285. S. DARLING AND P. O. LARSEN, Acta Chem. Scan&, 15 (1961) 743. A. KJAER AND P. O. LARSEN, A a a Chem. Scan&, 15 (I961) 75 o.
Received August 2oth, 1965 * Present address: Third Department of Internal Medicine, Osaka University Medical School, Osaka, Japan. Biochim. Biophys. Acta, i i i (1965) 346-348