[27]
Bacillus PYRUVATE KINASE
[27] P y r u v a t e K i n a s e o f B a c i l l u s
157
lichenilormis
By F. WILLIAM TUOMINEN and ROBERT W. BERNLOHR Phosphoenolpyruvate + ADP + H+ ~ pyruv~te + ATP The pyruvate kinase ATP:pyruvate phosphotransferase, (EC 2.7.1.40) of Bacillus licheni]ormis is an example of an AMP-activated, ATP-inhibited glycolytic enzyme that can exist in a number of kinetieally differentiable activity states. 1,2 Unless proper precautions are exercised with this enzyme during extract preparation, purification, and assay, the formation of inactive enzyme states will result in low or erroneous specific activity values, gross losses of activity, and misleading kinetic data. The methodology presented here emphasizes those details that allow the experimentalist to understand and deal with the difficulties inherent in this highly permutable pyruvate kinase. Culture and Harvest of Cells. The organism used is the genetieally stable (with respect to colonial morphology), rough colony-forming strain of B. licheni]ormis A-5. Spore stocks are prepared by inoculating 0.5 liter of 0.4% (w/v) peptone and 0.1% yeast extract contained in a 2.8-liter Fernbach flask with either vegetative or spore seed that has been derived from an isolated colony. Greater than 95% of the microscopically recognizable cells exist as free spores after 24 hr of incubation at 37 ° on a rotary shaker. The minimal medium used routinely for the production of vegetative cells contains (per liter) 65 mmoles of K2HPO4-KH~P04 (pH 7.2), 0.8 mmole of MgS04, 0.02 mmole of MnCI=,, 0.5 mmole of CaCh, 15 mmoles of NH4C1, and 15 mmoles of glucose. The glucose, NH~C1, MgS04, MnC12, and NH4C1 are added from sterile, concentrated stocks to autoclaved K_~HPO4-KH2P04 to yield a nutritionally complete, unprecipitated growth medium. Cultures are started by adding approximately 10TM spores to 1 liter of minimal medium supplemented with 0.5 mM L-alanine. Cultures are aerated by shaking on a Gyrotory shaker (New Brunswick Scientific Co.) at 37 °. When cultures reach a turbidity of 150-170 Klett units (540 ran), 1 liter is transferred to aerated fermentation vessels of a New Brunswick Scientific Co. fermentor (Model FS-307) at 38 °. Each vessel contains 10 liters of sterile, prewarmed medium. Vegetative cells are harvested for enzyme studies from volumes of culF. W. Tuominen and R. W. Bernlohr, J. Biol. Chem. 9,,46, 1732 (1971). 2F. W. Tuominen and R. W. Bernlohr, J. Biol. Chem. 246, 1746 (1971).
158
KINASES
[27]
ture up to 3 liters by centrifugation (13,000 g for 10 rain at 0 °) in a Sorvall RC2-B centrifuge after being cooled by the addition of ice. Volumes greater than 3 liters are harvested in a Lourdes continuous flow centrifuge (12,000 rpm) after the addition of ice. Cells are washed with a 0.1 M Tris.HC1 (pH 7.6) buffer that contains 10 mM MgCl_~ and 0.1 M KC1. The washed-cell pellets are either immediately used to prepare cell-free extracts or stored at --20 ° . Preparation o] Cell.Free Extracts. Cells are suspended in a 0.1 M Tris.HC1 (pH 7.6) buffer that contains 10 mM MgC12 and 0.1 M KC1 (Buffer TMK) on the basis of 1.0 ml buffer per gram, wet weight. Cells are broken by sonic disruption when the suspension volume is less than 5 ml and in a French pressure cell (at 12,000 psi) when larger volumes are employed. Sonic disruption is performed in a MSE sonicator (Measuring and Scientific Equipment, Ltd., London) at 110 V and 2 A with a 1.9-cm probe at 4-10 °. The total time of sonic disruption is 3 min, i.e. six, 30-see bursts that are interrupted by 30-see cooling periods (ice bath). This sonic disruption time (3 min) is twice that required to release a maximum amount of pyruvate kinase from a suspension of late exponential phase cells. Assay System. Pyruvate kinase ]s assayed by employing the linked lactic dehydrogenase (EC 1.1.1.27) reaction as modified from B~icher and Pfleiderer2 The standard assay system (1.0 ml) contains 50 ~moles imidazole-HC1 (pH 7.1, the optimum), 50 ~moles of KC1 (also an optimum concentration), 7 ~moles of MgCl~_, 2 ~moles of tricyclohexylammonium phosphoenolpyruvate (PEP) (TCHA-PEP), 4 ~moles NaADP (pH 7.1), 0.12 ~mole NaNADH and 40 ~g lactic dehydrogenase. Reactions are initiated by the addition of 2-20 ~l of enzyme preparation to the reaction mixture at 30 ° in a 10-ram quartz cuvette. The time course of NADH oxidation is followed at 340 nm in a Zeiss PMQII spectrophotometer connected to a Sargent Model SRL recorder. Initial rates are converted to micromo]es of pyruvate formed per minute by dividing the change in absorbance per minute by 6.22. All rates are corrected for blank rates obtained by the omission of ADP. One unit of activity is defined as the amount of pyruvate kinase yielding 1 ~mole of pyruvate per minute in the standard assay system. Freshly prepared solutions of NaADP (P-L Biochemicals, Inc.), pH 7.1, and TCHA-PEP (Sigma) are used for kinetic studies and stored on ice for up to 16 hr. Stock solutions of 0.1 M NaADP (pH 7.1) and 0.1 M TCHA-PEP, for use in the standard assay system, are stored for up to 2 weeks a~ --60 °. The concentrations of the PEP and ADP solutions are determined by assaying PEP and ADP in a modified standard assay s T. Biicher and G. Pfleiderer, this series, Vol. 1, p. 435.
Bacillus PYRUVATE KINASE
[27]
0.6
159
i &
E x
0.4 >I>
0.2 (.) ,'7
U I.u O. U')
%
zo
40
PROTEIN
~o
80
(mg/ml)
Fla. 1. Effect of dilution on the apparent specific activity of pyruvate kinase in crude extracts. A crude extract (80 mg of protein per milliliter) was prepared in 0.1 M Tris.HC1, ptI 7.6, containing 10 mM MgCh and 0.1 M KC1 at 22°. Aliquots of this extract were diluted to the indicated levels in the same buffer at 22 °. Samples of each dilution were assayed in the standard assay system at 1 min ( 0 O), 2 hr (O O), and 4 hr (A A) postdilution. The amount of extract protein was approximately the same in all assays. mixture containing 0.25 t~mole N A D H , 0.5 unit rabbit muscle p y r u v a t e kinase and 0.1-0.2 t~mole of either P E P or ADP. The change in absorbance after the addition of the p y r u v a t e kinase is divided by 6.22 to give the ~moles of P E P or A D P in the volume of solution assayed. Beef heart lactic dehydrogenase ( L D H ) is obtained as a crystalline suspension in 55% saturated (NH,)~SO~. This preparation is diluted 5-fold in 50 m M Tris.HC1, p H 7.5 and dialyzed against three changes of t h e same Tris.HC1 buffer at 4% The dialyzed preparation is diluted another 2-fold in glycerol and stored at 4 °. The final L D H stock solution contains 4 m g / m l L D H (350 I U / m g ) and 50% ( v / v ) glycerol. N a N A D H ( P - L Biochemicals, Inc.) stock solutions (8-15 raM) are stored at 2-4 ° in 10 m M Na(HCO:,-C03) buffer, p H 10.5, in accord with published recommendations2 Determination of Specific Activity in Crude Extracts. Crude extracts must contain greater than 40 mg per milliliter of protein in order to oblain meaningful p y r u v a t e kinase specific activities. The reason for this precaution against greater dilution is illustrated in Fig. 1. The data presented in Fig. 1 show t h a t p y r u v a t e kinase in crude extracts is rapidly inactivated by dilution of these extracts to less than 40 m g / m l of protein at 22 °. In addition to being sensitive to dilution at 22 °, p y r u v a t e kinase 40. H. Lawry, J. V. Passonneau, and M. K. Rock, J. Biol. Chem. 236, 2756 (1961).
160
KINASES
[27]
is reversibly inactivated at 0°. 1 As the extent of dilution is increased, at 0 °, both the amount of residual activity and reversibility of inactivation decrease. However, no cold inactivation can be detected in parallel experiments which employ warm pipettes (22°), rather than ice-cold pipettes, for removal of samples (2-10 ul) from the dilutions incubated near 0 °. These results suggest that the undiluted enzyme is rapidly reactirated during the resident time (about 20-30 sec) in the pipettes. The specific activity of pyruvate kinase in freshly prepared crude extracts, containing greater than 40 mg/ml of protein, has been found to be independent of the stage of growth (on glucose) or sporulation. 1 Similar results are observed when extracts are dialyzed against a buffer containing KPi, MgCL and PEP. The specific activities generally range between 0.55 and 0.65 unit per milligram of protein. The specific activity present in crude extracts of exponential-phase cells grown on malate range from 0.53. to 0.61 unit per milligram of protein. Values of 0.50 and 0.59 (unit per milligram of protein) have been observed with exponential-phase cells grown on pyruvate. Purification. The otherwise labile pyruvate kinase activity of B. lichemformis is stabilized throughout the following purification scheme by avoiding extensive dilution of the enzyme and by the inclusion of a stabilizing ligand mixture (PEP, Pi, and Mg ions) in the supporting buffers when dilution is unavoidable. Protein concentrations through the penultimate purification step are determined by the method of Lowry et al. 5 The protein concentrations of the most highly purified enzyme preparations are estimated spectrophotometrically at 280 nm by employing an ~,0.1 ~-~lcm~ = 0.65 which is the constant obtained by Hunsley and Suelter for crystalline yeast pyruvate kinase. Thirty grams of late exponential-phase cells are suspended at 0 ° in 45 ml of 0.1 M Tris.HC1 (pH 7.6) that contains 10 mM MgC12 and 0.1 M KC1. Cells are ruptured by two passes through a French pressure cell at 12,000 psi. The extract is sonicated for 2 rain in order to reduce the viscosity and centrifuged (0 °) at 78,000 g for 2 hr in a Spinco Model L centrifuge. The upper two-thirds of the resulting supernatants are combined to yield 40 ml of a solution that contains 2100 mg protein and 1200 units of pyruvate kinase activity. This supernatant fluid is designated as $78 (see the table). The pH of $78 is adjusted to 8.0, at 4 °, by the addition of 1 M N H , O H from a capillary-tipped pipette. The extract is diluted 2-fold by the dropwise addition of a saturated solution (490 g/l; pH 8.0 at 50. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). "J. R. Hunsley and C. H. Suelter, J. Biol. Chem. 244, 4815 (1969).
[27]
Bacillus
PYRUVATE KINASE
161
4 °) of (NH4)~SO~ at 4 °, stirred another 20 min, and then centrifuged for 1 hr at 15,000 g. Sufficient powdered (NH4)2S04 is then added slowly to the supernatant until 0.8 saturation is achieved, based on an observed saturation of 490 g/liter at 4 °. After 20 min of additional stirring and 1 hr of centrifugation, a precipitate is obtained which contains proteins precipitating between 0.5 and 0.8 saturation. The precipitate is then dissolved in 0.1 M KPi, pH 6.5 (4°), to a final volume of 34 ml. This solution, designated (NH4)2S04 I, contains 1300 mg of protein and 1100 units of pyruvate kinase activity. A saturated solution of (NH4)2S0, (pH 6.5, 4 °) is added, with stirring at 4 ° , to the above fraction until 0.30 saturation is achieved. After 20 rain, the suspension is centrifuged for 1 hr at 15,000 g and 4 °. Saturated (NH4)2SO~ is added to the resulting supernatant fluid to a concentration of 0.55 saturation. The precipitate (0.30-0.55 saturation) is then sedimented by 1 hr of centrifugation and dissolved in 40 mM KPi (pH 7.0 at 22 °) buffer that contains 5 mM MgCl~ and 3 mM PEP. This produces 25 ml of a solution, designated (NH4)2SO~ II, that contains 410 mg of protein and 660 units of pyruvate kinase activity. The (NH,)~S04 II fraction is added to a 5 X 12 cm hydroxyapatite (Bio-Gel-HT Bio-Rad Laboratories, Richmond, California) column at 4 °, that has been previously washed with 2 liters of 40 mM KPi buffer (pH 7.0), 5 mM MgC12, and 2 mM PEP. Two-milliliter fractions are colinto the gel, the column is washed with 370 ml of a buffer that contains 40 mM KPi (pH 7.0 at 22°), 5 mM MgC1._,, and 2 mM PEP. The bulk of the pyruvate kinase activity is eluted from the column by 210 ml of a solution that contains 75 mM KPi (pH 7.0), 5 mM MgC1._, and 2 mM PEP. The fractions collected between 380 and 480 ml of effluent are pooled and precipitated by adding solid (NH~)~SO, to 0.9 saturation. The precipitate is sedimented by centrifugation as before and suspended in 0.9 saturated (NH4)~S04. The suspension contains 17 mg of protein and 300 units of pyruvate kinase. This preparation is stable for at least 5 months when stored at 4 °. The (NH,).,S04 precipitate of the pooled, hydroxyapatite fractions (380-480 ml) is sedimented by centrifugation as before and dissolved in 20 mM KP~ (pH 7.0), containing 5 mM MgC1.., and 2 mM PEP, to a final volume of 1 ml. This solution is passed through a 1.7 X 65 cm, Bio-Gel Ao.~n, (Bio-Rad) column at a flow rate of l0 ml/hr after the column is equilibrated (at 4 °) with a buffer that contains 20 mM KPi (pH 7.0), 5 mM MgCl~, and 2 mM PEP. Two-milliter fractions are collected after discarding the first 60 ml of eluent which represented the void volume as determined with Blue-Dextran 2000 (Pharmacia). The first six fractions contain a total of 210 units of pyruvate kinase and an
162
KINASES
[27]
PURIFICATION Of PYRUVATE KINASE FROM Bacillus licheniformis
Fraction
Protein (mg)
Units (umoles/min)
Specific activity (units/mg)
Yield (%)
$78 (NH4) 2SO4 I (NH4)~S04 II Hydroxyapatite Agarose
2100 1300 410 17 1.0
1200 1100 660 300 210
0. 570 0. 845 1.62 17.6 210
92 55 25 17
estimated protein content of 1 mg. Each fraction is diluted 2-fold with glycerol and stored at --20% No loss of pyruvate kinase activity is observed over a 5-month period under these conditions. A summary of the purification scheme is presented in the table, which shows a yield of approximately 1 mg (17%) of pyruvate kinase at a specific activity of 210 units/mg. This specific activity compares favorably with that of the crystalline yeast enzyme2 This preparation yielded one protein band when a 50-~g aliquot was subjected to polyaerylamide electrophoresis according to the method described by MaizelJ A significant loss of activity is observed in a comparable purification scheme which does not include PEP during the hydroxyapatite and agarose chromatography steps. The rate of catalysis is proportional to the concentration of both purified and unpurified enzyme. ADP, PEP, and Mg(II) are absolutely required for catalysis by the purified enzyme. The purified pyruvate kinase preparation contains less than 0.08 unit of adenylate kinase ~ and less than 0.04 unit of ATPase 9 per milligram. The Kinetic Detection o] Dif]erent Activity States. The progress curves shown in Fig. 2 illustrate that the order in which the reaction components are mixed together has a dramatic effect on the rate of catalysis by pyruvate kinase. Maximum activities and linear progress curves are observed when reactions are initiated with enzyme (curve a, Fig. 2), ADP (curve b), or by the simultaneous addition of ADP and PEP (curve d). However, 80-90% lower initial rates and nonlinear progress curves are observed when reactions are initiated by PEP addition (curve c). A comparison of curves c, d, and e demonstrates that such depressed initial rates are due to the inactivation of enzyme diluted in the absence 7j. V. Maizel, in "Fundamental Techniques in Virology" (K. Habel and N. P. Salzman, eds.), p. 334. Academic Press, New York, 1969. 8 Assayed according to H. U. Bergmeyer, in "Methods of Enzymatic Analysis," p. 989. Academic Press, New York, 1963. gAssayed by the adenylate kinase assay system, modified by excluding AMP.
[27]
Bacillus
1.0
PK
ADP
PYRUVATE
PEP
ADP PEP
KINASE
163
ADPl PREINCUBATED PEP3 +AMP
0.8-E = 0.6
O qr i¢)
ow 0.4 Z m ¢
O 0.2 (Io
i
i
i
A
i
i
i
i
~
i
TIME
FI~. 2. Reaction time course and the detection of catalytically inactive enzyme states. Progress curves are shown for the standard assay system containing 0.05 unit of 50-fold purified pyruvate kinase. The method for initiating the reaction was the variable employed in this experiment. (a) Reaction initiated by the addition of enzyme; (b) initiated by ADP 2 rain after addition of enzyme; (c) initiated by PEP 2 min after the addition of enzyme; (d) initiated by the simultaneous addition of ADP and PEP 2 min after the addition of enzyme; (e) initiated by the simultaneous addition of ADP and PEP 2 min after the addition of enzyme to a reaction system containing 60 #M AMP. All operations were conducted at 30°. of P E P and the presence of the low levels (1-2%) of A M P commonly found contaminating commercial A D P preparations. The increase in rate in reactions initiated with P E P shown in Fig. 2 is due to a reactivation process dependent upon P E P and M g ( I I ) ions. 1 The nature of the inactivation and reactivation processes can be revealed by employing the following kinetic method. First, enzyme is inactivated by dilution in the absence of P E P and presence of AMP. Second, assays containing various amounts of inactive enzyme are initiated with P E P to produce curves resembling curves c and e in Fig. 2. The gain in activity, determined from reaction rate approximations over successive 10-, 20-, or 30-sec intervals, is then plotted against time. Finally, a rate of activity gain is calculated for each level of enzyme used. Such rates of reactivation for the B. licheni]ormis pyruvate kinase plotted against the log of inactive enzyme concentration yield a reaction order of two. 1 Thus, it is reasoned that the reactivation dependent on M g ( I I ) and P E P involves the association of the subunits that are produced by the dissociation induced by A M P and dilution. A second inactive enzyme state can also be detected kinetically. 1 This state can be produced by diluting the enzyme in buffers lacking all li-
164
KINASE8
[27]
gands. This form cannot be directly reactivated by PEP and Mg(II). This inactive state can, however, be converted to the activatable state via a time-dependent reaction facilitated by AMP. Thus, incubation in the presence of AMP will, with time, yield the subunits that are in turn subject to the Mg 27 PEP-mediated association to the fully active enzyme state. Studies similar to those described above have established that the complete stabilization of the active enzyme state at 0-37 ° can be achieved in the presence of 2 mM PEP, 0.5 mM AMP, and 5 mM Mg(II) ions. 1 Ten millimolar Pi (an inhibitor of catalysis) can be substituted for AMP (an activator of catalysis) in this protecting ligand mixture. Partial stabilization of the active enzyme can be achieved by 3 mM Mg ATP, 50 uM of either NAD or NADH, 50% glycerol, or 0.5 M sucrose. Kinetic Characterization. Many of the ligands that influence stability also effect the rate of catalysis of the pyruvate kinase of B. licheni]ormis. 2 Unlike the time-dependent effects on enzyme stability, the effects of ligands on the rate of catalysis occur almost instantaneously. Unfortunately, estimations of initial velocity are made over relatively lengthy periods of time (0.5-2 min) during which substantial losses of enzyme activity can occur. Such losses of activity become significant and unpredictable when the effect of varying (subsaturating) concentrations of a stabilizing ligand on initial velocity is being assessed. Therefore, the stability of the enzyme during assay must be followed closely in order to establish whether an apparent effect on the rate of catalysis might not be due to a hidden effect on enzyme stability. Enzyme stability during initial velocity determinations can be assessed conveniently by employing the following general method. Initial rates at different concentrations of the variable ligand are determined from a continuous recording of the reaction time course over a 40-sec interval beginning 10 sec after the addition of enzyme. After the 40-sec rate determination, enzyme stability in the different reaction mixtures is estimated by adding (approximately 60 sec after the addition of enzyme) an amount of the variable ligand that is sufficient to yield a constant and saturating concentration. The rate of catalysis for the ligandsaturated system is then determined over a 60-sec interval. This new rate is expressed as a percentage of the initial rate that is observed when a ligand-saturated reaction mixture is initiated directly by the addition of enzyme. The application of these methods with PEP as the variable ligand (substrate) is illustrated in Fig. 3. The experiment illustrated in the upper portion of Fig. 3 shows apparently sigmoid saturation curves for PEP and an apparent activation of catalysis by AMP. However, the stability data (inset) suggest that
[27]
Bacillus PYRUVATE KINASE
165
90
6C
u~ 3C z mM PEP
~J
J
¢
3 .J I.- 4C z
I 80[
2C
0
0
0.5
STABILITY
0.5 rnM PEP
I
1.0
PEP(mM)
FIa. 3. Relationship between initial velocity and phosphoenolpyruvate (PEP) concentration in the presence and in the absence of AMP and ATP. The assay mixtures (1.0 ml) for the upper illustration contained 50 tmloles of imidazole-HC] (pH 7.1), 50 t~moles of KC1, 7 t~nloles of MgC12, 0.25 tmmle NADH, 40 t~g of lactate dehydrogenase, 0.5 ]zg of pyruvate kinase (115 units/rag), 2 ~moles ADP and a variable concentration of PEP in the absence ( 0 ) and in the presence (O) of 1 t~mole of AMP. The assay mixtures for the lower illustration were as above but for the presence of 2.8 #moles MgATP, 0.8/zmole ADP, and 1.0 #g pyruvate kinase (PK). Initial rates were estimated from a continuous recording of the reaction time course over a 40-sec interval beginning 10 sec after the addition of enzyme. After the 40-sec rate determination, the stability of PK in the different reaction mixtures was estimated by adding sufficient PEP to yield a final concentration of 2 mM and obtaining a new reaction rate. This new rate is expressed as a percentage of the initial rate observed when a reaction, containing 2 mM PEP, was initiated by the addition of enzyme. both the sigmoid n a t u r e of the s a t u r a t i o n curves a n d the s t i m u l a t i o n by A M P are caused by a v a r i a t i o n of e n z y m e s t a b i l i t y . I n contrast, the sigmoidal n a t u r e of the P E P s a t u r a t i o n curve in the presence of A T P a n d the a c t i v a t i n g influence of A M P are clearly established by the experim e n t i l l u s t r a t e d in the lower p o r t i o n of Fig. 3. T h e complete s t a b i l i z a t i o n of the e n z y m e is achieved here at all c o n c e n t r a t i o n s of P E P b y the stabilizing effect of A T P .
166
KINASES
[28]
The kinetic characterization of the pyruvate kinase of B. licheni]ormis, conducted with a constant assessment of the effects of reaction conditions on enzyme stability, reveals the following properties. 2 Catalysis by this enzyme proceeds optimally at pH 7.0-7.4. The initial rate of catalysis is modulated by substrate activation (both PEP and ADP), activation by AMP, and inhibition by ATP, Pi and carbamyl phosphate. Positive cooperatively is manifested by PEP, ADP, Pi and ATP in the presence of Mg(II) ions. AMP relieves the inhibition by ATP, yielding hyperbolic saturation curves for both substrates. The apparent substrate Km values for the fully activated enzyme, 0.2 mM for PEP, and 0.7 mM for ADP, in the presence of Mg(II) are independent of the concentration of the second substrate. In the presence of Mn(II) as the required divalent cation, the Km for PEP is 7-fold lower than that obtained with the Mg(II)-activated enzyme. Finally, the inhibition of catalysis by ATP in the presence of Mn(II) is not reversed by AMP and does not effect the hyperbolic nature of the PEP saturation curve observed under these conditions. Thus, the K-type 1° allosteric properties manifested in the presence of Mg(II) are obliterated when Mn(II) is employed as the activating divalent cation. loj. Monod, J. Wyman, and J.-P. Changeux, J. Mol. Biol. 12, 88 (1965).
[28] Isozymes of Pyruvate Kinase from the Grassfrog 1 B y L. E. FLANDERS, L. H. SCHLOEN, and H. J. SALLACH Phosphoeno/pyruvate + ADP ~ pyruvate + ATP
The occurrence of multiple forms of pyruvate kinase in different tissues of the rat was first reported by Tanaka et al. 2,3 These investigators detected from one to four isozymes in a given tissue as did Susor and Rutter. 4 Early work with human tissues demonstrated three different isozymes. ~ On the other hand, work in this laboratory 6 demonstrated that 1This work was supported by Research Contract No. AT(ll-1)-1631 from the U.S. Atomic Energy Commission and by National Institutes of Health Grant No. NS10287. T. Tanaka, Y. Harano, H. Morimura, and R. Mori, Bioehem. Biophys. Res. Commun. 21, 55 (1965). 8 T. Tanaka, Y. Harano, F. Sue, and H. Morimura, J. Biochem. (Tokyo) 62, 71 (1967). W. A. Susor and W. J. Rutter, Biochem. Biophys. Res. Commun. 30, 14 (1968). 5 R. H. Bibley, P. Stanzel, R. T. Jones, J. P. Campas, and R. ]5. Koler, Enzyme Biol. CIin. 9, 10 (1968). 8L. H. Schloen, J. R. Bamburg, and H. J. SaUach, Biochem. Biophys. Res. Commun. 36, 823 (1969).
Bacillus PYRUVATE KINASE
[27] P y r u v a t e K i n a s e o f B a c i l l u s
157
lichenilormis
By F. WILLIAM TUOMINEN and ROBERT W. BERNLOHR Phosphoenolpyruvate + ADP + H+ ~ pyruv~te + ATP The pyruvate kinase ATP:pyruvate phosphotransferase, (EC 2.7.1.40) of Bacillus licheni]ormis is an example of an AMP-activated, ATP-inhibited glycolytic enzyme that can exist in a number of kinetieally differentiable activity states. 1,2 Unless proper precautions are exercised with this enzyme during extract preparation, purification, and assay, the formation of inactive enzyme states will result in low or erroneous specific activity values, gross losses of activity, and misleading kinetic data. The methodology presented here emphasizes those details that allow the experimentalist to understand and deal with the difficulties inherent in this highly permutable pyruvate kinase. Culture and Harvest of Cells. The organism used is the genetieally stable (with respect to colonial morphology), rough colony-forming strain of B. licheni]ormis A-5. Spore stocks are prepared by inoculating 0.5 liter of 0.4% (w/v) peptone and 0.1% yeast extract contained in a 2.8-liter Fernbach flask with either vegetative or spore seed that has been derived from an isolated colony. Greater than 95% of the microscopically recognizable cells exist as free spores after 24 hr of incubation at 37 ° on a rotary shaker. The minimal medium used routinely for the production of vegetative cells contains (per liter) 65 mmoles of K2HPO4-KH~P04 (pH 7.2), 0.8 mmole of MgS04, 0.02 mmole of MnCI=,, 0.5 mmole of CaCh, 15 mmoles of NH4C1, and 15 mmoles of glucose. The glucose, NH~C1, MgS04, MnC12, and NH4C1 are added from sterile, concentrated stocks to autoclaved K_~HPO4-KH2P04 to yield a nutritionally complete, unprecipitated growth medium. Cultures are started by adding approximately 10TM spores to 1 liter of minimal medium supplemented with 0.5 mM L-alanine. Cultures are aerated by shaking on a Gyrotory shaker (New Brunswick Scientific Co.) at 37 °. When cultures reach a turbidity of 150-170 Klett units (540 ran), 1 liter is transferred to aerated fermentation vessels of a New Brunswick Scientific Co. fermentor (Model FS-307) at 38 °. Each vessel contains 10 liters of sterile, prewarmed medium. Vegetative cells are harvested for enzyme studies from volumes of culF. W. Tuominen and R. W. Bernlohr, J. Biol. Chem. 9,,46, 1732 (1971). 2F. W. Tuominen and R. W. Bernlohr, J. Biol. Chem. 246, 1746 (1971).
158
KINASES
[27]
ture up to 3 liters by centrifugation (13,000 g for 10 rain at 0 °) in a Sorvall RC2-B centrifuge after being cooled by the addition of ice. Volumes greater than 3 liters are harvested in a Lourdes continuous flow centrifuge (12,000 rpm) after the addition of ice. Cells are washed with a 0.1 M Tris.HC1 (pH 7.6) buffer that contains 10 mM MgCl_~ and 0.1 M KC1. The washed-cell pellets are either immediately used to prepare cell-free extracts or stored at --20 ° . Preparation o] Cell.Free Extracts. Cells are suspended in a 0.1 M Tris.HC1 (pH 7.6) buffer that contains 10 mM MgC12 and 0.1 M KC1 (Buffer TMK) on the basis of 1.0 ml buffer per gram, wet weight. Cells are broken by sonic disruption when the suspension volume is less than 5 ml and in a French pressure cell (at 12,000 psi) when larger volumes are employed. Sonic disruption is performed in a MSE sonicator (Measuring and Scientific Equipment, Ltd., London) at 110 V and 2 A with a 1.9-cm probe at 4-10 °. The total time of sonic disruption is 3 min, i.e. six, 30-see bursts that are interrupted by 30-see cooling periods (ice bath). This sonic disruption time (3 min) is twice that required to release a maximum amount of pyruvate kinase from a suspension of late exponential phase cells. Assay System. Pyruvate kinase ]s assayed by employing the linked lactic dehydrogenase (EC 1.1.1.27) reaction as modified from B~icher and Pfleiderer2 The standard assay system (1.0 ml) contains 50 ~moles imidazole-HC1 (pH 7.1, the optimum), 50 ~moles of KC1 (also an optimum concentration), 7 ~moles of MgCl~_, 2 ~moles of tricyclohexylammonium phosphoenolpyruvate (PEP) (TCHA-PEP), 4 ~moles NaADP (pH 7.1), 0.12 ~mole NaNADH and 40 ~g lactic dehydrogenase. Reactions are initiated by the addition of 2-20 ~l of enzyme preparation to the reaction mixture at 30 ° in a 10-ram quartz cuvette. The time course of NADH oxidation is followed at 340 nm in a Zeiss PMQII spectrophotometer connected to a Sargent Model SRL recorder. Initial rates are converted to micromo]es of pyruvate formed per minute by dividing the change in absorbance per minute by 6.22. All rates are corrected for blank rates obtained by the omission of ADP. One unit of activity is defined as the amount of pyruvate kinase yielding 1 ~mole of pyruvate per minute in the standard assay system. Freshly prepared solutions of NaADP (P-L Biochemicals, Inc.), pH 7.1, and TCHA-PEP (Sigma) are used for kinetic studies and stored on ice for up to 16 hr. Stock solutions of 0.1 M NaADP (pH 7.1) and 0.1 M TCHA-PEP, for use in the standard assay system, are stored for up to 2 weeks a~ --60 °. The concentrations of the PEP and ADP solutions are determined by assaying PEP and ADP in a modified standard assay s T. Biicher and G. Pfleiderer, this series, Vol. 1, p. 435.
Bacillus PYRUVATE KINASE
[27]
0.6
159
i &
E x
0.4 >I>
0.2 (.) ,'7
U I.u O. U')
%
zo
40
PROTEIN
~o
80
(mg/ml)
Fla. 1. Effect of dilution on the apparent specific activity of pyruvate kinase in crude extracts. A crude extract (80 mg of protein per milliliter) was prepared in 0.1 M Tris.HC1, ptI 7.6, containing 10 mM MgCh and 0.1 M KC1 at 22°. Aliquots of this extract were diluted to the indicated levels in the same buffer at 22 °. Samples of each dilution were assayed in the standard assay system at 1 min ( 0 O), 2 hr (O O), and 4 hr (A A) postdilution. The amount of extract protein was approximately the same in all assays. mixture containing 0.25 t~mole N A D H , 0.5 unit rabbit muscle p y r u v a t e kinase and 0.1-0.2 t~mole of either P E P or ADP. The change in absorbance after the addition of the p y r u v a t e kinase is divided by 6.22 to give the ~moles of P E P or A D P in the volume of solution assayed. Beef heart lactic dehydrogenase ( L D H ) is obtained as a crystalline suspension in 55% saturated (NH,)~SO~. This preparation is diluted 5-fold in 50 m M Tris.HC1, p H 7.5 and dialyzed against three changes of t h e same Tris.HC1 buffer at 4% The dialyzed preparation is diluted another 2-fold in glycerol and stored at 4 °. The final L D H stock solution contains 4 m g / m l L D H (350 I U / m g ) and 50% ( v / v ) glycerol. N a N A D H ( P - L Biochemicals, Inc.) stock solutions (8-15 raM) are stored at 2-4 ° in 10 m M Na(HCO:,-C03) buffer, p H 10.5, in accord with published recommendations2 Determination of Specific Activity in Crude Extracts. Crude extracts must contain greater than 40 mg per milliliter of protein in order to oblain meaningful p y r u v a t e kinase specific activities. The reason for this precaution against greater dilution is illustrated in Fig. 1. The data presented in Fig. 1 show t h a t p y r u v a t e kinase in crude extracts is rapidly inactivated by dilution of these extracts to less than 40 m g / m l of protein at 22 °. In addition to being sensitive to dilution at 22 °, p y r u v a t e kinase 40. H. Lawry, J. V. Passonneau, and M. K. Rock, J. Biol. Chem. 236, 2756 (1961).
160
KINASES
[27]
is reversibly inactivated at 0°. 1 As the extent of dilution is increased, at 0 °, both the amount of residual activity and reversibility of inactivation decrease. However, no cold inactivation can be detected in parallel experiments which employ warm pipettes (22°), rather than ice-cold pipettes, for removal of samples (2-10 ul) from the dilutions incubated near 0 °. These results suggest that the undiluted enzyme is rapidly reactirated during the resident time (about 20-30 sec) in the pipettes. The specific activity of pyruvate kinase in freshly prepared crude extracts, containing greater than 40 mg/ml of protein, has been found to be independent of the stage of growth (on glucose) or sporulation. 1 Similar results are observed when extracts are dialyzed against a buffer containing KPi, MgCL and PEP. The specific activities generally range between 0.55 and 0.65 unit per milligram of protein. The specific activity present in crude extracts of exponential-phase cells grown on malate range from 0.53. to 0.61 unit per milligram of protein. Values of 0.50 and 0.59 (unit per milligram of protein) have been observed with exponential-phase cells grown on pyruvate. Purification. The otherwise labile pyruvate kinase activity of B. lichemformis is stabilized throughout the following purification scheme by avoiding extensive dilution of the enzyme and by the inclusion of a stabilizing ligand mixture (PEP, Pi, and Mg ions) in the supporting buffers when dilution is unavoidable. Protein concentrations through the penultimate purification step are determined by the method of Lowry et al. 5 The protein concentrations of the most highly purified enzyme preparations are estimated spectrophotometrically at 280 nm by employing an ~,0.1 ~-~lcm~ = 0.65 which is the constant obtained by Hunsley and Suelter for crystalline yeast pyruvate kinase. Thirty grams of late exponential-phase cells are suspended at 0 ° in 45 ml of 0.1 M Tris.HC1 (pH 7.6) that contains 10 mM MgC12 and 0.1 M KC1. Cells are ruptured by two passes through a French pressure cell at 12,000 psi. The extract is sonicated for 2 rain in order to reduce the viscosity and centrifuged (0 °) at 78,000 g for 2 hr in a Spinco Model L centrifuge. The upper two-thirds of the resulting supernatants are combined to yield 40 ml of a solution that contains 2100 mg protein and 1200 units of pyruvate kinase activity. This supernatant fluid is designated as $78 (see the table). The pH of $78 is adjusted to 8.0, at 4 °, by the addition of 1 M N H , O H from a capillary-tipped pipette. The extract is diluted 2-fold by the dropwise addition of a saturated solution (490 g/l; pH 8.0 at 50. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). "J. R. Hunsley and C. H. Suelter, J. Biol. Chem. 244, 4815 (1969).
[27]
Bacillus
PYRUVATE KINASE
161
4 °) of (NH4)~SO~ at 4 °, stirred another 20 min, and then centrifuged for 1 hr at 15,000 g. Sufficient powdered (NH4)2S04 is then added slowly to the supernatant until 0.8 saturation is achieved, based on an observed saturation of 490 g/liter at 4 °. After 20 min of additional stirring and 1 hr of centrifugation, a precipitate is obtained which contains proteins precipitating between 0.5 and 0.8 saturation. The precipitate is then dissolved in 0.1 M KPi, pH 6.5 (4°), to a final volume of 34 ml. This solution, designated (NH4)2S04 I, contains 1300 mg of protein and 1100 units of pyruvate kinase activity. A saturated solution of (NH4)2S0, (pH 6.5, 4 °) is added, with stirring at 4 ° , to the above fraction until 0.30 saturation is achieved. After 20 rain, the suspension is centrifuged for 1 hr at 15,000 g and 4 °. Saturated (NH4)2SO~ is added to the resulting supernatant fluid to a concentration of 0.55 saturation. The precipitate (0.30-0.55 saturation) is then sedimented by 1 hr of centrifugation and dissolved in 40 mM KPi (pH 7.0 at 22 °) buffer that contains 5 mM MgCl~ and 3 mM PEP. This produces 25 ml of a solution, designated (NH4)2SO~ II, that contains 410 mg of protein and 660 units of pyruvate kinase activity. The (NH,)~S04 II fraction is added to a 5 X 12 cm hydroxyapatite (Bio-Gel-HT Bio-Rad Laboratories, Richmond, California) column at 4 °, that has been previously washed with 2 liters of 40 mM KPi buffer (pH 7.0), 5 mM MgC12, and 2 mM PEP. Two-milliliter fractions are colinto the gel, the column is washed with 370 ml of a buffer that contains 40 mM KPi (pH 7.0 at 22°), 5 mM MgC1._,, and 2 mM PEP. The bulk of the pyruvate kinase activity is eluted from the column by 210 ml of a solution that contains 75 mM KPi (pH 7.0), 5 mM MgC1._, and 2 mM PEP. The fractions collected between 380 and 480 ml of effluent are pooled and precipitated by adding solid (NH~)~SO, to 0.9 saturation. The precipitate is sedimented by centrifugation as before and suspended in 0.9 saturated (NH4)~S04. The suspension contains 17 mg of protein and 300 units of pyruvate kinase. This preparation is stable for at least 5 months when stored at 4 °. The (NH,).,S04 precipitate of the pooled, hydroxyapatite fractions (380-480 ml) is sedimented by centrifugation as before and dissolved in 20 mM KP~ (pH 7.0), containing 5 mM MgC1.., and 2 mM PEP, to a final volume of 1 ml. This solution is passed through a 1.7 X 65 cm, Bio-Gel Ao.~n, (Bio-Rad) column at a flow rate of l0 ml/hr after the column is equilibrated (at 4 °) with a buffer that contains 20 mM KPi (pH 7.0), 5 mM MgCl~, and 2 mM PEP. Two-milliter fractions are collected after discarding the first 60 ml of eluent which represented the void volume as determined with Blue-Dextran 2000 (Pharmacia). The first six fractions contain a total of 210 units of pyruvate kinase and an
162
KINASES
[27]
PURIFICATION Of PYRUVATE KINASE FROM Bacillus licheniformis
Fraction
Protein (mg)
Units (umoles/min)
Specific activity (units/mg)
Yield (%)
$78 (NH4) 2SO4 I (NH4)~S04 II Hydroxyapatite Agarose
2100 1300 410 17 1.0
1200 1100 660 300 210
0. 570 0. 845 1.62 17.6 210
92 55 25 17
estimated protein content of 1 mg. Each fraction is diluted 2-fold with glycerol and stored at --20% No loss of pyruvate kinase activity is observed over a 5-month period under these conditions. A summary of the purification scheme is presented in the table, which shows a yield of approximately 1 mg (17%) of pyruvate kinase at a specific activity of 210 units/mg. This specific activity compares favorably with that of the crystalline yeast enzyme2 This preparation yielded one protein band when a 50-~g aliquot was subjected to polyaerylamide electrophoresis according to the method described by MaizelJ A significant loss of activity is observed in a comparable purification scheme which does not include PEP during the hydroxyapatite and agarose chromatography steps. The rate of catalysis is proportional to the concentration of both purified and unpurified enzyme. ADP, PEP, and Mg(II) are absolutely required for catalysis by the purified enzyme. The purified pyruvate kinase preparation contains less than 0.08 unit of adenylate kinase ~ and less than 0.04 unit of ATPase 9 per milligram. The Kinetic Detection o] Dif]erent Activity States. The progress curves shown in Fig. 2 illustrate that the order in which the reaction components are mixed together has a dramatic effect on the rate of catalysis by pyruvate kinase. Maximum activities and linear progress curves are observed when reactions are initiated with enzyme (curve a, Fig. 2), ADP (curve b), or by the simultaneous addition of ADP and PEP (curve d). However, 80-90% lower initial rates and nonlinear progress curves are observed when reactions are initiated by PEP addition (curve c). A comparison of curves c, d, and e demonstrates that such depressed initial rates are due to the inactivation of enzyme diluted in the absence 7j. V. Maizel, in "Fundamental Techniques in Virology" (K. Habel and N. P. Salzman, eds.), p. 334. Academic Press, New York, 1969. 8 Assayed according to H. U. Bergmeyer, in "Methods of Enzymatic Analysis," p. 989. Academic Press, New York, 1963. gAssayed by the adenylate kinase assay system, modified by excluding AMP.
[27]
Bacillus
1.0
PK
ADP
PYRUVATE
PEP
ADP PEP
KINASE
163
ADPl PREINCUBATED PEP3 +AMP
0.8-E = 0.6
O qr i¢)
ow 0.4 Z m ¢
O 0.2 (Io
i
i
i
A
i
i
i
i
~
i
TIME
FI~. 2. Reaction time course and the detection of catalytically inactive enzyme states. Progress curves are shown for the standard assay system containing 0.05 unit of 50-fold purified pyruvate kinase. The method for initiating the reaction was the variable employed in this experiment. (a) Reaction initiated by the addition of enzyme; (b) initiated by ADP 2 rain after addition of enzyme; (c) initiated by PEP 2 min after the addition of enzyme; (d) initiated by the simultaneous addition of ADP and PEP 2 min after the addition of enzyme; (e) initiated by the simultaneous addition of ADP and PEP 2 min after the addition of enzyme to a reaction system containing 60 #M AMP. All operations were conducted at 30°. of P E P and the presence of the low levels (1-2%) of A M P commonly found contaminating commercial A D P preparations. The increase in rate in reactions initiated with P E P shown in Fig. 2 is due to a reactivation process dependent upon P E P and M g ( I I ) ions. 1 The nature of the inactivation and reactivation processes can be revealed by employing the following kinetic method. First, enzyme is inactivated by dilution in the absence of P E P and presence of AMP. Second, assays containing various amounts of inactive enzyme are initiated with P E P to produce curves resembling curves c and e in Fig. 2. The gain in activity, determined from reaction rate approximations over successive 10-, 20-, or 30-sec intervals, is then plotted against time. Finally, a rate of activity gain is calculated for each level of enzyme used. Such rates of reactivation for the B. licheni]ormis pyruvate kinase plotted against the log of inactive enzyme concentration yield a reaction order of two. 1 Thus, it is reasoned that the reactivation dependent on M g ( I I ) and P E P involves the association of the subunits that are produced by the dissociation induced by A M P and dilution. A second inactive enzyme state can also be detected kinetically. 1 This state can be produced by diluting the enzyme in buffers lacking all li-
164
KINASE8
[27]
gands. This form cannot be directly reactivated by PEP and Mg(II). This inactive state can, however, be converted to the activatable state via a time-dependent reaction facilitated by AMP. Thus, incubation in the presence of AMP will, with time, yield the subunits that are in turn subject to the Mg 27 PEP-mediated association to the fully active enzyme state. Studies similar to those described above have established that the complete stabilization of the active enzyme state at 0-37 ° can be achieved in the presence of 2 mM PEP, 0.5 mM AMP, and 5 mM Mg(II) ions. 1 Ten millimolar Pi (an inhibitor of catalysis) can be substituted for AMP (an activator of catalysis) in this protecting ligand mixture. Partial stabilization of the active enzyme can be achieved by 3 mM Mg ATP, 50 uM of either NAD or NADH, 50% glycerol, or 0.5 M sucrose. Kinetic Characterization. Many of the ligands that influence stability also effect the rate of catalysis of the pyruvate kinase of B. licheni]ormis. 2 Unlike the time-dependent effects on enzyme stability, the effects of ligands on the rate of catalysis occur almost instantaneously. Unfortunately, estimations of initial velocity are made over relatively lengthy periods of time (0.5-2 min) during which substantial losses of enzyme activity can occur. Such losses of activity become significant and unpredictable when the effect of varying (subsaturating) concentrations of a stabilizing ligand on initial velocity is being assessed. Therefore, the stability of the enzyme during assay must be followed closely in order to establish whether an apparent effect on the rate of catalysis might not be due to a hidden effect on enzyme stability. Enzyme stability during initial velocity determinations can be assessed conveniently by employing the following general method. Initial rates at different concentrations of the variable ligand are determined from a continuous recording of the reaction time course over a 40-sec interval beginning 10 sec after the addition of enzyme. After the 40-sec rate determination, enzyme stability in the different reaction mixtures is estimated by adding (approximately 60 sec after the addition of enzyme) an amount of the variable ligand that is sufficient to yield a constant and saturating concentration. The rate of catalysis for the ligandsaturated system is then determined over a 60-sec interval. This new rate is expressed as a percentage of the initial rate that is observed when a ligand-saturated reaction mixture is initiated directly by the addition of enzyme. The application of these methods with PEP as the variable ligand (substrate) is illustrated in Fig. 3. The experiment illustrated in the upper portion of Fig. 3 shows apparently sigmoid saturation curves for PEP and an apparent activation of catalysis by AMP. However, the stability data (inset) suggest that
[27]
Bacillus PYRUVATE KINASE
165
90
6C
u~ 3C z mM PEP
~J
J
¢
3 .J I.- 4C z
I 80[
2C
0
0
0.5
STABILITY
0.5 rnM PEP
I
1.0
PEP(mM)
FIa. 3. Relationship between initial velocity and phosphoenolpyruvate (PEP) concentration in the presence and in the absence of AMP and ATP. The assay mixtures (1.0 ml) for the upper illustration contained 50 tmloles of imidazole-HC] (pH 7.1), 50 t~moles of KC1, 7 t~nloles of MgC12, 0.25 tmmle NADH, 40 t~g of lactate dehydrogenase, 0.5 ]zg of pyruvate kinase (115 units/rag), 2 ~moles ADP and a variable concentration of PEP in the absence ( 0 ) and in the presence (O) of 1 t~mole of AMP. The assay mixtures for the lower illustration were as above but for the presence of 2.8 #moles MgATP, 0.8/zmole ADP, and 1.0 #g pyruvate kinase (PK). Initial rates were estimated from a continuous recording of the reaction time course over a 40-sec interval beginning 10 sec after the addition of enzyme. After the 40-sec rate determination, the stability of PK in the different reaction mixtures was estimated by adding sufficient PEP to yield a final concentration of 2 mM and obtaining a new reaction rate. This new rate is expressed as a percentage of the initial rate observed when a reaction, containing 2 mM PEP, was initiated by the addition of enzyme. both the sigmoid n a t u r e of the s a t u r a t i o n curves a n d the s t i m u l a t i o n by A M P are caused by a v a r i a t i o n of e n z y m e s t a b i l i t y . I n contrast, the sigmoidal n a t u r e of the P E P s a t u r a t i o n curve in the presence of A T P a n d the a c t i v a t i n g influence of A M P are clearly established by the experim e n t i l l u s t r a t e d in the lower p o r t i o n of Fig. 3. T h e complete s t a b i l i z a t i o n of the e n z y m e is achieved here at all c o n c e n t r a t i o n s of P E P b y the stabilizing effect of A T P .
166
KINASES
[28]
The kinetic characterization of the pyruvate kinase of B. licheni]ormis, conducted with a constant assessment of the effects of reaction conditions on enzyme stability, reveals the following properties. 2 Catalysis by this enzyme proceeds optimally at pH 7.0-7.4. The initial rate of catalysis is modulated by substrate activation (both PEP and ADP), activation by AMP, and inhibition by ATP, Pi and carbamyl phosphate. Positive cooperatively is manifested by PEP, ADP, Pi and ATP in the presence of Mg(II) ions. AMP relieves the inhibition by ATP, yielding hyperbolic saturation curves for both substrates. The apparent substrate Km values for the fully activated enzyme, 0.2 mM for PEP, and 0.7 mM for ADP, in the presence of Mg(II) are independent of the concentration of the second substrate. In the presence of Mn(II) as the required divalent cation, the Km for PEP is 7-fold lower than that obtained with the Mg(II)-activated enzyme. Finally, the inhibition of catalysis by ATP in the presence of Mn(II) is not reversed by AMP and does not effect the hyperbolic nature of the PEP saturation curve observed under these conditions. Thus, the K-type 1° allosteric properties manifested in the presence of Mg(II) are obliterated when Mn(II) is employed as the activating divalent cation. loj. Monod, J. Wyman, and J.-P. Changeux, J. Mol. Biol. 12, 88 (1965).
[28] Isozymes of Pyruvate Kinase from the Grassfrog 1 B y L. E. FLANDERS, L. H. SCHLOEN, and H. J. SALLACH Phosphoeno/pyruvate + ADP ~ pyruvate + ATP
The occurrence of multiple forms of pyruvate kinase in different tissues of the rat was first reported by Tanaka et al. 2,3 These investigators detected from one to four isozymes in a given tissue as did Susor and Rutter. 4 Early work with human tissues demonstrated three different isozymes. ~ On the other hand, work in this laboratory 6 demonstrated that 1This work was supported by Research Contract No. AT(ll-1)-1631 from the U.S. Atomic Energy Commission and by National Institutes of Health Grant No. NS10287. T. Tanaka, Y. Harano, H. Morimura, and R. Mori, Bioehem. Biophys. Res. Commun. 21, 55 (1965). 8 T. Tanaka, Y. Harano, F. Sue, and H. Morimura, J. Biochem. (Tokyo) 62, 71 (1967). W. A. Susor and W. J. Rutter, Biochem. Biophys. Res. Commun. 30, 14 (1968). 5 R. H. Bibley, P. Stanzel, R. T. Jones, J. P. Campas, and R. ]5. Koler, Enzyme Biol. CIin. 9, 10 (1968). 8L. H. Schloen, J. R. Bamburg, and H. J. SaUach, Biochem. Biophys. Res. Commun. 36, 823 (1969).
