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Effect of fluoroacetate poisoning on the glycogen content of rat heart and skeletal muscle
Life Sciences Vol . 7, Part II, pp . 847-854, 1968 . Printed in Great Britain .
Pergamon Press
EFFI?CT OF FLTi0R0ACETATE POISONIN(: ON Tf1F CLYC(~f:FAT CONTENT OF RAT HEART AN11 SK.FT.FTAL MTf~CLT? Héctor M . C>odoy, Elida V . CiRnoli and .iosé A . CaaLro Laboratorio de (Mimics Bio-Toxicolôgica, Institi~to de Investigaciones Cientificas y Técnicas de las FP .AA . 3 de Fehrero 1250, San Martin, Pcia . Buenos Aires, Argentina
(Received 22 April 1968 ; in final form 7 June 1968) it was found that in alloxan diabetic or starved rats heart P,lycogen content is increased (1,2,3) . This effect is reversed by anoxia (4) . These findings were explained on the basis of the interplay of metabolic factors which are known to affect the rate of g: ;~colysia at the phosphofructokinase (PFK) stet (4,5,6,7,8) . It is known that fluoroacetate (FAc) poisoning in the intact raf produces a diabetic-like syndrome (9), while at the cellular level there occur what we should call "anoxic-type" effects, such as creatine phosphate and ATP breakdown, and increases of inorganic phosphate, APp and AMP levels (10,11,12) . 7n other words, FAc is unique in producing simultaneously two opposite effects nn carbohydrate metabolism : diabetes and anoxia . So, we thoyght that it could be interesting to observe how those comnetinp, effects are reflected in the glycogen content in heart and skeletal muscle, especially because the known time-dependence of the process of lethal
synthesis
(10) anticipated the possibility of changing situations when time studies were made . Accordingly, in our experiments we found a two-staP,e variation in plyco~en content in heart, showing an initial sudden decrease to almost zero levels, followed by a slow increase after 6 hours of intoxication . 7n contrast, skeletal muscle does not show pronounced changes in glycogen levels . Materials and Methods In most experiments 24 hr . fasted male albino rats, weighin(t from 220 to 340 g were used, with the exception of the experiments with adrenalectomized rats,
847
848
EFFECT OF FLUOROACETATE
Vol . 7, No. 18
in which the weight of the animals was between 120 and 7.40 g . Bilateral adrenalectamy was performed in our laboratory ; the glands were submitted to a careful visual inspection to ensure the completeness of the extraction . After operation the adrenalectomized rats were kept under saline-glu core treatment during 3 days ; on the next day they received saline alone . A saline solution of FAc was injected intraperitoneally at a dose of 6 mg/Kq to previously anesthetized rats . The controls received an equal amount of saline . All the animals were kept under Nembutal anesthesia during the first 6 hr . after FAc injection, to prevent the appearance of the characteristic FAc convulsive state ; the initial dose was 30 mg/Kg, but successive injections were employed when necessary . In the 12 and 18 hr . experiments another injection was given 30 minutes before sacrifice . Adrenalectomized rats were ,treated similarly but using half of the dose of Nembutal employed in normal animals . 7n a].1 cases the control rats followed the same treatment . Blood samples for sugar analysis were obtained from the portal vein and received in heparinized tubes . A sample of heart tissue was immediately cut and divided in two portions . One of diem was rapidly immersed in a pre weighed vial containing 337 K071 and used for glycogen analysis (13) . The other one was tutted further in smaller pieces and placed in a pre-weighed Potter-Elvehjem homogenizer containing some cold 12 .52 trichloroacetic acid (TCA) . After the sample was weighed and homogenized, the volume was adjusted with cold TCA and centrifuged at 3000 rpm for 10 minutes . Citrate was determined in a portion of the supernatant according to Natelaon et al (14) . Hind-leq muscle was processed aimilarly . Results In Fig . 1 (A, B and C) are plotted heart glycogen and citrate content as well as blood sugar levels as a function of the time after FAc injection . Ae can he seen, citrate rises very sharply, reaching at 30 minutes a 2-fold and at 60 minutes a 3 to 4-fold increased level . Inversely, glycogen levels rapidly fell
Vol. 7, No . 18
1500
EFFECT OF FT;UOROACETATE
CITRATE FA c
849
A
300
G 07 V
200
S.
m~ 100 th
FIG . 1 Heart citrate (A), glycogen (B) and blood Rlucose (C) levels after FAç injection (i .p . 6 mg/Kg) . Each point is the mean value for at least 6 animals in the FAc curve, and 3 animals in the control curve . Increases of citrate levels were significative from 0.5 hr . on (p<0 .001) . Glycogen levels were significantly decreased (p<0 .001) during the first 6 hr ., while at lA hr . the differences were not eignificative (p>0 .05), with respect to the untreated controls . Blood sugar levels were significantly increased at 12 and 18 hr . (p<0 .001), All data are means ± S .D .
850
EFFECT OF FLUOROACETATE
Vol . 7, No . 16
to near-zero values and then they remained unaltered during the first 6 hours, increasing later to almost fasting control values, it is interesting, to remark that the increases in plvcogen content are produced simultaneously with the rises in blood sugar and also that citrate levels reach very early their maximum values (4 to 6 hr . after FAc in jjection) . In Fig . 2 (A and ß) are shown the changes in the same Parameters observed in skeletal muscle . Sign ificative increases in citrate content were observed after 6 hr . of FAc injection ; although they were less Pronounced than those ohser200
A
CITRATE FAc
y 0!
3
100
GLYCOGEN
û 3
B ."
8-
"
","
coNTROL .-
""
FA c
4
4 Hours
8
12
A FEer FAc
16 InjecEi,on
Fir, . z Skeletal muscle citrate (A) and R1ycoQen (R) levels after FAc injection (i .P ., 6 mß/Kg) . Fach point is the mean value for at least 6 animals in the FAc curve and 3 animals in the control curve . hardly significative changes in plvcoRen content were found only at 6 hr . after FAc (n<0 .02) . Citrate increases were siRnificative (p<0 .001) after 6 hr . of poisonin~t . All data are means + S .D .
Vol . 7, No . 1 6
EFFECT OF FLUOROACETATE
851
ved in cardiac tissue . Changes in glycogen content in muscle were small and hardly significative . In adrenalectomized rats, cardiac citrate increases and glycogen breakdown 1 hr . after FAc injection were produced (see Table 1), whereas skeletal muscle citrate and glycogen were not appreciably altered . These results are similar to those obtained with normal poisoned animals . TARTE
I
Citrate and glycogen content in heart and skeletal muscle of adrenalectomized rats 1 hr . after FAc administration Glycogen mg/q wet tissue
Treatment
}leart
Muscle
Adrenalectanized
4 .3 t 1 .7
4 .1 ± 0,6
Adrenalect . + FAc
0,0*
4 .4 t 1 .0
Citrate Ng/g wet tissue }Ieart 63 ± 6 508 ± 40*
wtuscle 88 .5 ± 15 29 t 2
Each value is the mean of 5 experiments ± S,D . *p c 0 .001 Discussion It is known that FAc is metabolised in the tissues to fluorocitrate, which ie an inhib~tor of aconitase . As a conseavence, citric acid cycle is blocked and citrate accumulates (10) . Furthermore, citric acid cycle inhibition results i.n decreased energy reserves of the cell in the form of ATp and creatine phosphate, and increased levels of inorganic phosphate, AMP and APP (1(1,1] .,12,15), These facts make FAc an useful tool for studying the control . of g).~icose metahol .ism in tissue, since it is believed that citrate, as well as AT?', AMf and inorganic phosphate might act effectively
in viyo
as reeulators of the rate of s~hicose
utilization at the PFK step (6,8,16,17) . The PFK inhibition by citrate was supposed to he responsible fir the heart glycogen increases observed in al.loxan diabetic or starved rats (7) . That inhihition was reversed by anoxia, presiunahly by increasen¢ the AMP and inorganic nhos-
852
EFFECT OF FLUOROACETATE
Vol. 7, No . 18
phate levels (S,lA) . So, if this hypothesis is correct, similar effects on cardisc glycogen levels should be expected in the case of FAc poisoning . Moreover, even the interplay between citrate and anoxia could he nrobably observed . In effect, our results in Fiq . 1 (A, B and C) show that there is an initial rapid depletion of the heart glycogen followed several hours later by a marked glycogen accumulation, which is accompanied by a simultaneous increase in blood sugar levels . These changes occur in spite of the fact that increased citrate levels were observed since the very beginning (they reach a maximum at 4-6 hr . and remain almost unaltered therefrom) . Our experiments strongly suggest that in the first period after FAc poisonine there is a marked activation of rat-heart PFR . The fact that PFK is activated in the presence of high citrate levels might imp ].y that the citrate inhihi tion is being antagonized by high levels of inorganic phosphate and AMP or alternatively that low levels of ATP are present . That is, the situation could he very similar to the one described by Williamson in his experiments on changes in PFK during FAc action (15), where he found that PFR was inhibited in hearts from FAc-treated rats but activated in perfused hearts treated
in vitro
with FAc,
despite a large rise of the citrate content in both cases . In the set of experimenta in which PFK was found activated, simultaneous increases in inorganic phosphate, AMP and ADP were observed, which were taken as suggesting that inorganic phosphate and AMP might act effectively
in vivc
as antagonists of the citrate
inhibition of PFK . In agreement with this possibility, Williamson also showed that in perfused rat heart it is possible to reverse partially the inhibition with anoxia . In order to exclude the possibility that the initial decrease in glvcoqen content were due to a sudden adrenaline discharge as could he suspected after the suggestions made by Annision et al (19), we checked if the adrenalectomized rat showed the same decrease in the heart glycogen content 1 hr . after FAc administration . 7n agreement with our previous assumption, we found that in the
Vol . 7, No . 1 8
EFFECT OF FLUOROACETATE
853
adrenalectomized rat the heart glycogen content fell very rapidly, while citrate levels were highly elevated 1 hr . after FAc, like in the treated normal rat (see Table I) . In the second stage of the intoxication the heart glvcopen levels, after remaining several hours at almost zero values, slow].y increase to the original fasting control levels (see Fig . 1-B) . This situation should correspond to a decreased PFK activity, which might fie due to changes in the ratio ATP to AMP + inorganic phosphate, since total citrate remained unchanged after the third hour of the intoxication . Alternatively, a slow process of diffusion of citrate from mitochondria to the cytoplasmic compartment might offer an explanation for the delay in PFR inhibition . Another possibility would be that other factor or factors, not being those mentioned above, had a regulatory action on PFK activity . At the present time, an evaluation of these different possibilities is in progress in our laboratory . Skeletal muscle citrate levels were not affected during the first 6 hr . after FAc injection, whereas glycogen content was only slightly decreased . These results are in contrast with those obtained in cardiac tissue . However, citrate increases were observed after 6 hr, of intoxication, and it map be of interest to remark that this change is paralleled fiy the rise in blood sugar levels (see Fig . 2-A and Fig . 1-C) . A clear-cut interpretation of these findings can net be drawn on the basis of our experiments . Acknowled~tement We are indebted to Miss M .C . Villarruel for skillful technical assigtance . Refercncea 1.
R.W . LA('.RFY, C .A . BUNDE and L .C . HARRIS, Amer . J . Physiol . 145, 470 (1946) .
2.
R .H . BOWMAN, Amer . J . Phvaiol . 197, 1017 (1959) .
854
Vol. 7, No. 16
EFFECT OF FLUOROACETATE
3.
A .A . Ii.LiNGWORTH and ,T,A . RTiSSRl.T., Endocrinology 48, 423 (1951) .
4.
E .A . NRT~TSHOi .MT? and P,j,
5.
E .A . NFWSHOT.ME and P .J . RANDLE, Riochem . J . 93, 641 (] .964) .
6.
D.M . REGEN, W.W . RAVIS, H .R . MORGAN and C. .R . PARK, J . Aial . . Chem . 239, 43 (1964) .
7.
E .A . NEWSHOLME, P .,T . RANT)T.R and R .T.. MANCHESTER, Nature 193, 270 (1962) .
8.
P .B . GARLAND and P.J, RANIIT.R, Aiochem . J . 93 , 678 (1964) .
9.
F.L . ENGEL, K. HEWSON and B .T . COLE, Amer . J . Phvsiol . 179,
RANOLR, Aiochem, .T . RO, 65 .5 (196]) .
324 (1Q54) .
10 .
R.A . PETERS, Advances in Ensymol . 18, 113 (1957) .
11 .
P. BUFFA, Chem . and Ind ., p . 1080 (1952) .
12 .
G . FAWAZ, Naunyn-SchmiedeberQ Arch . F.xptl . Pathol . Pharmakol ., (1956) .
13 .
C .R . KRISMAN, Analvt . Aiochem . 4,
14,
S . NATELSON, J.B . PINCTTS and ,T .R . T.IK:OBOV, J . Riol . ('.hem . 17~5, 745 (1948) .
15 .
J .R . WILLIAMSON, J . Biol . Chem . 24~2, 4476 (1967) .
16 .
P .B . GARLAND, P .J . RANDT.R and E .A . NEWSHOLME, Nature 2~, l69 (1963) .
17 .
O.H . LOWRY and J .V . PASSONNF.ATT, J . Riol, Chem . 2s
18 .
2 0, R3 (1961) . D.M . REGEN, W,W . DAVIS and H. .R . MORf*AN, Fed . proc . y
19 .
E.F . ANNISON, K . NihL, D. LINT1SAV and R .A . PRTRRS, J . ('.omp . Pathol . and Therap . 70, 145 (1960) .
228,
377
17 (1962) .
31 (1964),
Pergamon Press
EFFI?CT OF FLTi0R0ACETATE POISONIN(: ON Tf1F CLYC(~f:FAT CONTENT OF RAT HEART AN11 SK.FT.FTAL MTf~CLT? Héctor M . C>odoy, Elida V . CiRnoli and .iosé A . CaaLro Laboratorio de (Mimics Bio-Toxicolôgica, Institi~to de Investigaciones Cientificas y Técnicas de las FP .AA . 3 de Fehrero 1250, San Martin, Pcia . Buenos Aires, Argentina
(Received 22 April 1968 ; in final form 7 June 1968) it was found that in alloxan diabetic or starved rats heart P,lycogen content is increased (1,2,3) . This effect is reversed by anoxia (4) . These findings were explained on the basis of the interplay of metabolic factors which are known to affect the rate of g: ;~colysia at the phosphofructokinase (PFK) stet (4,5,6,7,8) . It is known that fluoroacetate (FAc) poisoning in the intact raf produces a diabetic-like syndrome (9), while at the cellular level there occur what we should call "anoxic-type" effects, such as creatine phosphate and ATP breakdown, and increases of inorganic phosphate, APp and AMP levels (10,11,12) . 7n other words, FAc is unique in producing simultaneously two opposite effects nn carbohydrate metabolism : diabetes and anoxia . So, we thoyght that it could be interesting to observe how those comnetinp, effects are reflected in the glycogen content in heart and skeletal muscle, especially because the known time-dependence of the process of lethal
synthesis
(10) anticipated the possibility of changing situations when time studies were made . Accordingly, in our experiments we found a two-staP,e variation in plyco~en content in heart, showing an initial sudden decrease to almost zero levels, followed by a slow increase after 6 hours of intoxication . 7n contrast, skeletal muscle does not show pronounced changes in glycogen levels . Materials and Methods In most experiments 24 hr . fasted male albino rats, weighin(t from 220 to 340 g were used, with the exception of the experiments with adrenalectomized rats,
847
848
EFFECT OF FLUOROACETATE
Vol . 7, No. 18
in which the weight of the animals was between 120 and 7.40 g . Bilateral adrenalectamy was performed in our laboratory ; the glands were submitted to a careful visual inspection to ensure the completeness of the extraction . After operation the adrenalectomized rats were kept under saline-glu core treatment during 3 days ; on the next day they received saline alone . A saline solution of FAc was injected intraperitoneally at a dose of 6 mg/Kq to previously anesthetized rats . The controls received an equal amount of saline . All the animals were kept under Nembutal anesthesia during the first 6 hr . after FAc injection, to prevent the appearance of the characteristic FAc convulsive state ; the initial dose was 30 mg/Kg, but successive injections were employed when necessary . In the 12 and 18 hr . experiments another injection was given 30 minutes before sacrifice . Adrenalectomized rats were ,treated similarly but using half of the dose of Nembutal employed in normal animals . 7n a].1 cases the control rats followed the same treatment . Blood samples for sugar analysis were obtained from the portal vein and received in heparinized tubes . A sample of heart tissue was immediately cut and divided in two portions . One of diem was rapidly immersed in a pre weighed vial containing 337 K071 and used for glycogen analysis (13) . The other one was tutted further in smaller pieces and placed in a pre-weighed Potter-Elvehjem homogenizer containing some cold 12 .52 trichloroacetic acid (TCA) . After the sample was weighed and homogenized, the volume was adjusted with cold TCA and centrifuged at 3000 rpm for 10 minutes . Citrate was determined in a portion of the supernatant according to Natelaon et al (14) . Hind-leq muscle was processed aimilarly . Results In Fig . 1 (A, B and C) are plotted heart glycogen and citrate content as well as blood sugar levels as a function of the time after FAc injection . Ae can he seen, citrate rises very sharply, reaching at 30 minutes a 2-fold and at 60 minutes a 3 to 4-fold increased level . Inversely, glycogen levels rapidly fell
Vol. 7, No . 18
1500
EFFECT OF FT;UOROACETATE
CITRATE FA c
849
A
300
G 07 V
200
S.
m~ 100 th
FIG . 1 Heart citrate (A), glycogen (B) and blood Rlucose (C) levels after FAç injection (i .p . 6 mg/Kg) . Each point is the mean value for at least 6 animals in the FAc curve, and 3 animals in the control curve . Increases of citrate levels were significative from 0.5 hr . on (p<0 .001) . Glycogen levels were significantly decreased (p<0 .001) during the first 6 hr ., while at lA hr . the differences were not eignificative (p>0 .05), with respect to the untreated controls . Blood sugar levels were significantly increased at 12 and 18 hr . (p<0 .001), All data are means ± S .D .
850
EFFECT OF FLUOROACETATE
Vol . 7, No . 16
to near-zero values and then they remained unaltered during the first 6 hours, increasing later to almost fasting control values, it is interesting, to remark that the increases in plvcogen content are produced simultaneously with the rises in blood sugar and also that citrate levels reach very early their maximum values (4 to 6 hr . after FAc in jjection) . In Fig . 2 (A and ß) are shown the changes in the same Parameters observed in skeletal muscle . Sign ificative increases in citrate content were observed after 6 hr . of FAc injection ; although they were less Pronounced than those ohser200
A
CITRATE FAc
y 0!
3
100
GLYCOGEN
û 3
B ."
8-
"
","
coNTROL .-
""
FA c
4
4 Hours
8
12
A FEer FAc
16 InjecEi,on
Fir, . z Skeletal muscle citrate (A) and R1ycoQen (R) levels after FAc injection (i .P ., 6 mß/Kg) . Fach point is the mean value for at least 6 animals in the FAc curve and 3 animals in the control curve . hardly significative changes in plvcoRen content were found only at 6 hr . after FAc (n<0 .02) . Citrate increases were siRnificative (p<0 .001) after 6 hr . of poisonin~t . All data are means + S .D .
Vol . 7, No . 1 6
EFFECT OF FLUOROACETATE
851
ved in cardiac tissue . Changes in glycogen content in muscle were small and hardly significative . In adrenalectomized rats, cardiac citrate increases and glycogen breakdown 1 hr . after FAc injection were produced (see Table 1), whereas skeletal muscle citrate and glycogen were not appreciably altered . These results are similar to those obtained with normal poisoned animals . TARTE
I
Citrate and glycogen content in heart and skeletal muscle of adrenalectomized rats 1 hr . after FAc administration Glycogen mg/q wet tissue
Treatment
}leart
Muscle
Adrenalectanized
4 .3 t 1 .7
4 .1 ± 0,6
Adrenalect . + FAc
0,0*
4 .4 t 1 .0
Citrate Ng/g wet tissue }Ieart 63 ± 6 508 ± 40*
wtuscle 88 .5 ± 15 29 t 2
Each value is the mean of 5 experiments ± S,D . *p c 0 .001 Discussion It is known that FAc is metabolised in the tissues to fluorocitrate, which ie an inhib~tor of aconitase . As a conseavence, citric acid cycle is blocked and citrate accumulates (10) . Furthermore, citric acid cycle inhibition results i.n decreased energy reserves of the cell in the form of ATp and creatine phosphate, and increased levels of inorganic phosphate, AMP and APP (1(1,1] .,12,15), These facts make FAc an useful tool for studying the control . of g).~icose metahol .ism in tissue, since it is believed that citrate, as well as AT?', AMf and inorganic phosphate might act effectively
in viyo
as reeulators of the rate of s~hicose
utilization at the PFK step (6,8,16,17) . The PFK inhibition by citrate was supposed to he responsible fir the heart glycogen increases observed in al.loxan diabetic or starved rats (7) . That inhihition was reversed by anoxia, presiunahly by increasen¢ the AMP and inorganic nhos-
852
EFFECT OF FLUOROACETATE
Vol. 7, No . 18
phate levels (S,lA) . So, if this hypothesis is correct, similar effects on cardisc glycogen levels should be expected in the case of FAc poisoning . Moreover, even the interplay between citrate and anoxia could he nrobably observed . In effect, our results in Fiq . 1 (A, B and C) show that there is an initial rapid depletion of the heart glycogen followed several hours later by a marked glycogen accumulation, which is accompanied by a simultaneous increase in blood sugar levels . These changes occur in spite of the fact that increased citrate levels were observed since the very beginning (they reach a maximum at 4-6 hr . and remain almost unaltered therefrom) . Our experiments strongly suggest that in the first period after FAc poisonine there is a marked activation of rat-heart PFR . The fact that PFK is activated in the presence of high citrate levels might imp ].y that the citrate inhihi tion is being antagonized by high levels of inorganic phosphate and AMP or alternatively that low levels of ATP are present . That is, the situation could he very similar to the one described by Williamson in his experiments on changes in PFK during FAc action (15), where he found that PFR was inhibited in hearts from FAc-treated rats but activated in perfused hearts treated
in vitro
with FAc,
despite a large rise of the citrate content in both cases . In the set of experimenta in which PFK was found activated, simultaneous increases in inorganic phosphate, AMP and ADP were observed, which were taken as suggesting that inorganic phosphate and AMP might act effectively
in vivc
as antagonists of the citrate
inhibition of PFK . In agreement with this possibility, Williamson also showed that in perfused rat heart it is possible to reverse partially the inhibition with anoxia . In order to exclude the possibility that the initial decrease in glvcoqen content were due to a sudden adrenaline discharge as could he suspected after the suggestions made by Annision et al (19), we checked if the adrenalectomized rat showed the same decrease in the heart glycogen content 1 hr . after FAc administration . 7n agreement with our previous assumption, we found that in the
Vol . 7, No . 1 8
EFFECT OF FLUOROACETATE
853
adrenalectomized rat the heart glycogen content fell very rapidly, while citrate levels were highly elevated 1 hr . after FAc, like in the treated normal rat (see Table I) . In the second stage of the intoxication the heart glvcopen levels, after remaining several hours at almost zero values, slow].y increase to the original fasting control levels (see Fig . 1-B) . This situation should correspond to a decreased PFK activity, which might fie due to changes in the ratio ATP to AMP + inorganic phosphate, since total citrate remained unchanged after the third hour of the intoxication . Alternatively, a slow process of diffusion of citrate from mitochondria to the cytoplasmic compartment might offer an explanation for the delay in PFR inhibition . Another possibility would be that other factor or factors, not being those mentioned above, had a regulatory action on PFK activity . At the present time, an evaluation of these different possibilities is in progress in our laboratory . Skeletal muscle citrate levels were not affected during the first 6 hr . after FAc injection, whereas glycogen content was only slightly decreased . These results are in contrast with those obtained in cardiac tissue . However, citrate increases were observed after 6 hr, of intoxication, and it map be of interest to remark that this change is paralleled fiy the rise in blood sugar levels (see Fig . 2-A and Fig . 1-C) . A clear-cut interpretation of these findings can net be drawn on the basis of our experiments . Acknowled~tement We are indebted to Miss M .C . Villarruel for skillful technical assigtance . Refercncea 1.
R.W . LA('.RFY, C .A . BUNDE and L .C . HARRIS, Amer . J . Physiol . 145, 470 (1946) .
2.
R .H . BOWMAN, Amer . J . Phvaiol . 197, 1017 (1959) .
854
Vol. 7, No. 16
EFFECT OF FLUOROACETATE
3.
A .A . Ii.LiNGWORTH and ,T,A . RTiSSRl.T., Endocrinology 48, 423 (1951) .
4.
E .A . NRT~TSHOi .MT? and P,j,
5.
E .A . NFWSHOT.ME and P .J . RANDLE, Riochem . J . 93, 641 (] .964) .
6.
D.M . REGEN, W.W . RAVIS, H .R . MORGAN and C. .R . PARK, J . Aial . . Chem . 239, 43 (1964) .
7.
E .A . NEWSHOLME, P .,T . RANT)T.R and R .T.. MANCHESTER, Nature 193, 270 (1962) .
8.
P .B . GARLAND and P.J, RANIIT.R, Aiochem . J . 93 , 678 (1964) .
9.
F.L . ENGEL, K. HEWSON and B .T . COLE, Amer . J . Phvsiol . 179,
RANOLR, Aiochem, .T . RO, 65 .5 (196]) .
324 (1Q54) .
10 .
R.A . PETERS, Advances in Ensymol . 18, 113 (1957) .
11 .
P. BUFFA, Chem . and Ind ., p . 1080 (1952) .
12 .
G . FAWAZ, Naunyn-SchmiedeberQ Arch . F.xptl . Pathol . Pharmakol ., (1956) .
13 .
C .R . KRISMAN, Analvt . Aiochem . 4,
14,
S . NATELSON, J.B . PINCTTS and ,T .R . T.IK:OBOV, J . Riol . ('.hem . 17~5, 745 (1948) .
15 .
J .R . WILLIAMSON, J . Biol . Chem . 24~2, 4476 (1967) .
16 .
P .B . GARLAND, P .J . RANDT.R and E .A . NEWSHOLME, Nature 2~, l69 (1963) .
17 .
O.H . LOWRY and J .V . PASSONNF.ATT, J . Riol, Chem . 2s
18 .
2 0, R3 (1961) . D.M . REGEN, W,W . DAVIS and H. .R . MORf*AN, Fed . proc . y
19 .
E.F . ANNISON, K . NihL, D. LINT1SAV and R .A . PRTRRS, J . ('.omp . Pathol . and Therap . 70, 145 (1960) .
228,
377
17 (1962) .
31 (1964),