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Regulatory properties of pyruvate kinase in human cells grown in tissue culture
Lüe Sciences Vol . 10, Part II, pp . 1183-1171, 1971 . printed in Great Britain
Pergamon Press
REGtTLATORY PROPERTIES OF PYRUVATE KINASE IN HDMAN CELLS GROWN IN TISSUE CULTURE George A. Dunaway, Jr . and Sddie C. Smith Department of Chemistry, University of Oklahoma, Norman, Oklahoma
73069
(Received 8 July 1971; in final form 22 September 1971) Summary Pyruvate kinase (E . C . 2.7 .1 .40) from human embryonic lung tissue, WI-38, has a pA optimum range of 7.0 to 7.2 . This sesame was found to be activated 2.1, 5.0 and 2.5 fold by fructose-l,6 diphoephate, &C1, and NHyCl, respectively, and unaffected by NaCl . Eves though the degree of activation by these effectors is different, they all cause an increase in maximal velocity and increase the affinity of the enzyme for phoephoenolpyruvate . ATP and L-alanine were found to inhibit maaímal velocity by 40X and 70X, respectively, and is a non-competitive meaner with respect to both substrates . Speculation is offered on the physiological effect of these modulators on glycolgsis and glyconeogeaesis . Introduction The cells used in this atudq were the cell strain, WI-38, which were originally obtained from human embryonic lung tissue (1) .
It has been shown
that WI-38 maintains a stable diploid karyotype and ezhibita a limited life span (1,2) .
The glucose flux for WI-38 has been determined by several in-
vestigators, and their results indicate that the major fate of glucose is lactate (3,4) . Regulatory properties of pyruvate kinase (E . C . 2.7 .1 .40) have bean determined for rat liver (5,6,7,8), human erythrocytes (9), mouse liver (10), yeast (11), and Escherichia cola (12) .
Such studies concerning regulatioa of py
ruvate kinase in human calls grows in tissue culture have not bees reported . Thus, the objective of this paper ie to preeeat the results of work performed to determine the regulation of WI-38 pyruvate kinase by small molecular weight effectora ía order to speculate on Chair contribution to glycolytic regulation and to creating conditions conducive to glyconeogeaesis .
1183
1164
Regulatory Properties ad Pyruvate Kinase
Vol. 10, No. 20
Materials and Methode Original starter cultures of WI-38 were obtained from Dr . Paul F . Rruse Jr .
Maintenance and harvest of cells were as previously described (4) .
Har-
vested cells were sonicated at maximum cavitation for 2 min . i n O .OSM tris buffer pH 7 .5 and then cooled in a salt-ice bath from a maximum temperature of 20°C to 0°C .
The supernatant fluid resulting from centrifugation at 27,000
x g for 20 min . was dialyzed overnight against 0 .05M trie buffer pH 7 .5 .
This
dialyzed solution, which was used in the study of pyruvate kinasa, contained insignificant ATPase and myokiaase activity .
The dialysis tubing was boiled
in water for 15 min . before being used for dialysis . The pyruvate kinasa activity was measured by a slightly modified method of Shonk and Boxer (13) .
The assay solution (3 .0 ml) contained 150 umoles
tris pH 7 .5, 30 umoles MgC12 , 0 .9 umolas NADH, an excess of pre-dialyzed lactate dehydrogenase, and 9 umolas of phoaphoenolpyruvate (PEP) and/or 9 umolea of ADP .
Both substrates were maintained at 3 .0 mM unless substrate
saturations curves were determined .
The reaction was started with supernatant
fluid and the reaction velocity was followed by measuring the decrease in abeorbance at 340 nm at room temperature . Protein concentration was measured using a modified biuret method (14) . The pH curve was determined using tris and imidazole buffers, and the pH was measured before and after assaying using the expanded scale of a Corning pH meter .
All substrates and lactate dehydrogenase were purchased from Sigma
Chemical Co ., St . Louis, Mo . and used without further purification . Results Effect of pH .
The pyruvate kinase activity had an optimal pH range of
7 .0 to 7 .2 (see Fig . 1) .
The activity decreased to zero at pH 5 .5 and decreased
to 20x mammal velocity at pH 9 .5 .
Examination of the overlapping points in-
dicated that the activity is the same in imidazole or tris buffers . Effect of Activators on Pyruvata Kinase Activity . and NaCl on pyruvate kinase activity ie shown in Fig . 2 .
The effect of KC1, NH4 C1, NaCl had no effect up
Vol. 10, No. 20
Regulatory Properties od Pyruvate Kinase
1185
to a concentration of 150 mM, while NH 4 C1 displayed a mazímum activation of approximately 2 .5 fold at 1 mM .
However, at a concentration of 100 mM, NH4C1
inhibited maximum activity by 20Z, an inhibition which could ba a pH effect . KC1, which was the best activator,
increased activity by 5 fold .
RC1 activa-
tion was detected only above 1 mM with full activation occurring at 40 mM .
~vm
pH Profile of Pyruvate &mass No concentration of RC1 studied inhibited eazymn activity .
Also, 1.0 mM FDP
increased pyruvate kinase maximal velocity by approximately 2 .1 fold (see Fig . 3) . Ia the abéanca of them activators (Fig . 3)
the PEP saturation curve was aigmoidal
in nature with as apparent Rm for PSP of 0 .3 mM .
Data in the farm of
a Hill plot (15), yielded as iataraction coefficient of 2 which is indicative of interactions betaraea PSP biadiag sites and thus verifying the sigmoidal nature of the saturation curve .
However, in addition to increasing the maximal
Regulatory Properties ad Pyruvate Kinase
1188
Vol. 10, No . 20
W F
Effect of Monovalent Cations on Pyruvate Kinase Activity . Away Conditions are in Materials and Mathoda Section . Represents the Activity of Pyruvate Rinaee in the Absence of NaCl, RC1 and NH 4C1 . velocity 1 mM FDP, 50 mM NH 4 C1, or RC1 decreased the PEP apparent Km to 0 .15 mM, 0 .10 mM, and 0 .10 mlS, respectively .
As a consequence of the effect of
these activators the PEP saturation curve (Fig . 3) was transformed to a hyperbolic type curve characteristic of Michaelis-Meaten type curves and the PEP biding site interactions decreased (the interaction coefficient becomes 1) . Effect of Inhibitors on PEP Saturation .
The effect of 1 .0 mM L-alanine
and ATP on the PEP saturation curve is shown in Fig . 4 .
These data indicated
that L-alanine (70x inhibitioc~ was a more potent inhibitor than ATP (40Z in hibition) .
The apparent Km was not altered by either inhibitor which implied
that both inhibitors are non-competitive type inhibitors .
The interaction co-
efficient of 2 was unchanged and thus, the aigmoidal nature of the saturation curves was not altered . Effect of Inhibitors on ADP Saturation . on ADP saturation ie ehwn in Fig . 5 .
The effect of ATP and L-alanine
In the absence of inhibitors the ADP
Vol . 10, No. 20
Regulatory Properties ad Pyruvate Einase
1187
w E
P E P (mI~
nlc . 3 Effect of Activators on PEP Saturation of Pyruvate Rinses Activity . Assay Conditions are in Materials aad Methods Section. The Concentration of ADP is 3 .0 mM . apparent Rm was 0.38 mM, aad the ADP saturation curve was éigmoidal with the ADP binding site interactioa coefficient being 2.
ATP and Iralanine the apparent competitive typa inhibition .
m
In the presence of 1.0 mM
was unchanged which wee. indicative of non-
Also, the ADP saturation curve in the presence
of these inhibitors remained sigmoidal as well as the Interaction coefficient for ADP binding remaiaiag unaltered at 2. Discussion
The apparent Rm for PEP for WI-38 pyruvate kinase (0 .3 mM) compares with that of rat liver (5,6), pig liver (6), rat adipose tissue (16), and humas erythrocyte (9) pyruvate kinase .
The apparent
m
for ADP for wI-38 pynivate
kíaase (0 .38 mM) ie of the same order of magnitude as that for human erythrocytes
1188
Regulatory Properties ~ Pyruvate Kinase
Vol. 10, No. 20
FIG . 4 Effect of Inhibitors on PEP Saturation of Pyruvatn Kinase Activity . Assay Conditions are in Materials and Methods Section . The Concentration of ADP ie 3 .0 mM.
(9) . This enzyme, which ie unaffected by NaCl and is activated by RC1 and NH 4 C1, does not seem to have ,an absolute requirement for R+ or NH4+ .
Mono-
valent cation activation ie mediated by increasing the affinity of the enzyme for PEP as wall as increasing Vim .
RC1 i.e a 2 fold greatnr stimulator than
NH 4C1, but NH 4 C1 activation is fully active at a concentration where RC1 has little or no activating effect .
Pyruvate kinases from a variety of sources
are affected by monovalent cations .
Erythrocyte pyruvate kinase was found to
be activated by RC1 and NH 4C1 (17) .
Mouse liver pyruvate kinase has been re-
ported to require a monovalent cation with NH4 being more efficient than K+ (10) .
Yeast pyruvate kinase is strongly activated by R+ and NH4 with R+ having
twice the activating potential of NHy (11) .
Both rabbit muscle and Ehrlich
Vol . 10, No . 20
Regulatory Properties ad Pyruvate Kiva,se
1189
FIG . 5 Effect of Inhibitors on ADP Saturation of Pyruvate Kinase Activity . Assay Conditions are in Materials and Methods Section . The Concentration of PEP is 3 .0 mM . aecities calls have an absolute requíremeat for R+ and NH4 both of which are equally affective (18) . FDP activation of WI-38 pyruvate kinase causes not only an increase inth~ mazimal velocity and the affinity of the enzyme for PEP but also changes the eigmoidal type PEP saturation curve to a hyperbolic type curve .
A similar
type of activation has been reported for pyruvate kinase frac rat liver (6,7,8) rat kidney (7), human erythrocytes (9), and mouse liver (10) . vats kinass FDP lowers the apparent
For yeast pyru-
for PEP and transforme the eigmoidal
m
type curve to hyperbolic, but the mammal velocity is unchanged (11) .
E . cola
pyruvate kinase ie activated by FDP, but the PEP apparent Km and aigmoidal type curves are not altered (12) . Inhibition of enzyme activity by ATP sad Iralanine is strictly noncompetitive with respect to both substrates . for rat liver pyruvate kinase (5,8) .
Comparable studies have been made
ATP inhibition, reported to be 40-60x,
1170
Regulatory Properties of Pyruvate Kina.ee
Vol. 10, No. 20
was mixed type inhibition with respect to ADP and noncompetitive with respect to PEP (8) .
Also, L-alanine inhibition was found to be competitive with ie-
spect to PEP (5) . The possible physiological effect on glycolyais of FDP activation and ATP inhibition of pyruvate kinase is straightforward .
FDP activation of pyruvate
kinase serves as a forward feed type of activator which could stimulate a sluggish glycolyais when FDP levels increase above their steady state levels . ATP inhibition is easily integrated with the Atkinson adenylate hypothesis of the control of glycolyais (19) sad can ba interpreted as one of the adeaylate control points involved in the overall regulation of ATP formation . Eves though glucose is primarily converted to lactate, measurable quantitie~ of glycogen have been reported in WI-38 cells which were approaching the end of their log phase of growth (3) .
Probably, the majority of the gly
cogen is produced directly from the glucose in the tissue culture media .
How-
ever, the potential for these cells to produce glycogen from PEP may not have been lost as a result of these cells adapting to tissue culture .
In order
for WI-38 to produce glycogen from PEP, pyruvate kinase must be strongly inhibited to prevent channeling of PEP in an opposing direction to pyruvate . Such a condition could be created separately or together by ATP and L-elanine inhibition of pyruvate kinase . References 1.
Hayflick, L ., Exp . Cell Ree ., }~, 614 (1965) .
2.
Hayflick, L . and Moorehead, P . S ., Ezp . Cell Res ., ~,~, 585 (1961) .
3.
Cristofalo, V . J . and Rritchevaty, David, J . Call . Phyeiol ., ~, 125 (1966) .
4.
Runaway, Jr ., George A . sad Smith, E . C ., Aan . Okla . Aced . Sci ., ~, 84 (1970) .
5.
Weber, G ., Lea, M . A ., Stamm, N . B ., Advances in Enzyme Regulation , ¢, 101 (1968) .
6.
Bailey, E ., Stripe, F ., and Taylor, C . B ., Biochem. J ., ~, 427 (1968) .
7.
Llorente, P ., Marco, R ., and Sola, A ., Eur . J . Biochem ., ~, 45 (1970) .
Vol . 10, No. 20
Regulatory Properties ad Pyruvate Einase
1171
8.
Weber, G., Lea, M. A., Coavery, H. J. H., and Stmm, N. B ., Advances in Enzyme Regulation , ~,, 257 (1967) .
9.
Staal, G. E. J., Roster, J. F., Ramp . H., van Millignr-Boerema, L., and Benger, C ., Biochim . Biophye . Acta , 227, 86 (1971) .
10 .
Carainati, H., DaAsua, L. J ., Reco~o, E., Passeron, J., snd Rosengurt, E., J. Biol . Ch®., ~, 3051 (1968) .
11 .
Hunsley, J. R. and Suelter, C. H ., J . Biol . Chem ., ~,, 4819 (1969) .
12 :
Maeba, P. and Sanwal, B . D., J . Biol . Chem ., ~,, 448 (1968) .
13 .
Shook, C . E . and Bozer, G. E ., Cascar Ree ., ~, 709 (1964) .
14 .
Itahaka, R. F. aad Gill, D. M., Anal . Biochem., ~, 401 (1964) .
15 .
Atkinaon, D . E ., Hathaway, J . A., and Smith, E. C., J . Biol . Chem ., ~, 2682 (1965) .
16 .
Levnille, G. A., Comp . Biochem. Physiol., ~, 773 (1969) .
17 .
Ibenn, R. H ., Schiller, R. N., Venn-Watson, E . A., Arch . Bioch~. Biophys ., ,~,, 583 (1968) .
18 .
Bygrave, F. J ., Bioch®. J., ~, 488 (1966) .
Pergamon Press
REGtTLATORY PROPERTIES OF PYRUVATE KINASE IN HDMAN CELLS GROWN IN TISSUE CULTURE George A. Dunaway, Jr . and Sddie C. Smith Department of Chemistry, University of Oklahoma, Norman, Oklahoma
73069
(Received 8 July 1971; in final form 22 September 1971) Summary Pyruvate kinase (E . C . 2.7 .1 .40) from human embryonic lung tissue, WI-38, has a pA optimum range of 7.0 to 7.2 . This sesame was found to be activated 2.1, 5.0 and 2.5 fold by fructose-l,6 diphoephate, &C1, and NHyCl, respectively, and unaffected by NaCl . Eves though the degree of activation by these effectors is different, they all cause an increase in maximal velocity and increase the affinity of the enzyme for phoephoenolpyruvate . ATP and L-alanine were found to inhibit maaímal velocity by 40X and 70X, respectively, and is a non-competitive meaner with respect to both substrates . Speculation is offered on the physiological effect of these modulators on glycolgsis and glyconeogeaesis . Introduction The cells used in this atudq were the cell strain, WI-38, which were originally obtained from human embryonic lung tissue (1) .
It has been shown
that WI-38 maintains a stable diploid karyotype and ezhibita a limited life span (1,2) .
The glucose flux for WI-38 has been determined by several in-
vestigators, and their results indicate that the major fate of glucose is lactate (3,4) . Regulatory properties of pyruvate kinase (E . C . 2.7 .1 .40) have bean determined for rat liver (5,6,7,8), human erythrocytes (9), mouse liver (10), yeast (11), and Escherichia cola (12) .
Such studies concerning regulatioa of py
ruvate kinase in human calls grows in tissue culture have not bees reported . Thus, the objective of this paper ie to preeeat the results of work performed to determine the regulation of WI-38 pyruvate kinase by small molecular weight effectora ía order to speculate on Chair contribution to glycolytic regulation and to creating conditions conducive to glyconeogeaesis .
1183
1164
Regulatory Properties ad Pyruvate Kinase
Vol. 10, No. 20
Materials and Methode Original starter cultures of WI-38 were obtained from Dr . Paul F . Rruse Jr .
Maintenance and harvest of cells were as previously described (4) .
Har-
vested cells were sonicated at maximum cavitation for 2 min . i n O .OSM tris buffer pH 7 .5 and then cooled in a salt-ice bath from a maximum temperature of 20°C to 0°C .
The supernatant fluid resulting from centrifugation at 27,000
x g for 20 min . was dialyzed overnight against 0 .05M trie buffer pH 7 .5 .
This
dialyzed solution, which was used in the study of pyruvate kinasa, contained insignificant ATPase and myokiaase activity .
The dialysis tubing was boiled
in water for 15 min . before being used for dialysis . The pyruvate kinasa activity was measured by a slightly modified method of Shonk and Boxer (13) .
The assay solution (3 .0 ml) contained 150 umoles
tris pH 7 .5, 30 umoles MgC12 , 0 .9 umolas NADH, an excess of pre-dialyzed lactate dehydrogenase, and 9 umolas of phoaphoenolpyruvate (PEP) and/or 9 umolea of ADP .
Both substrates were maintained at 3 .0 mM unless substrate
saturations curves were determined .
The reaction was started with supernatant
fluid and the reaction velocity was followed by measuring the decrease in abeorbance at 340 nm at room temperature . Protein concentration was measured using a modified biuret method (14) . The pH curve was determined using tris and imidazole buffers, and the pH was measured before and after assaying using the expanded scale of a Corning pH meter .
All substrates and lactate dehydrogenase were purchased from Sigma
Chemical Co ., St . Louis, Mo . and used without further purification . Results Effect of pH .
The pyruvate kinase activity had an optimal pH range of
7 .0 to 7 .2 (see Fig . 1) .
The activity decreased to zero at pH 5 .5 and decreased
to 20x mammal velocity at pH 9 .5 .
Examination of the overlapping points in-
dicated that the activity is the same in imidazole or tris buffers . Effect of Activators on Pyruvata Kinase Activity . and NaCl on pyruvate kinase activity ie shown in Fig . 2 .
The effect of KC1, NH4 C1, NaCl had no effect up
Vol. 10, No. 20
Regulatory Properties od Pyruvate Kinase
1185
to a concentration of 150 mM, while NH 4 C1 displayed a mazímum activation of approximately 2 .5 fold at 1 mM .
However, at a concentration of 100 mM, NH4C1
inhibited maximum activity by 20Z, an inhibition which could ba a pH effect . KC1, which was the best activator,
increased activity by 5 fold .
RC1 activa-
tion was detected only above 1 mM with full activation occurring at 40 mM .
~vm
pH Profile of Pyruvate &mass No concentration of RC1 studied inhibited eazymn activity .
Also, 1.0 mM FDP
increased pyruvate kinase maximal velocity by approximately 2 .1 fold (see Fig . 3) . Ia the abéanca of them activators (Fig . 3)
the PEP saturation curve was aigmoidal
in nature with as apparent Rm for PSP of 0 .3 mM .
Data in the farm of
a Hill plot (15), yielded as iataraction coefficient of 2 which is indicative of interactions betaraea PSP biadiag sites and thus verifying the sigmoidal nature of the saturation curve .
However, in addition to increasing the maximal
Regulatory Properties ad Pyruvate Kinase
1188
Vol. 10, No . 20
W F
Effect of Monovalent Cations on Pyruvate Kinase Activity . Away Conditions are in Materials and Mathoda Section . Represents the Activity of Pyruvate Rinaee in the Absence of NaCl, RC1 and NH 4C1 . velocity 1 mM FDP, 50 mM NH 4 C1, or RC1 decreased the PEP apparent Km to 0 .15 mM, 0 .10 mM, and 0 .10 mlS, respectively .
As a consequence of the effect of
these activators the PEP saturation curve (Fig . 3) was transformed to a hyperbolic type curve characteristic of Michaelis-Meaten type curves and the PEP biding site interactions decreased (the interaction coefficient becomes 1) . Effect of Inhibitors on PEP Saturation .
The effect of 1 .0 mM L-alanine
and ATP on the PEP saturation curve is shown in Fig . 4 .
These data indicated
that L-alanine (70x inhibitioc~ was a more potent inhibitor than ATP (40Z in hibition) .
The apparent Km was not altered by either inhibitor which implied
that both inhibitors are non-competitive type inhibitors .
The interaction co-
efficient of 2 was unchanged and thus, the aigmoidal nature of the saturation curves was not altered . Effect of Inhibitors on ADP Saturation . on ADP saturation ie ehwn in Fig . 5 .
The effect of ATP and L-alanine
In the absence of inhibitors the ADP
Vol . 10, No. 20
Regulatory Properties ad Pyruvate Einase
1187
w E
P E P (mI~
nlc . 3 Effect of Activators on PEP Saturation of Pyruvate Rinses Activity . Assay Conditions are in Materials aad Methods Section. The Concentration of ADP is 3 .0 mM . apparent Rm was 0.38 mM, aad the ADP saturation curve was éigmoidal with the ADP binding site interactioa coefficient being 2.
ATP and Iralanine the apparent competitive typa inhibition .
m
In the presence of 1.0 mM
was unchanged which wee. indicative of non-
Also, the ADP saturation curve in the presence
of these inhibitors remained sigmoidal as well as the Interaction coefficient for ADP binding remaiaiag unaltered at 2. Discussion
The apparent Rm for PEP for WI-38 pyruvate kinase (0 .3 mM) compares with that of rat liver (5,6), pig liver (6), rat adipose tissue (16), and humas erythrocyte (9) pyruvate kinase .
The apparent
m
for ADP for wI-38 pynivate
kíaase (0 .38 mM) ie of the same order of magnitude as that for human erythrocytes
1188
Regulatory Properties ~ Pyruvate Kinase
Vol. 10, No. 20
FIG . 4 Effect of Inhibitors on PEP Saturation of Pyruvatn Kinase Activity . Assay Conditions are in Materials and Methods Section . The Concentration of ADP ie 3 .0 mM.
(9) . This enzyme, which ie unaffected by NaCl and is activated by RC1 and NH 4 C1, does not seem to have ,an absolute requirement for R+ or NH4+ .
Mono-
valent cation activation ie mediated by increasing the affinity of the enzyme for PEP as wall as increasing Vim .
RC1 i.e a 2 fold greatnr stimulator than
NH 4C1, but NH 4 C1 activation is fully active at a concentration where RC1 has little or no activating effect .
Pyruvate kinases from a variety of sources
are affected by monovalent cations .
Erythrocyte pyruvate kinase was found to
be activated by RC1 and NH 4C1 (17) .
Mouse liver pyruvate kinase has been re-
ported to require a monovalent cation with NH4 being more efficient than K+ (10) .
Yeast pyruvate kinase is strongly activated by R+ and NH4 with R+ having
twice the activating potential of NHy (11) .
Both rabbit muscle and Ehrlich
Vol . 10, No . 20
Regulatory Properties ad Pyruvate Kiva,se
1189
FIG . 5 Effect of Inhibitors on ADP Saturation of Pyruvate Kinase Activity . Assay Conditions are in Materials and Methods Section . The Concentration of PEP is 3 .0 mM . aecities calls have an absolute requíremeat for R+ and NH4 both of which are equally affective (18) . FDP activation of WI-38 pyruvate kinase causes not only an increase inth~ mazimal velocity and the affinity of the enzyme for PEP but also changes the eigmoidal type PEP saturation curve to a hyperbolic type curve .
A similar
type of activation has been reported for pyruvate kinase frac rat liver (6,7,8) rat kidney (7), human erythrocytes (9), and mouse liver (10) . vats kinass FDP lowers the apparent
For yeast pyru-
for PEP and transforme the eigmoidal
m
type curve to hyperbolic, but the mammal velocity is unchanged (11) .
E . cola
pyruvate kinase ie activated by FDP, but the PEP apparent Km and aigmoidal type curves are not altered (12) . Inhibition of enzyme activity by ATP sad Iralanine is strictly noncompetitive with respect to both substrates . for rat liver pyruvate kinase (5,8) .
Comparable studies have been made
ATP inhibition, reported to be 40-60x,
1170
Regulatory Properties of Pyruvate Kina.ee
Vol. 10, No. 20
was mixed type inhibition with respect to ADP and noncompetitive with respect to PEP (8) .
Also, L-alanine inhibition was found to be competitive with ie-
spect to PEP (5) . The possible physiological effect on glycolyais of FDP activation and ATP inhibition of pyruvate kinase is straightforward .
FDP activation of pyruvate
kinase serves as a forward feed type of activator which could stimulate a sluggish glycolyais when FDP levels increase above their steady state levels . ATP inhibition is easily integrated with the Atkinson adenylate hypothesis of the control of glycolyais (19) sad can ba interpreted as one of the adeaylate control points involved in the overall regulation of ATP formation . Eves though glucose is primarily converted to lactate, measurable quantitie~ of glycogen have been reported in WI-38 cells which were approaching the end of their log phase of growth (3) .
Probably, the majority of the gly
cogen is produced directly from the glucose in the tissue culture media .
How-
ever, the potential for these cells to produce glycogen from PEP may not have been lost as a result of these cells adapting to tissue culture .
In order
for WI-38 to produce glycogen from PEP, pyruvate kinase must be strongly inhibited to prevent channeling of PEP in an opposing direction to pyruvate . Such a condition could be created separately or together by ATP and L-elanine inhibition of pyruvate kinase . References 1.
Hayflick, L ., Exp . Cell Ree ., }~, 614 (1965) .
2.
Hayflick, L . and Moorehead, P . S ., Ezp . Cell Res ., ~,~, 585 (1961) .
3.
Cristofalo, V . J . and Rritchevaty, David, J . Call . Phyeiol ., ~, 125 (1966) .
4.
Runaway, Jr ., George A . sad Smith, E . C ., Aan . Okla . Aced . Sci ., ~, 84 (1970) .
5.
Weber, G ., Lea, M . A ., Stamm, N . B ., Advances in Enzyme Regulation , ¢, 101 (1968) .
6.
Bailey, E ., Stripe, F ., and Taylor, C . B ., Biochem. J ., ~, 427 (1968) .
7.
Llorente, P ., Marco, R ., and Sola, A ., Eur . J . Biochem ., ~, 45 (1970) .
Vol . 10, No. 20
Regulatory Properties ad Pyruvate Einase
1171
8.
Weber, G., Lea, M. A., Coavery, H. J. H., and Stmm, N. B ., Advances in Enzyme Regulation , ~,, 257 (1967) .
9.
Staal, G. E. J., Roster, J. F., Ramp . H., van Millignr-Boerema, L., and Benger, C ., Biochim . Biophye . Acta , 227, 86 (1971) .
10 .
Carainati, H., DaAsua, L. J ., Reco~o, E., Passeron, J., snd Rosengurt, E., J. Biol . Ch®., ~, 3051 (1968) .
11 .
Hunsley, J. R. and Suelter, C. H ., J . Biol . Chem ., ~,, 4819 (1969) .
12 :
Maeba, P. and Sanwal, B . D., J . Biol . Chem ., ~,, 448 (1968) .
13 .
Shook, C . E . and Bozer, G. E ., Cascar Ree ., ~, 709 (1964) .
14 .
Itahaka, R. F. aad Gill, D. M., Anal . Biochem., ~, 401 (1964) .
15 .
Atkinaon, D . E ., Hathaway, J . A., and Smith, E. C., J . Biol . Chem ., ~, 2682 (1965) .
16 .
Levnille, G. A., Comp . Biochem. Physiol., ~, 773 (1969) .
17 .
Ibenn, R. H ., Schiller, R. N., Venn-Watson, E . A., Arch . Bioch~. Biophys ., ,~,, 583 (1968) .
18 .
Bygrave, F. J ., Bioch®. J., ~, 488 (1966) .