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On the lack of acetoacetate formation in some amphibian livers
SHORT COMMUNICATIONS
On the lack of acetoacetate formation
417
in some amphibian livers
The formation of acetoacetate in mammalian liver is believed to proceed by way of the “/?-hydroxy-I-methylgluta.ryl-CoA cycle” of 1,YNEN et al.‘. The cycle consists of two enzymatic steps, the condensation of acetoacetyl-CoA and acetylCoA to form ~-hydra~y-~-methylglutaryl~co~~ and its subsequent splitting into free acetoacetate and acetyl-CoA. F&UMMOND AND STERN2 have suggested a pathway involving direct deacylation of acetoacetyl-CoA. Their results can be explained by the finding that mammalian liver contains a strong deacylase activity for acetoacetylglutathione and that commercial preparations of CoA contain appreciable amounts of glutathione3. SAUKR .4ND ERFLE~ found no evidence of the existence of a deacylase for purified acetoacetyl-C.oA in guinea pig liver. To the best of the author’s knowledge no work has been done on acetoacetate formation in any vertebrate lower than birds. The present paper reports the lack of formation of acetoacetate by the livers of two amphibians, an adult anuran (2&a ~~~~~~~~~.~~~ and a neotenic urodele ~~~~~~~~~0~~~~ ~~~~~~~~~~~~, before and after triidothyronine-induced metamorphosis. The livers of these amphibia show no activity of the @-hydroxy-/Y-methylglutaryl-CoA condensing enzyme* but contain p-hydroxy/3-methylglutaryl-CoA cleavage enzyme” activity. Livers of a bony fish and a reptile show rates of acetoacetate formation comparable to those of pigeon (B. PREISS, unpublished results), rat and beef livers.
The Axolotls [A. ~~x~~~~~~~ were obtained from the lake of Xochimilco (south of Mexico City) and were kept in the laboratory for 2-5 months. ~~etarnorph~sis was induced by adding 5 krgjl t~idot~l~~onine to the water for 5 days, followed by IO ,ctg/l for 15 days, with the water being changed daily. R. montezuma frogs were maintained on a chicken-liver diet. The snake, X%oztpLis deppei de~~ei, was captured near Mexico City and fed mice for about 2 months. The gold fish, Carassizcs auratus (Linneaus), were sacrificed
on the day of arrival in CoA, glutathione, biochem, and dried cells tate was determined by
the laboratory, carnitine and acetyl phosphate were purchased from Calof ~~Qs~~~~~~~~;~ &&q.~~~were obtained from Sigma. Acetoacethe method of WALKER@.
Formation of acetoacetate was first measured with liver homogenates and butyrate as substrate. Under these conditions Axolotl liver shows no measurable accumulation of acetoacetate (Table I). Carnitine did not influence considerably the formation of acetoacetate from butyrate by the rat liver homogenate. It seems to be needed for
the utilization of internal substrates. Later liver slices were employed as a test system with butyrate as substrate. It was thought that by using tissue slices, the possibility of inactivating a labile enzyme system would be minimized. Incubations were run in triplicate, and the experiments always included rat liver slices as a reference. It can be seen from Table II that frog liver and the livers from the axolotl before and after metamorphosis produce negligible amounts of acetoacetate as determined by the method of WALKE@. Rat, sna.ke and goldfish livers produce much higher levels. It was of interest to find out whether the activities of the ~-hydrox~-~-methyl-
SHORT COMMUNICATIONS
418 TABLE
I
ACETOACETATE
FORMATION
BY
RAT AND
AXOLOTL
LIVER
HOMOGENATES
The complete system contained: 40 pmoles phosphate buffer (pH 7.4), 3 pmoles ATP, 15 ~moles MgCI,, IOO ymoles KCI, 8 ,umoles EDTA, 0.8 mg bovine serum albumin, 5 pmoles potassium butyrate and r ymole carnitine in a total volume of 2 ml. Each vial contained 0.5 ml liver homogenate (a IO?; homogenatein 0.25 M sucrose spun down at 7ooxg for IO mm). The vials were incubated at 37” for 30 min with shaking. Protein was precipitated by adding HCIO, to a final concentration of 59b. Acetoacetate was determined according to the method of WALKER@. Liver homogenate
SysteP?z
A cetoacetate formed (nmoles) .-. _-..._
Rat
Complete -carnitine ~ butyrate -carnitine,
590 5ro
AxolotI
TABLE
Complete --carnitine - butyrate ~ carnitine,
180
-butyrate
90 IO -0 0
- butyrate
0
.~
II
ACETOACETATE
FORMATION
BY LIVER
SLICES FROM DIFFERENTVERTEBRATES
The medium was the same as in Table I without carnitine. The homogenate was replaced by 0.5 ml 0.25 M sucrose and zoo mg liver slices. Incubations were done in triplicate, with shaking for I h at 37”. Precipitated protein was homogenized and acetoacetate determined in the supernatant according to the procedure of WALXER~. -..-.-_-___ Liver slims n c&a&ate Liver slices Acetoacetate formed (nmoles) formed (nmoles) Rat Snake Goldfish
Axolotl Metamorphosed Frog
370 480 270
IO
axolotl
5 30
glutaryf-CoA condensing enzyme and cleavage enzyme could be demonstrated in the amphibian livers. The method used was that of LVNEN et aE.i. The test system includes acetyl phosphate, CoA and an extract from C. kluyveri as a source of phosphotransacetylase and thiolase. In the presence of both /%hydroxy-/Lmethylglutaryl-CoA condensing enzyme and fl-hydroxy$-methylglutaryl-CoA cleavage enzyme, the acetyl-CoA and acetoacetyl-CoA supplied by the system give rise to free acetoacetate: A crude extract from yeast which contains only condensing enzyme activity (before adsorbtion on AI( gel’) was used as a source of that enzyme (enzyme B). A crude extract from beef liver acetone powder contains both activities and loses its ~-hydroxy-~-meth~rlglutaryl-CoA condensing enzyme activity after heat treatment. Heat treated beef liver extract was used as a source of ,&hydroxy-@-methylglutarylCoA cleavage enzyme (enzyme A). Acetone powders were prepared from the livers of beef, snake, frog and axolotl before and after metamorphosis, and the extracts (without heat treatment) were tested for the activities of both enzymes. It can be seen from Table III that all three amphibian livers show /?-hydroxy~-methylglutaryl-CoA cleavage enzyme activity but no activity of the condensing enzyme. Since the Axolotl incorporates label from /r-14C]acetate into cholesterol (J. GUZMkN-GARCIA AND J. D~Az-NAGOYA, personal communication) and squalene (B. PREISS, unpublished results), it could be suggested that it only has a pathway to Biochim. Riophys. Acta, 176
(1969)
417-419
SHORT COMMUNICATIONS TABLE
419
III
A TEST FOR THE ACTIVITIES OF ~-HYDROXY-~-h~ET~YL~LUTARSL-COA ~-HYDRoxY-~-METHYLGL~~ARYL-CO~ VERTEBRATE LIVERS
CLEAVAGE
ENZYME
IN ACETONE
CONDENSING ENZYME AND POWDERS FROM DIFFERENT
Each tube contained: 70 /
---I___
Beef (0.2 ml) Snake Axolotl
metamorphosed
Auxiliary enzyme
Acetoacetate jovmed (nmoles)
None
290
None
67 7
None A B None
axolotl
A
Frog
Beef (0.2 ml) + axolotl Beef (0.2 ml) +metamorphosed None None
axolotl
B None A B None None A B
IO
80 IO
30 70 0
3 56 220 210 20
4
mevalonate which does not involve free intermediates, similar to the one found by BRODIE, WASSON AND PORTER' in pigeon liver. The Axolotl extracts do not seem to contain an inhibitor to the beef liver system, since addition of axolotl liver extracts does not diminish considerably the activity of the beef liver extract. The results also testify to the absence of a deacylase for acetoacetyl-CoA in the three amphibian livers. The author wishes to express his gratitude to Dr. JESSE Guz~irANGARCIA of this department for his helpful criticism of the work. Departamenta de Bioquimica, Falcultad de Medicina, C&dad Universitaria, M.hico, (M&co) I I?.LYNEN, 2
3 4 5
6 7
BENJAMIN
PREISS
D.F.
U. HENNING,C.BUBLITZ,B.S~RBOAND
L. KRGPLIN-REUFF
~~oc~e~.Z.,33O(Ig58)
269. G.L.DRUMMOND AND J.R.STERN,J. BioLChem.,z35 (x960)318. F. SAUER AND J. D. ERFLE,J.B~~Z.C~C~., 241 (1966) 30. J, J. FERGUSON, JR. AND H. RUDNEY, j. Bid. Chem., 234 (1959) 1072. B. K.BACHHAWAT,W.G.ROBINSON AND M.J. COON, J.Biol.Chem., 216(1955)7s7. P. G. WALKER, Biochem. J., $3 (1954) 699. J.D. BRODIE,G.WASSONAND J.W.PORTER, J.Biol.Chem.,238(1g63) 1294.
Received September rgth, 1968 Biochim.
Biophys.
Acta,
176 (rgbg) 417-419
On the lack of acetoacetate formation
417
in some amphibian livers
The formation of acetoacetate in mammalian liver is believed to proceed by way of the “/?-hydroxy-I-methylgluta.ryl-CoA cycle” of 1,YNEN et al.‘. The cycle consists of two enzymatic steps, the condensation of acetoacetyl-CoA and acetylCoA to form ~-hydra~y-~-methylglutaryl~co~~ and its subsequent splitting into free acetoacetate and acetyl-CoA. F&UMMOND AND STERN2 have suggested a pathway involving direct deacylation of acetoacetyl-CoA. Their results can be explained by the finding that mammalian liver contains a strong deacylase activity for acetoacetylglutathione and that commercial preparations of CoA contain appreciable amounts of glutathione3. SAUKR .4ND ERFLE~ found no evidence of the existence of a deacylase for purified acetoacetyl-C.oA in guinea pig liver. To the best of the author’s knowledge no work has been done on acetoacetate formation in any vertebrate lower than birds. The present paper reports the lack of formation of acetoacetate by the livers of two amphibians, an adult anuran (2&a ~~~~~~~~~.~~~ and a neotenic urodele ~~~~~~~~~0~~~~ ~~~~~~~~~~~~, before and after triidothyronine-induced metamorphosis. The livers of these amphibia show no activity of the @-hydroxy-/Y-methylglutaryl-CoA condensing enzyme* but contain p-hydroxy/3-methylglutaryl-CoA cleavage enzyme” activity. Livers of a bony fish and a reptile show rates of acetoacetate formation comparable to those of pigeon (B. PREISS, unpublished results), rat and beef livers.
The Axolotls [A. ~~x~~~~~~~ were obtained from the lake of Xochimilco (south of Mexico City) and were kept in the laboratory for 2-5 months. ~~etarnorph~sis was induced by adding 5 krgjl t~idot~l~~onine to the water for 5 days, followed by IO ,ctg/l for 15 days, with the water being changed daily. R. montezuma frogs were maintained on a chicken-liver diet. The snake, X%oztpLis deppei de~~ei, was captured near Mexico City and fed mice for about 2 months. The gold fish, Carassizcs auratus (Linneaus), were sacrificed
on the day of arrival in CoA, glutathione, biochem, and dried cells tate was determined by
the laboratory, carnitine and acetyl phosphate were purchased from Calof ~~Qs~~~~~~~~;~ &&q.~~~were obtained from Sigma. Acetoacethe method of WALKER@.
Formation of acetoacetate was first measured with liver homogenates and butyrate as substrate. Under these conditions Axolotl liver shows no measurable accumulation of acetoacetate (Table I). Carnitine did not influence considerably the formation of acetoacetate from butyrate by the rat liver homogenate. It seems to be needed for
the utilization of internal substrates. Later liver slices were employed as a test system with butyrate as substrate. It was thought that by using tissue slices, the possibility of inactivating a labile enzyme system would be minimized. Incubations were run in triplicate, and the experiments always included rat liver slices as a reference. It can be seen from Table II that frog liver and the livers from the axolotl before and after metamorphosis produce negligible amounts of acetoacetate as determined by the method of WALKE@. Rat, sna.ke and goldfish livers produce much higher levels. It was of interest to find out whether the activities of the ~-hydrox~-~-methyl-
SHORT COMMUNICATIONS
418 TABLE
I
ACETOACETATE
FORMATION
BY
RAT AND
AXOLOTL
LIVER
HOMOGENATES
The complete system contained: 40 pmoles phosphate buffer (pH 7.4), 3 pmoles ATP, 15 ~moles MgCI,, IOO ymoles KCI, 8 ,umoles EDTA, 0.8 mg bovine serum albumin, 5 pmoles potassium butyrate and r ymole carnitine in a total volume of 2 ml. Each vial contained 0.5 ml liver homogenate (a IO?; homogenatein 0.25 M sucrose spun down at 7ooxg for IO mm). The vials were incubated at 37” for 30 min with shaking. Protein was precipitated by adding HCIO, to a final concentration of 59b. Acetoacetate was determined according to the method of WALKER@. Liver homogenate
SysteP?z
A cetoacetate formed (nmoles) .-. _-..._
Rat
Complete -carnitine ~ butyrate -carnitine,
590 5ro
AxolotI
TABLE
Complete --carnitine - butyrate ~ carnitine,
180
-butyrate
90 IO -0 0
- butyrate
0
.~
II
ACETOACETATE
FORMATION
BY LIVER
SLICES FROM DIFFERENTVERTEBRATES
The medium was the same as in Table I without carnitine. The homogenate was replaced by 0.5 ml 0.25 M sucrose and zoo mg liver slices. Incubations were done in triplicate, with shaking for I h at 37”. Precipitated protein was homogenized and acetoacetate determined in the supernatant according to the procedure of WALXER~. -..-.-_-___ Liver slims n c&a&ate Liver slices Acetoacetate formed (nmoles) formed (nmoles) Rat Snake Goldfish
Axolotl Metamorphosed Frog
370 480 270
IO
axolotl
5 30
glutaryf-CoA condensing enzyme and cleavage enzyme could be demonstrated in the amphibian livers. The method used was that of LVNEN et aE.i. The test system includes acetyl phosphate, CoA and an extract from C. kluyveri as a source of phosphotransacetylase and thiolase. In the presence of both /%hydroxy-/Lmethylglutaryl-CoA condensing enzyme and fl-hydroxy$-methylglutaryl-CoA cleavage enzyme, the acetyl-CoA and acetoacetyl-CoA supplied by the system give rise to free acetoacetate: A crude extract from yeast which contains only condensing enzyme activity (before adsorbtion on AI( gel’) was used as a source of that enzyme (enzyme B). A crude extract from beef liver acetone powder contains both activities and loses its ~-hydroxy-~-meth~rlglutaryl-CoA condensing enzyme activity after heat treatment. Heat treated beef liver extract was used as a source of ,&hydroxy-@-methylglutarylCoA cleavage enzyme (enzyme A). Acetone powders were prepared from the livers of beef, snake, frog and axolotl before and after metamorphosis, and the extracts (without heat treatment) were tested for the activities of both enzymes. It can be seen from Table III that all three amphibian livers show /?-hydroxy~-methylglutaryl-CoA cleavage enzyme activity but no activity of the condensing enzyme. Since the Axolotl incorporates label from /r-14C]acetate into cholesterol (J. GUZMkN-GARCIA AND J. D~Az-NAGOYA, personal communication) and squalene (B. PREISS, unpublished results), it could be suggested that it only has a pathway to Biochim. Riophys. Acta, 176
(1969)
417-419
SHORT COMMUNICATIONS TABLE
419
III
A TEST FOR THE ACTIVITIES OF ~-HYDROXY-~-h~ET~YL~LUTARSL-COA ~-HYDRoxY-~-METHYLGL~~ARYL-CO~ VERTEBRATE LIVERS
CLEAVAGE
ENZYME
IN ACETONE
CONDENSING ENZYME AND POWDERS FROM DIFFERENT
Each tube contained: 70 /
---I___
Beef (0.2 ml) Snake Axolotl
metamorphosed
Auxiliary enzyme
Acetoacetate jovmed (nmoles)
None
290
None
67 7
None A B None
axolotl
A
Frog
Beef (0.2 ml) + axolotl Beef (0.2 ml) +metamorphosed None None
axolotl
B None A B None None A B
IO
80 IO
30 70 0
3 56 220 210 20
4
mevalonate which does not involve free intermediates, similar to the one found by BRODIE, WASSON AND PORTER' in pigeon liver. The Axolotl extracts do not seem to contain an inhibitor to the beef liver system, since addition of axolotl liver extracts does not diminish considerably the activity of the beef liver extract. The results also testify to the absence of a deacylase for acetoacetyl-CoA in the three amphibian livers. The author wishes to express his gratitude to Dr. JESSE Guz~irANGARCIA of this department for his helpful criticism of the work. Departamenta de Bioquimica, Falcultad de Medicina, C&dad Universitaria, M.hico, (M&co) I I?.LYNEN, 2
3 4 5
6 7
BENJAMIN
PREISS
D.F.
U. HENNING,C.BUBLITZ,B.S~RBOAND
L. KRGPLIN-REUFF
~~oc~e~.Z.,33O(Ig58)
269. G.L.DRUMMOND AND J.R.STERN,J. BioLChem.,z35 (x960)318. F. SAUER AND J. D. ERFLE,J.B~~Z.C~C~., 241 (1966) 30. J, J. FERGUSON, JR. AND H. RUDNEY, j. Bid. Chem., 234 (1959) 1072. B. K.BACHHAWAT,W.G.ROBINSON AND M.J. COON, J.Biol.Chem., 216(1955)7s7. P. G. WALKER, Biochem. J., $3 (1954) 699. J.D. BRODIE,G.WASSONAND J.W.PORTER, J.Biol.Chem.,238(1g63) 1294.
Received September rgth, 1968 Biochim.
Biophys.
Acta,
176 (rgbg) 417-419