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Laboratory experiments on the regulation of pyruvate kinase isoenzymes
42 V~
Experimental Section
Laboratory Experiments on the Regulation of Pyruvate Kinase Isoenzymes E HANNAPPEL, W FISCHER AND K BRAND lnstitut ftir Physiologische Chemie Universitiit Erlangen-Nt'irnberg 8520 Erlangen, F R G
~" ATP
. .
I.15
2'.0
Introduction Since 1982 some 1800 medical students have carried out a very simple and impressive experiment on the regulation of liver pyruvate kinase (PK). Muscle PK is available from commercial suppliers such as Boehringer Mannheim (FRG). On the other hand the isoenzyme from rat liver has to be enriched by a fractional ammonium sulfate precipitation. Because of our large number of students this enrichment was carried out by technicians, but it is also possible for thc students to carry ()tit this procedure themselves. The cxpcrintcnt will bc dcscribcd in its most simple form and should provide some information on isocnzymcs, allostcric sitcs, and the regulation of glycolysis. The experiment can easily be expanded, for cxantple in order t(} show the stereospccificity of regulatory sites :rod activation of an allostcrically-inhibitcd enzyme. I|ackground Most of tile retell(ms of glycolysis and gluconcogcncsis arc catalyzed by the same enzymes. 11owcver, there are three irreversible steps in glycolysis which cannot bc utilized in gluconcogcncsis front pyruvatc. Examining these reactions closely leads to the conclusion that wasteful hydrolysis of ATP ('futile cycling') is possible if glycolysis and gluconcogcncsis proceed uncontr(fllcd at the same time. Such a futile cycle cannot occur in cells such as muscle cells which are glycolytic but not gluconcogcnetic, lit hepatocytcs, on the other hand, which - dcpcnding on the metabolic requirements - - may be glycolytic or gluconcogcnctic, the activities of the kcy enzymes of these pathways arc carefully regulated. One key enzyme of glycolysis is pyruvatc kinase (EC 2.7.1.40) which catalyzes the transfer of the phosphatc group from phosphoenolpyruvatc (PEP) to ADP yielding ATP and pyruvate. The reaction can be easily measured if a largtc antount of lactate dchydrogenasc (LDI I) and NADI 1 is added. Different isocnzymcs of PK exist in mammalian tissues-" but we restricted ourselves to the two major isoenzymcs namely type L from livcr and type M from rnusclc. PK type L possesses allostcric binding sites for at least two inhibitors (ATP and t.All) ~''~ and one activator (fructose-l.6-bisphospate; Fru-l,6p,).5-7 PK type 1. is a positive homotropic enzyme with a sigmoid saturation curve relating initial velocity to the concentration of the substrate PEP. A great technical advantage of PK type L is that its K,.s for PEP is well in the millimolar range. Therefl)re, only minute amounts of the substrate are consuntcd during the photometrically observed part of the reaction, and the absorbance ver.sus time plot is linear until the NADI I, necessary for the indicator reaction, is exhausted. Although PK type L is inhibited by ATP, the amount of ATP accumulated during the reaction is so little that no noticeable inhibition is observed. The regulatory properties of PK type L arc shown in Fig I. PK typc M possesses no allosteric binding sites for ATP. Ala, or Fru-1.6Pz. The binding of PEP carl bc dcscribcd by the MichaclisMenten equation and can be represented by a curve similar to the curve of PK type L at saturation with an activator. These much simpler kinctic propcrtics of pyruvate kinasc type M arc sufficient because only glycolysis and no gh, concogcnesis can o c c u r in nluscle.
B I O C H E M I C A L E D U C A T I O N 15(!) 1987
0.5
1.0 mM
/
2'.5
PEP
Figure 1 Activity vers,s /PEP/plot of pyruvate kinase type L without an effector (middh" curve), in the presence of an inhibitor (lower curve) and an activator (upper curve). The arrows indicate the concentration of PEP used in the experbnent Materials
Enrichment o] I'K tylw L A Sprague-Dawlcy rat on a carbohydratc rich diet is sacrificed by CO, asphyxiation and the liver removed. All following steps arc carried out at 4°C. The liver is homogenized in 4 volumes of 10-times dilutcd buffer A (0.1 M trietlmnolanfinc/13 mM MgSO4/fl7 mM K(?I. pll = 7.6). The homogcnate is centrifuged at 100 (}00 x g for 45 rain and then 11.7 nil of satt, ratcd amntonium sull'atc solution in buffer A is added to each ml of the supernatant. The solution is stirred for 15 rain and then ccntrifugcd at 2500 x g for 10 rain. The pellet is washed once and rcsuspcnded with 1.6 M a n l n l o n i u n l sulfate in buffer A. i'K type I. is stable at 4"(? for at least one week. The enzyme from one rat liver is sufficient for about 300 students to work with. I'K type AI and lype L working sol,tio,s PK type M was from Boehringcr Mannheint. The activitics of the solution of PK type L and Nl are adjusted daily so that AE was between 0.04 and (LOS m i n - t after the "first' start of the assay described below. Pyrm'ate Kinase assay Tile studies on the regulation of PK are carried out in one cuvette by sequential addition of substrate and effectors (Table 1). Assay conditions: wavclcngth 365 ms; light path 1 cm; temperature 25°C. Results A typical absorbancc vers,s time plot fl)r PK type L and M is shown in Fig 2. A v c r y similar cxperimcnt can bc carried out using 50 mM i.-Ala instead of ATP. In order to show that the binding of alaninc to PK type L is stcrcospecific one can try to use i)-Ala instead of L-AI,t. In order to dentonstrate the concentration dependence of the inhibition by l.-Ala it is possible to add i.-Ala several times instead of ATP at the "second" start and record the change in absorbance each time. Art absorbance versus time plot is shown in Fig 3.
Evaluation of the results (I) Summarize tile results as shown in Table 2. The values in brackets are those obtained by the students. (2) The relative activity of PK type L as a function of [PEP] is shown in Fig 1 (K, 5 = 1.5 raM). By how many times has the concentration of PEP to be raised to change the velocity from 0. I to 1).9 Vm:,(? (answer: 0.86 mM and 2.6 raM: ie about 3 times). (3) Calculate for a Michaelis-Mcnten type enzyme by how many times the concentration of the substrate has to be raised in order to change the velocity front 0. I to (1.9 V,,,:,~. (answer: IS l = 0.11 K,,, to 9 K,,; ie abot, t ,~2 times).
43
Table 1 Assay o f pyruvate kinase pipette successively into cuvettes
E365
]inal concentrations PK type L PK O,'pe M (raM) 750 p.l 20 ~.1 20 O.I 20 p.I 20 p.I --
buffer A NADH (10 mM) PEP (50 mM) LDH (550 U/ml) PK type L PK type M
750 p.I 20 ~.1 20 gl 20 p.I -20 ILl
K ÷, 59; big -'+, 11 0.24 1.2
0.8 0.7 0.6 0.5 0.4
• mix; record change in absorbance for up to 3 min; 0.3 (1) start: ADP (60 mM)
20 p.I
ADP
20 p.I
I !
"Ll0~'ll20u.l
[~20/dl ~l/ FI'U.
1.6 Pz
1.4 0.2
• mix; record change in absorbance for up to 3 rain; 0.1 (2) start: ATP (120 raM)
20 p.I
20 p.I
2.8
• mix; record change in absorbancc for up to 3 rain; (3) start: Fru-l,f-P, (5 raM)
211 gl
PK type L
20 p.I
Figure 3 Time versus absorbance plot of PK O'pe L after addition of A D P and repeated additions of 50 mM t.-A la. Afterwards the en:yme wtts" activated by adding Fru-l,6-P,
0.12
1!3¢,~
I'K type M
0.8
0.0
~
0.2 L
I
J
6 - I'2
J
3 rain
3 min Time
Fig((re 2 77me verstts absorhance plot of PK type Lattd At after successive addition of A D P, e1TP, attd fructose-I, 6-b isphosphate (Fru-l,6-P,.) Tahle 2 Evaluation of Results
(I) start (ADI') (2) start (ATP) (3) start (Fru-1,6-P,)
I'K type L At'/min V,~h,,,,,
PK type AI A E / m i n v,,t,,,,,.,
(0.066) ((1.(X)7) (11.155)
((1.(R'-,4) ((1.()(,~5) (11.064)
I.(N) (0.111) (2.35)
1.(~1 ( 1.01 ) (I.(X))
(4) Explain the regulation of PK typc L by Fru-l,6-P, in vivo. Under which conditions does the concentration of Fru-l,6-P, incrcase? What is the role of Fru-2, 6-P,? (for answer sec rcfs 8, 9). (5) Convert the results shown in Fig 3 into a plot of reaction rate versus concentration of t=Ala. Determine the concentration of ~.Ala necessary for 50% inhibition and explain the result in terms of regulation of glycolysis. BIOCHEMICAL
EDUCATION
1 5 ( I ) 1987
Of course these are only a few points which arc discussed by the students (luring their experiments and they serve only to stimulate discussion of the regulation of glucose-producing and utilizing pathways inchtding phosphorylation and dcphosphorylation of enzymes. It is our intention to present sonic of the possible questions which can bc raised in the contcxt of these cxpcriments. Acknowledgement '[iris work is supported by l~ochringcr Mannhcim. FR(L The authors thank Ms M [ Icrgcnhahn for stylistic advice.
6 - I' 2
0.4
L Time
• mix; record change in absorbance for up to 3 rain
l..~,5
3 min
References I Biichcr, T and Pfleiderer, G (1955) Methods in Enzymology l, 341-353 21ma my ra, K and Tanaka, T (1982) Alethod.~in En :ymology 90, 150-1¢~5 ~Carminatli, 1I, dc Astia, L J, Rccondo. E. Passer(m, S and Rozcngurt, F (1968) J Biol Chem 243, 3051-3056 4Scubcrt, W, Ilcnning, II V, Schoncr, W and L'age, M (1968) ,,tdv Enzyme Regul 6, 153-178 ~tlcss, B, Ilacckcl. R and Brand, K (1966) Biodtem Biophys Res Commun 24, 824-831 "'r:maka, T, Suc, F and Morimura, I I (1967) Biochem Biophys Res Commun 29. 444-449 7Weber, G, l.ca, M A, Convcry, II J II and Stature, N B (1967) Adv Enzyme Regul 5,257-298 Sllcrs, |1 G. Iluc. L and v:m Schaftingcn. E (1982) TII~S 7. 329-331 "llcrs, |I G and [luc, L (1983) Ann Rev Biochem 52. 617-653
Announcement C o m p u t e r s in U n i v e r s i t y E d u c a t i o n A workshop for the physical and life sciences will be held under the auspices o f the U G C / C B U R C Computers in Teaching Initiative. Titles and abstracts ;,ire invited k)r contributions to a meeting to be held at the University of Liverpool on 6-7 April 1987. Coverage will include general aspects of software implementation in tertiary education as well as subject-specific sessions for chemistry, physics and the life sciences. Intending participants should contact D. Margerison, Donnan Laboratories, University of Liverpool, PO Box 147, Liverpool L69 3BX, U K , as soon as possible.
Experimental Section
Laboratory Experiments on the Regulation of Pyruvate Kinase Isoenzymes E HANNAPPEL, W FISCHER AND K BRAND lnstitut ftir Physiologische Chemie Universitiit Erlangen-Nt'irnberg 8520 Erlangen, F R G
~" ATP
. .
I.15
2'.0
Introduction Since 1982 some 1800 medical students have carried out a very simple and impressive experiment on the regulation of liver pyruvate kinase (PK). Muscle PK is available from commercial suppliers such as Boehringer Mannheim (FRG). On the other hand the isoenzyme from rat liver has to be enriched by a fractional ammonium sulfate precipitation. Because of our large number of students this enrichment was carried out by technicians, but it is also possible for thc students to carry ()tit this procedure themselves. The cxpcrintcnt will bc dcscribcd in its most simple form and should provide some information on isocnzymcs, allostcric sitcs, and the regulation of glycolysis. The experiment can easily be expanded, for cxantple in order t(} show the stereospccificity of regulatory sites :rod activation of an allostcrically-inhibitcd enzyme. I|ackground Most of tile retell(ms of glycolysis and gluconcogcncsis arc catalyzed by the same enzymes. 11owcver, there are three irreversible steps in glycolysis which cannot bc utilized in gluconcogcncsis front pyruvatc. Examining these reactions closely leads to the conclusion that wasteful hydrolysis of ATP ('futile cycling') is possible if glycolysis and gluconcogcncsis proceed uncontr(fllcd at the same time. Such a futile cycle cannot occur in cells such as muscle cells which are glycolytic but not gluconcogcnetic, lit hepatocytcs, on the other hand, which - dcpcnding on the metabolic requirements - - may be glycolytic or gluconcogcnctic, the activities of the kcy enzymes of these pathways arc carefully regulated. One key enzyme of glycolysis is pyruvatc kinase (EC 2.7.1.40) which catalyzes the transfer of the phosphatc group from phosphoenolpyruvatc (PEP) to ADP yielding ATP and pyruvate. The reaction can be easily measured if a largtc antount of lactate dchydrogenasc (LDI I) and NADI 1 is added. Different isocnzymcs of PK exist in mammalian tissues-" but we restricted ourselves to the two major isoenzymcs namely type L from livcr and type M from rnusclc. PK type L possesses allostcric binding sites for at least two inhibitors (ATP and t.All) ~''~ and one activator (fructose-l.6-bisphospate; Fru-l,6p,).5-7 PK type 1. is a positive homotropic enzyme with a sigmoid saturation curve relating initial velocity to the concentration of the substrate PEP. A great technical advantage of PK type L is that its K,.s for PEP is well in the millimolar range. Therefl)re, only minute amounts of the substrate are consuntcd during the photometrically observed part of the reaction, and the absorbance ver.sus time plot is linear until the NADI I, necessary for the indicator reaction, is exhausted. Although PK type L is inhibited by ATP, the amount of ATP accumulated during the reaction is so little that no noticeable inhibition is observed. The regulatory properties of PK type L arc shown in Fig I. PK typc M possesses no allosteric binding sites for ATP. Ala, or Fru-1.6Pz. The binding of PEP carl bc dcscribcd by the MichaclisMenten equation and can be represented by a curve similar to the curve of PK type L at saturation with an activator. These much simpler kinctic propcrtics of pyruvate kinasc type M arc sufficient because only glycolysis and no gh, concogcnesis can o c c u r in nluscle.
B I O C H E M I C A L E D U C A T I O N 15(!) 1987
0.5
1.0 mM
/
2'.5
PEP
Figure 1 Activity vers,s /PEP/plot of pyruvate kinase type L without an effector (middh" curve), in the presence of an inhibitor (lower curve) and an activator (upper curve). The arrows indicate the concentration of PEP used in the experbnent Materials
Enrichment o] I'K tylw L A Sprague-Dawlcy rat on a carbohydratc rich diet is sacrificed by CO, asphyxiation and the liver removed. All following steps arc carried out at 4°C. The liver is homogenized in 4 volumes of 10-times dilutcd buffer A (0.1 M trietlmnolanfinc/13 mM MgSO4/fl7 mM K(?I. pll = 7.6). The homogcnate is centrifuged at 100 (}00 x g for 45 rain and then 11.7 nil of satt, ratcd amntonium sull'atc solution in buffer A is added to each ml of the supernatant. The solution is stirred for 15 rain and then ccntrifugcd at 2500 x g for 10 rain. The pellet is washed once and rcsuspcnded with 1.6 M a n l n l o n i u n l sulfate in buffer A. i'K type I. is stable at 4"(? for at least one week. The enzyme from one rat liver is sufficient for about 300 students to work with. I'K type AI and lype L working sol,tio,s PK type M was from Boehringcr Mannheint. The activitics of the solution of PK type L and Nl are adjusted daily so that AE was between 0.04 and (LOS m i n - t after the "first' start of the assay described below. Pyrm'ate Kinase assay Tile studies on the regulation of PK are carried out in one cuvette by sequential addition of substrate and effectors (Table 1). Assay conditions: wavclcngth 365 ms; light path 1 cm; temperature 25°C. Results A typical absorbancc vers,s time plot fl)r PK type L and M is shown in Fig 2. A v c r y similar cxperimcnt can bc carried out using 50 mM i.-Ala instead of ATP. In order to show that the binding of alaninc to PK type L is stcrcospecific one can try to use i)-Ala instead of L-AI,t. In order to dentonstrate the concentration dependence of the inhibition by l.-Ala it is possible to add i.-Ala several times instead of ATP at the "second" start and record the change in absorbance each time. Art absorbance versus time plot is shown in Fig 3.
Evaluation of the results (I) Summarize tile results as shown in Table 2. The values in brackets are those obtained by the students. (2) The relative activity of PK type L as a function of [PEP] is shown in Fig 1 (K, 5 = 1.5 raM). By how many times has the concentration of PEP to be raised to change the velocity from 0. I to 1).9 Vm:,(? (answer: 0.86 mM and 2.6 raM: ie about 3 times). (3) Calculate for a Michaelis-Mcnten type enzyme by how many times the concentration of the substrate has to be raised in order to change the velocity front 0. I to (1.9 V,,,:,~. (answer: IS l = 0.11 K,,, to 9 K,,; ie abot, t ,~2 times).
43
Table 1 Assay o f pyruvate kinase pipette successively into cuvettes
E365
]inal concentrations PK type L PK O,'pe M (raM) 750 p.l 20 ~.1 20 O.I 20 p.I 20 p.I --
buffer A NADH (10 mM) PEP (50 mM) LDH (550 U/ml) PK type L PK type M
750 p.I 20 ~.1 20 gl 20 p.I -20 ILl
K ÷, 59; big -'+, 11 0.24 1.2
0.8 0.7 0.6 0.5 0.4
• mix; record change in absorbance for up to 3 min; 0.3 (1) start: ADP (60 mM)
20 p.I
ADP
20 p.I
I !
"Ll0~'ll20u.l
[~20/dl ~l/ FI'U.
1.6 Pz
1.4 0.2
• mix; record change in absorbance for up to 3 rain; 0.1 (2) start: ATP (120 raM)
20 p.I
20 p.I
2.8
• mix; record change in absorbancc for up to 3 rain; (3) start: Fru-l,f-P, (5 raM)
211 gl
PK type L
20 p.I
Figure 3 Time versus absorbance plot of PK O'pe L after addition of A D P and repeated additions of 50 mM t.-A la. Afterwards the en:yme wtts" activated by adding Fru-l,6-P,
0.12
1!3¢,~
I'K type M
0.8
0.0
~
0.2 L
I
J
6 - I'2
J
3 rain
3 min Time
Fig((re 2 77me verstts absorhance plot of PK type Lattd At after successive addition of A D P, e1TP, attd fructose-I, 6-b isphosphate (Fru-l,6-P,.) Tahle 2 Evaluation of Results
(I) start (ADI') (2) start (ATP) (3) start (Fru-1,6-P,)
I'K type L At'/min V,~h,,,,,
PK type AI A E / m i n v,,t,,,,,.,
(0.066) ((1.(X)7) (11.155)
((1.(R'-,4) ((1.()(,~5) (11.064)
I.(N) (0.111) (2.35)
1.(~1 ( 1.01 ) (I.(X))
(4) Explain the regulation of PK typc L by Fru-l,6-P, in vivo. Under which conditions does the concentration of Fru-l,6-P, incrcase? What is the role of Fru-2, 6-P,? (for answer sec rcfs 8, 9). (5) Convert the results shown in Fig 3 into a plot of reaction rate versus concentration of t=Ala. Determine the concentration of ~.Ala necessary for 50% inhibition and explain the result in terms of regulation of glycolysis. BIOCHEMICAL
EDUCATION
1 5 ( I ) 1987
Of course these are only a few points which arc discussed by the students (luring their experiments and they serve only to stimulate discussion of the regulation of glucose-producing and utilizing pathways inchtding phosphorylation and dcphosphorylation of enzymes. It is our intention to present sonic of the possible questions which can bc raised in the contcxt of these cxpcriments. Acknowledgement '[iris work is supported by l~ochringcr Mannhcim. FR(L The authors thank Ms M [ Icrgcnhahn for stylistic advice.
6 - I' 2
0.4
L Time
• mix; record change in absorbance for up to 3 rain
l..~,5
3 min
References I Biichcr, T and Pfleiderer, G (1955) Methods in Enzymology l, 341-353 21ma my ra, K and Tanaka, T (1982) Alethod.~in En :ymology 90, 150-1¢~5 ~Carminatli, 1I, dc Astia, L J, Rccondo. E. Passer(m, S and Rozcngurt, F (1968) J Biol Chem 243, 3051-3056 4Scubcrt, W, Ilcnning, II V, Schoncr, W and L'age, M (1968) ,,tdv Enzyme Regul 6, 153-178 ~tlcss, B, Ilacckcl. R and Brand, K (1966) Biodtem Biophys Res Commun 24, 824-831 "'r:maka, T, Suc, F and Morimura, I I (1967) Biochem Biophys Res Commun 29. 444-449 7Weber, G, l.ca, M A, Convcry, II J II and Stature, N B (1967) Adv Enzyme Regul 5,257-298 Sllcrs, |1 G. Iluc. L and v:m Schaftingcn. E (1982) TII~S 7. 329-331 "llcrs, |I G and [luc, L (1983) Ann Rev Biochem 52. 617-653
Announcement C o m p u t e r s in U n i v e r s i t y E d u c a t i o n A workshop for the physical and life sciences will be held under the auspices o f the U G C / C B U R C Computers in Teaching Initiative. Titles and abstracts ;,ire invited k)r contributions to a meeting to be held at the University of Liverpool on 6-7 April 1987. Coverage will include general aspects of software implementation in tertiary education as well as subject-specific sessions for chemistry, physics and the life sciences. Intending participants should contact D. Margerison, Donnan Laboratories, University of Liverpool, PO Box 147, Liverpool L69 3BX, U K , as soon as possible.