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Inhibition of gluconeogenesis in the kidney cortex by γ-hydroxyglutamate and γ-hydroxy-α-ketoglutarate
Biochimica et Biophysica Acta, 338 (1974) 78--82 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in the Netherlands
BBA 27289
INHIBITION OF GLUCONEOGENESIS IN THE KIDNEY CORTEX BY 7-HYDROXYGLUTAMATE AND 7-HYDROXY-a-KETOGLUTARATE
ROBERT ROGNSTAD
Cedars-Sinai Medical Research Institute, Los Angeles, Calif. 90029 (U.S.A.) (Received April 16th, 1973) (Revised manuscript received August 24th, 1973)
Summary 7-Hydroxyglutamate, 7-hydroxy-~-ketoglutarate and glyoxylate inhibit kidney gluconeogenesis from pyruvate and lactate strongly but have little effect on glucose synthesis from Krebs cycle intermediates. The inhibitory effect in the presence of pyruvate appears to result from a block in the Krebs cycle between citrate and a-ketoglutarate.
Introduction 7-Hydroxyglutamate has been isolated from plants [1] and has been established as an intermediate in the mammalian catabolism of L-hydroxyproline [2]. 7-Hydroxyglutamate can be converted by glutamate oxalacetate transaminase (and also glutamic dehydrogenase) to 7-hydroxy<~-ketoglutarate, which in turn can be cleaved by an aldolase in liver and kidney to pyruvate plus glyoxylate [3,4]. 7-Hydroxy~-ketoglutarate has been reported to inhibit several enzymes of the Krebs cycle [5,6]. We show here that 7-hydroxyglutamate, 7-hydroxy<~-ketoglutarate and glyoxylate all strongly inhibit gluconeogenesis from pyruvate and lactate in kidney cortex segments, while gluconeogenesis from Krebs cycle intermediates is relatively unaffected. Methods Rat kidney cortex segments were prepared by the m e t h o d of Guder et al. [7]. 125 mg of segments were incubated in 2 ml of a phosphate-salts buffer ]8] for 2 h at 3,7°C in 25 ml Ehrlenmeyer flasks with various substrates and inhibitors. When [1-14C]acetate was used as a substrate, flasks were capped with serum stoppers from which a plastic well was suspended. The incubations were terminated by injection of 0.5 ml of 1 M H2SO4 to the medium and 0.25 ml of phenethylamine--water (1:1, by vol.) to the plastic cup. 1 t C O 2 was collected by further shaking for 3 h.
79
L-allo-7-Hydroxyglutamate was obtained from Calbiochem. 7-Hydroxy~-ketoglutarate was synthesized nonenzymically from glyoxylate and oxalacetare according to Payes and Laties [6]. Glucose, lactate and Krebs cycle intermediates were estimated by enzymic methods [9]. Results and Discussion
7-Hydroxyglutamate, ~-hydroxy<~-ketoglutarate and glyoxylate strongly inhibited glucose synthesis from pyruvate and lactate in kidney cortex segments, while gluconeogenesis from malate, succinate and ~-ketoglutarate was, if TABLE I EFFECT OF 7-HYDROXYGLUTAMATE, ON GLUCONEOGENESIS Substrateconcentrationwasl0 Expt No.
Substrate
7-HYDROXY-~-KETOGLUTARATE
mM exceptinExptlwhercitwas5
~,-Hydroxyglutamate (mM)
~'-Hydroxy-a-ketoglutarate
AND GLYOXYLATE
m M . 10 m M a c e t a t e w a s a l s o p r e s e n t .
Glyoxylate (raM)
Glucose formation (pmoles/125 mg per 2 h)
(raM)
1
2
3
Pyruvate Pyruvate L-Lactate
-5
-
-
2.79
-
-
0.46
-
-
3.44
L-Lactate
5
--
-
0.90
Pyruvate
--
-
-
4.31
Pyruvate Pyruvate L-Lactate L-Lactate L-Lactate L-Malate L-Malate L-Malate
5
-
-
0
2
-
0.20
-
-
4.08
-
-
1.26
2
-
0.56
-
-
3.55
-
~
5.02
2
-
4.29
5
5
.
4
9
~-Ketoglut3xate
4.21
(~-Keto-
4
glutarate
5
Succinate Succinate
5
5.10 3.34 3.79
D-Glycer4.26
aldehyde D-Glyccraldehyde
5
2.85
DL-Glyeerate
--
2.28
DL-Glycerate
5
5
D-Fructose
6
Pyruvate
D-Fructose
5
8
Pyruvate/ Malate Pyruvate/ Malate Citrate Citrate
8.12 3.52
- -
Pyruvate L-Lactate L-Lactate L-Malate L-Malate 7
1.27 10.40
- -
1
1.60 2.94
1
1
0.80 3.47 3.97 6.67
5
6.32
--
1.78 1.73
5
80 anything, slightly increased by these same compounds (Table I). From substrates " a b o v e " phosphoenolpyruvate in the gluconeogenic pathway these compounds caused a moderate inhibition of gluconeogenesis, considerably less than that from pyruvate. Since 7-hydroxy~-ketoglutarate has been reported to inhibit several enzymes of the Krebs cycle [5,6], and since both ~-hydroxyglutamate and glyoxylate can be converted to 7-hydroxy~-ketoglutarate by enzymes present in the rat kidney, the inhibitory effects of these compounds on the Krebs cycle were examihed (Table II). Again a high inhibition of the Krebs cycle was found only when pyruvate or lactate was the substrate, with no inhibition when malate was the substrate. Also the rate of the Krebs cycle in the presence of acetate but absence of any added glucogenic substrate was unaffected by 7hydroxy~-ketoglutarate. In an attempt to localize the site of inhibitionfin the Krebs cycle a large scale incubation was carried out with pyruvate as substrate and certain Krebs cycle intermediates were measured {Table III). The results showed that both 7-hydroxyglutamate and glyoxylate caused an increase in the level of citrate and a marked depression of a-ketoglutarate, with a smaller decrease in malate. This suggests an inhibition at either (or both) aconitase or isocitrate dehydrogenase, both of which are inhibited by 7-hydroxy<~-ketoglutarate in vitro
[5,6]. We feel that the inhibition of gluconeogenesis caused by these compounds is probably related to this inhibition of the Krebs cycle as an indirect effect due to the lowering of ATP production. It should be stressed that a complete explanation of the data cannot be formulated at present. Our original inter-
TABLE
II
EFFECT OF 7-HYDROXYGLUTAMATE FROM [1-14C]ACETATE The concentration determined. Expt No.
10
Substrate
of
the
gluconeogenic
3'~Hydroxyglutamate (mM)
OR
7-HYDROXY-~-KETOGLUTARATE
substrate
and
that
of
ON
[1-14C]acetate
was
10
14CO 2 YIELD
raM.
n.d.,
9'-Hydroxy~-keto glutarate (raM)
14C yield in CO 2 (% added 14C)
Glucose formation (pmoles/125 mg per 2 h)
None None None
5 --
2
19.1 (lost) 18.8
n.d. n.d. n.d.
Pyruvate Pyruvate Pyruvate
-5 -
--2
27.0 9.8 7.4
3.67 0.37 0.18
L-Lactate L-Lactate L-Lactate
5 -
2
41.0 25.6 15.2
4, 52 0.56 0.27
None None
-5
--
36.6 39.4
n.d. n.d.
Pyruvate Pyruvate
5
---
32.6 6.6
n.d. n.d.
L-Malate L-Malate
-5
--
33.1 33.7
n.d. n.d.
not
81 TABLE
III
EFFECT OF 7-HYDROXYGLUTAMATE AND GLYOXYLATE TERMEDIATES AND PRODUCTS DURING GLUCONEOGENESIS
ON THE CONCENTRATION FROM PYRUVATE
OF IN-
20 mM pyruvate and 20 mM acetate were the substrates. 1 g of kidney cortex slices were incubated for 60 m i n i n a t o t a l v o l u m e o f 1 0 m l . A n a l y t i c a l v a l u e s a r e t o t a l ~zmoles p e r i n c u b a t i o n f l a s k . W h e n b o t h a - k e t o glutarate and 7-hydroxy-c~-ketoglutarate were present, ~-ketoglutarate was first estimated using excess alaninc plus glutamate pyruvate transarainase, NADH, and lactate debydrogenase. 7-Hydroxy-~-ketoglutarate was then estimated using excess aspartate plus glutamate oxalacetate transaminase, NADH and malate dehydrogenase. Inhibitor
Concn (mM)
Glucose
Lactate
Citrate
~-Ketoglutarate
Malate
7-Hydroxy -c~-ketoglutarate
None 7-HY droxyglutamate Glyoxylate
-5 2
18.7 1.8 0.6
16.0 9.6 7.9
1.37 2.48 2.62
2.12 0.30 0.15
1.04 0.26 0.28
-2.30 3.02
pretation of the results of Table I was that 7-hydroxy-~-ketoglutarate was causing a specific inhibition of pyruvate carboxylase. This would be consistent with the strong inhibition of gluconeogenesis from lactate or pyruvate, with no inhibition from the Krebs cycle intermediates. A number of a-keto acids have been shown to inhibit pyruvate carboxylase [10]. However, there is no independent evidence to support an effect on pyruvate carboxylase by the inhibitors used in this paper. Also in unpublished experiments L-malate restores 14 C incorporation into glucose from L-[1-14C] lactate in the presence of ~/-hydroxyglutamate. Rat liver pyruvate carboxylase is reported to be unaffected by L-malate and a number of other Krebs cycle intermediates. What remains to be explained is the great difference in the effects caused by these compounds depending upon the nature of the substrates used. It might be considered that pyruvate at high concentrations should keep the equilibrium of the aldolase reaction: glyoxylate + p y r u v a t e ~ 7 - h y d r o x y ~ ketoglutarate toward the probable inhibitor, 7-hydroxy~-ketoglutarate. The importance of this, however, is made somewhat dubious by two considerations: (1) lactate potentiates the inhibition nearly as much as does pyruvate, although the intracellular pyruvate level is probably about 10-fold lower when lactate is the substrate and (2) with malate and pyruvate as combined substrates, the high level of pyruvate now induces very little affect on gluconeogenesis with 7-hydroxyglutamate as the inhibitor (Expt 7, Table I). It would seem that the Krebs cycle intermediates in some way overcome the inhibition caused by 7-hydroxy-~-ketoglutarate, or they keep this c o m p o u n d away from the site of its inhibitory action, i.e. out of the mitochondria. Again however this falls short of a complete explanation in regard to the lack of inhibition of the Krebs cycle in the absence of any added gluconeogenic intermediate. Further experiments are required to fully understand the markedly different effects of this series of inhibitors dependent upon the added gluconeogenic substrates. Acknowledgement This work was supported by USPHS Grant Am-15297.
82
References 1 2 3 4 5 6 7 8 9 l0 11
V i r t a h z n , A . S . a n d H i e t a l a , P.K. ( 1 9 5 5 ) A c t a C h e m . S t a n d . 9 , 1 7 5 - 1 8 3 A d a m s , E. a n d G o l d s t o n e , A. ( 1 9 6 0 ) J. B i o l . C h e m . 2 3 5 , 3 4 9 2 - 3 4 9 8 G o l d s t o n e , A. a n d A d a m s , E. ( 1 9 6 2 ) J. B i o l . C h e m . 2 3 7 , 3 4 7 6 - 3 4 8 5 M a i t r a , U. a n d D e k k e r , E. ( 1 9 6 3 ) J. B i o l . C h e m . 2 3 8 , 3 6 6 0 - 3 6 6 9 R u f f o , A., A d i n o l f i , A., B u d i l l o n , G . a n d C a p o b i a n c o , G . ( 1 9 6 2 ) B i o c b e m . J . 8 5 , 5 9 3 - 6 0 0 P a y e s , B. a n d L a t i e s , G . G . ( 1 9 6 3 ) B i o c h e m . B i o p h y s . R e s . C o m m u n . 10, 4 6 0 - 4 6 6 G u d e r , W., Siess, E. a n d W i e l a n d , O. { 1 9 6 9 ) F E B S L e t t . 3, 31 - 3 7 K r e b s , H . A . , H e m s , R . a n d G a s c o y n e , T. { 1 9 6 3 ) A c t a B i o l . M e d . G e r . 1 1 , 6 0 7 - 6 1 5 B e r g m e y e r , H . U . ( 1 9 6 5 ) M e t h o d s o f E n z y m a t i c A n a l y s i s , A c a d e m i c Press, N e w Y o r k S e u b e r t , W. a n d H u t h ~ W. ( 1 9 6 5 ) B i o c h e m . Z. 3 4 3 , 1 7 6 - 1 9 1 M c C l u r e , W . R . a n d L a r d y , H . A . ( 1 9 7 1 ) J. B i o l . C h e m . 2 4 6 , 3 5 9 1 - 3 5 9 6
BBA 27289
INHIBITION OF GLUCONEOGENESIS IN THE KIDNEY CORTEX BY 7-HYDROXYGLUTAMATE AND 7-HYDROXY-a-KETOGLUTARATE
ROBERT ROGNSTAD
Cedars-Sinai Medical Research Institute, Los Angeles, Calif. 90029 (U.S.A.) (Received April 16th, 1973) (Revised manuscript received August 24th, 1973)
Summary 7-Hydroxyglutamate, 7-hydroxy-~-ketoglutarate and glyoxylate inhibit kidney gluconeogenesis from pyruvate and lactate strongly but have little effect on glucose synthesis from Krebs cycle intermediates. The inhibitory effect in the presence of pyruvate appears to result from a block in the Krebs cycle between citrate and a-ketoglutarate.
Introduction 7-Hydroxyglutamate has been isolated from plants [1] and has been established as an intermediate in the mammalian catabolism of L-hydroxyproline [2]. 7-Hydroxyglutamate can be converted by glutamate oxalacetate transaminase (and also glutamic dehydrogenase) to 7-hydroxy<~-ketoglutarate, which in turn can be cleaved by an aldolase in liver and kidney to pyruvate plus glyoxylate [3,4]. 7-Hydroxy~-ketoglutarate has been reported to inhibit several enzymes of the Krebs cycle [5,6]. We show here that 7-hydroxyglutamate, 7-hydroxy<~-ketoglutarate and glyoxylate all strongly inhibit gluconeogenesis from pyruvate and lactate in kidney cortex segments, while gluconeogenesis from Krebs cycle intermediates is relatively unaffected. Methods Rat kidney cortex segments were prepared by the m e t h o d of Guder et al. [7]. 125 mg of segments were incubated in 2 ml of a phosphate-salts buffer ]8] for 2 h at 3,7°C in 25 ml Ehrlenmeyer flasks with various substrates and inhibitors. When [1-14C]acetate was used as a substrate, flasks were capped with serum stoppers from which a plastic well was suspended. The incubations were terminated by injection of 0.5 ml of 1 M H2SO4 to the medium and 0.25 ml of phenethylamine--water (1:1, by vol.) to the plastic cup. 1 t C O 2 was collected by further shaking for 3 h.
79
L-allo-7-Hydroxyglutamate was obtained from Calbiochem. 7-Hydroxy~-ketoglutarate was synthesized nonenzymically from glyoxylate and oxalacetare according to Payes and Laties [6]. Glucose, lactate and Krebs cycle intermediates were estimated by enzymic methods [9]. Results and Discussion
7-Hydroxyglutamate, ~-hydroxy<~-ketoglutarate and glyoxylate strongly inhibited glucose synthesis from pyruvate and lactate in kidney cortex segments, while gluconeogenesis from malate, succinate and ~-ketoglutarate was, if TABLE I EFFECT OF 7-HYDROXYGLUTAMATE, ON GLUCONEOGENESIS Substrateconcentrationwasl0 Expt No.
Substrate
7-HYDROXY-~-KETOGLUTARATE
mM exceptinExptlwhercitwas5
~,-Hydroxyglutamate (mM)
~'-Hydroxy-a-ketoglutarate
AND GLYOXYLATE
m M . 10 m M a c e t a t e w a s a l s o p r e s e n t .
Glyoxylate (raM)
Glucose formation (pmoles/125 mg per 2 h)
(raM)
1
2
3
Pyruvate Pyruvate L-Lactate
-5
-
-
2.79
-
-
0.46
-
-
3.44
L-Lactate
5
--
-
0.90
Pyruvate
--
-
-
4.31
Pyruvate Pyruvate L-Lactate L-Lactate L-Lactate L-Malate L-Malate L-Malate
5
-
-
0
2
-
0.20
-
-
4.08
-
-
1.26
2
-
0.56
-
-
3.55
-
~
5.02
2
-
4.29
5
5
.
4
9
~-Ketoglut3xate
4.21
(~-Keto-
4
glutarate
5
Succinate Succinate
5
5.10 3.34 3.79
D-Glycer4.26
aldehyde D-Glyccraldehyde
5
2.85
DL-Glyeerate
--
2.28
DL-Glycerate
5
5
D-Fructose
6
Pyruvate
D-Fructose
5
8
Pyruvate/ Malate Pyruvate/ Malate Citrate Citrate
8.12 3.52
- -
Pyruvate L-Lactate L-Lactate L-Malate L-Malate 7
1.27 10.40
- -
1
1.60 2.94
1
1
0.80 3.47 3.97 6.67
5
6.32
--
1.78 1.73
5
80 anything, slightly increased by these same compounds (Table I). From substrates " a b o v e " phosphoenolpyruvate in the gluconeogenic pathway these compounds caused a moderate inhibition of gluconeogenesis, considerably less than that from pyruvate. Since 7-hydroxy~-ketoglutarate has been reported to inhibit several enzymes of the Krebs cycle [5,6], and since both ~-hydroxyglutamate and glyoxylate can be converted to 7-hydroxy~-ketoglutarate by enzymes present in the rat kidney, the inhibitory effects of these compounds on the Krebs cycle were examihed (Table II). Again a high inhibition of the Krebs cycle was found only when pyruvate or lactate was the substrate, with no inhibition when malate was the substrate. Also the rate of the Krebs cycle in the presence of acetate but absence of any added glucogenic substrate was unaffected by 7hydroxy~-ketoglutarate. In an attempt to localize the site of inhibitionfin the Krebs cycle a large scale incubation was carried out with pyruvate as substrate and certain Krebs cycle intermediates were measured {Table III). The results showed that both 7-hydroxyglutamate and glyoxylate caused an increase in the level of citrate and a marked depression of a-ketoglutarate, with a smaller decrease in malate. This suggests an inhibition at either (or both) aconitase or isocitrate dehydrogenase, both of which are inhibited by 7-hydroxy<~-ketoglutarate in vitro
[5,6]. We feel that the inhibition of gluconeogenesis caused by these compounds is probably related to this inhibition of the Krebs cycle as an indirect effect due to the lowering of ATP production. It should be stressed that a complete explanation of the data cannot be formulated at present. Our original inter-
TABLE
II
EFFECT OF 7-HYDROXYGLUTAMATE FROM [1-14C]ACETATE The concentration determined. Expt No.
10
Substrate
of
the
gluconeogenic
3'~Hydroxyglutamate (mM)
OR
7-HYDROXY-~-KETOGLUTARATE
substrate
and
that
of
ON
[1-14C]acetate
was
10
14CO 2 YIELD
raM.
n.d.,
9'-Hydroxy~-keto glutarate (raM)
14C yield in CO 2 (% added 14C)
Glucose formation (pmoles/125 mg per 2 h)
None None None
5 --
2
19.1 (lost) 18.8
n.d. n.d. n.d.
Pyruvate Pyruvate Pyruvate
-5 -
--2
27.0 9.8 7.4
3.67 0.37 0.18
L-Lactate L-Lactate L-Lactate
5 -
2
41.0 25.6 15.2
4, 52 0.56 0.27
None None
-5
--
36.6 39.4
n.d. n.d.
Pyruvate Pyruvate
5
---
32.6 6.6
n.d. n.d.
L-Malate L-Malate
-5
--
33.1 33.7
n.d. n.d.
not
81 TABLE
III
EFFECT OF 7-HYDROXYGLUTAMATE AND GLYOXYLATE TERMEDIATES AND PRODUCTS DURING GLUCONEOGENESIS
ON THE CONCENTRATION FROM PYRUVATE
OF IN-
20 mM pyruvate and 20 mM acetate were the substrates. 1 g of kidney cortex slices were incubated for 60 m i n i n a t o t a l v o l u m e o f 1 0 m l . A n a l y t i c a l v a l u e s a r e t o t a l ~zmoles p e r i n c u b a t i o n f l a s k . W h e n b o t h a - k e t o glutarate and 7-hydroxy-c~-ketoglutarate were present, ~-ketoglutarate was first estimated using excess alaninc plus glutamate pyruvate transarainase, NADH, and lactate debydrogenase. 7-Hydroxy-~-ketoglutarate was then estimated using excess aspartate plus glutamate oxalacetate transaminase, NADH and malate dehydrogenase. Inhibitor
Concn (mM)
Glucose
Lactate
Citrate
~-Ketoglutarate
Malate
7-Hydroxy -c~-ketoglutarate
None 7-HY droxyglutamate Glyoxylate
-5 2
18.7 1.8 0.6
16.0 9.6 7.9
1.37 2.48 2.62
2.12 0.30 0.15
1.04 0.26 0.28
-2.30 3.02
pretation of the results of Table I was that 7-hydroxy-~-ketoglutarate was causing a specific inhibition of pyruvate carboxylase. This would be consistent with the strong inhibition of gluconeogenesis from lactate or pyruvate, with no inhibition from the Krebs cycle intermediates. A number of a-keto acids have been shown to inhibit pyruvate carboxylase [10]. However, there is no independent evidence to support an effect on pyruvate carboxylase by the inhibitors used in this paper. Also in unpublished experiments L-malate restores 14 C incorporation into glucose from L-[1-14C] lactate in the presence of ~/-hydroxyglutamate. Rat liver pyruvate carboxylase is reported to be unaffected by L-malate and a number of other Krebs cycle intermediates. What remains to be explained is the great difference in the effects caused by these compounds depending upon the nature of the substrates used. It might be considered that pyruvate at high concentrations should keep the equilibrium of the aldolase reaction: glyoxylate + p y r u v a t e ~ 7 - h y d r o x y ~ ketoglutarate toward the probable inhibitor, 7-hydroxy~-ketoglutarate. The importance of this, however, is made somewhat dubious by two considerations: (1) lactate potentiates the inhibition nearly as much as does pyruvate, although the intracellular pyruvate level is probably about 10-fold lower when lactate is the substrate and (2) with malate and pyruvate as combined substrates, the high level of pyruvate now induces very little affect on gluconeogenesis with 7-hydroxyglutamate as the inhibitor (Expt 7, Table I). It would seem that the Krebs cycle intermediates in some way overcome the inhibition caused by 7-hydroxy-~-ketoglutarate, or they keep this c o m p o u n d away from the site of its inhibitory action, i.e. out of the mitochondria. Again however this falls short of a complete explanation in regard to the lack of inhibition of the Krebs cycle in the absence of any added gluconeogenic intermediate. Further experiments are required to fully understand the markedly different effects of this series of inhibitors dependent upon the added gluconeogenic substrates. Acknowledgement This work was supported by USPHS Grant Am-15297.
82
References 1 2 3 4 5 6 7 8 9 l0 11
V i r t a h z n , A . S . a n d H i e t a l a , P.K. ( 1 9 5 5 ) A c t a C h e m . S t a n d . 9 , 1 7 5 - 1 8 3 A d a m s , E. a n d G o l d s t o n e , A. ( 1 9 6 0 ) J. B i o l . C h e m . 2 3 5 , 3 4 9 2 - 3 4 9 8 G o l d s t o n e , A. a n d A d a m s , E. ( 1 9 6 2 ) J. B i o l . C h e m . 2 3 7 , 3 4 7 6 - 3 4 8 5 M a i t r a , U. a n d D e k k e r , E. ( 1 9 6 3 ) J. B i o l . C h e m . 2 3 8 , 3 6 6 0 - 3 6 6 9 R u f f o , A., A d i n o l f i , A., B u d i l l o n , G . a n d C a p o b i a n c o , G . ( 1 9 6 2 ) B i o c b e m . J . 8 5 , 5 9 3 - 6 0 0 P a y e s , B. a n d L a t i e s , G . G . ( 1 9 6 3 ) B i o c h e m . B i o p h y s . R e s . C o m m u n . 10, 4 6 0 - 4 6 6 G u d e r , W., Siess, E. a n d W i e l a n d , O. { 1 9 6 9 ) F E B S L e t t . 3, 31 - 3 7 K r e b s , H . A . , H e m s , R . a n d G a s c o y n e , T. { 1 9 6 3 ) A c t a B i o l . M e d . G e r . 1 1 , 6 0 7 - 6 1 5 B e r g m e y e r , H . U . ( 1 9 6 5 ) M e t h o d s o f E n z y m a t i c A n a l y s i s , A c a d e m i c Press, N e w Y o r k S e u b e r t , W. a n d H u t h ~ W. ( 1 9 6 5 ) B i o c h e m . Z. 3 4 3 , 1 7 6 - 1 9 1 M c C l u r e , W . R . a n d L a r d y , H . A . ( 1 9 7 1 ) J. B i o l . C h e m . 2 4 6 , 3 5 9 1 - 3 5 9 6