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A role of phosphofructokinase in pH-dependent regulation of glycolysis
31o
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
25580
A ROLE OF P H O S P H O F R U C T O K I N A S E IN p H - I ) E P E N D E N T REGULATION OF GLYCOLYSIS MICHIO uI Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido Univcrsi/3:, Sapporo (Japan)
(Received January 4th, t966
SUMMARY
i. Glycolysis in a cell-free extract from rat diaphragm was stimulated at elevated pH in the presence of higher concentrations of ATP. A similar situation was also observed in the case of intact-cell preparations of rat diaphragm. 2. It was found that the rate of inhibition of phosphofructokinase (ATP:Dfructose 6-phosphate I-phosphotransferase, EC 2.7.I.11) activity by excess NFP was strictly dependent upon the pH; at pH 7.3 an increase in ATP concentration from I to 1. 7 mM resulted in a sudden inhibition of phosphofructokinase activity, whereas at pH 7.6 such an effect was not observed until the ATP level was raised to 2 3 mM (o.o5 mM fructose 6-phosphate was employed as substrate). Consequently phosphofructokinase activity was profoundly influenced by quite a minute change in pH when larger amounts of ATP were present in the glycolytic system. 3-Since the glucose 6-phosphate which accumulated during phosphofructokinase inhibition was inhibitory to hexokinase (ATP : D-hexose 6-phosphotransferase, EC 2.7.1.1), the pH-induced stimulation of phosphofructokinase caused an exaggerated acceleration of overall glycolysis. 4. Preincubation of the cell-free extract without substrates (aging) rendered phosphofructokinase protein unresponsive both to the stimulation on raising pH and to the inhibition by ATP, It is tentatively proposed that the affinity of the inhibitory site(s) of phosphofructokinase for ATP is affected by hydrogen ion concentration and that aging causes a change of conformation of the enzyme, thus blocking the control mechanism of phosphofructokinase.
INTRODUCTION
Recently the present author has reported that the hyperglycemic action of epinephrine was abolished during alkalosis 1. This abolition was due to tile blockage of the epinephrine-induced inhibition of glucose utilization by muscle at higher wdues of body fluid pH (refs. I, 2). During the course of these studies, it was found that an increase of pH in the incubation medium resulted in a marked increase in glucose uptake by isolated rat diaphragm. It may be expected that enzymes in the metabolic chain responding to changes of environmental pH would also play a significant role in the mechanisms of hormonal control of carbohydrate metabolism. The purpose of Biochbm Biophys. Acta, ~z4 (lO0()) 3Io-3zz
pH-DEPENDENT GLYCOLYSIS
3II
the present study is to establish which enzymes or enzyme systems m a y be responsible for the apparent p H dependency of muscle glucose utilization. As will be seen, the results obtained indicate the significance of phosphofructokinase (ATP: D-fructose 6-phosphate I-phosphotransferase, EC 2.7.1.11) in the regulatory mechanisms of glycolysis. MATERIALS AND METHODS
The experiments employing the rat diaphragm preparations containing intact cells (Table I) were conducted as already descri~edl, 2. All of the data included in the present paper, other than those in Table I, were obtained in experiments with cell-free preparations of muscle glycolytic systems. These experiments were carried out as described below. Cell-free muscle extract was prepared by mincing the rat diaphragm in a m o r t a r at o ° in a medium containing 15o mM K +, 5 mM Mg2+, 12o mM CI-, 24 mM HC03-, 2 mM PO43-. The ratio of the amount of tissue to medium used was i : IO. The mince was centrifuged at 700 × g for IO min to remove the residual intact cells and nuclei. Substrate was added in a final concentration of 5 mM unless otherwise specified; NAD + (1.2 mM), nicotinamide (5.4 mM) and ATP at the concentration indicated were also added. When required, the extract was aged (preincubated without substrate) at 37 ° for a period indicated prior to the incubation. Incubation was carried out in an atmosphere of either 93 % 0 3 - 7 To COs or ioo To 03 at 22 ° or 37 ° for the period indicated. The p H of the cell-free preparation after incubation was 7.15-7.25 for an incubation in 93 % 0 3 - 7 To CO2 and 7.55-7.65 for IOO % 0 2. (As already reported, the pH of the medium buffered with 24 mM HCO 8was 7.3-7.4 in an atmosphere of 93 % 03--7 % COs (refs. I, 2), and introduction of Oo. into incubation flasks rendered the pH as high as p H 8.6. The presence of IOO o~ /o protein in the cell-free extract, however, prevents the increase of pH of the medium in the oxygenated flask.) The reaction was stopped by the addition of HC104 to a final concentration of 5 %. In order to determine glucose or 2-deoxyglucose, the deproteinized aliquot, after being neutralized, was further treated with Ba(OH)2 and Z n S Q to remove phosphorylated sugars and glycogen. Glucose was analyzed with an anthrone reagent. Colorimetric procedures were applied for the determination of 2-deoxyglucose a and lactate 4. Intermediates of the glycolytic pathway were enzymically assayed by following the change in absorbance at 340 m~ as described below: Glu-6-P, by the reduction of NADP + in the presence of glucose-6-phosphate dehydrogenase (EC 1.1.1.49); Fru-6-P, by the reduction of NADP + in the presence of glucosephosphate isomerase (EC 5.3.1,9) and glucose-6-phosphate dehydrogenase; Fru-I,6-P 2 by the oxidation of NADH in the presence of ketose-I-phosphate aldolase (EC 4.i.2.7), triosephosphate isomerase (EC 5.3.1.1) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8); triosephosphate, by the oxidation of NADH in the presence of triosephosphate isomerase and glycerol-3-phosphate dehydrogenase. The amount of the intermediates to be assayed was calculated according to the molar extinction of N A D H or N A D P H as well as from the standard curves obtained with known amounts of substrates. The following chemicals were obtained from Sigma Chemical Co.: ATP, ADP, NAD +, NADP+, 2-deoxyglucose, Glu-6-P, Fru-6-P, Fru-i,6-P2, yeast hexokinase Biochim. Biophys. Acta, 124 (1966) 31o- 322
312
~I. uI
(EC 2.7.I.~), glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, triosephosphate isomerase. Ketose-I-phosphate aldolase and glucosephosphate isomerase were prepared in this laboratory from rat skeletal muscle according to TAYLOR5 and SLEIX6. Phosphofructokinase was also prepared from rabbit skeletal muscle according to G.~'rT aND RAC~;ERv. The protein concentration in the reaction mixture was determined according to LOWRY et al. s and the metabolic activity of the cell-free extract was expressed, on the basis of mg protein, as the mean value of three or four observations. Standard deviations were calculated to substantiate the significance of the differences (or agreements) ; these are given, however, only when rather subtle differences (or agreements) are mentioned for the discussion of the results. RESULTS
Experiments with isolated rat diaphragm preparations containing intact cells In agreement with the previous report 2, glucose uptake by isolated rat quarterdiaphragm was markedly stimulated when the incubation was conducted at a higher p H (Table I). Prevention of this stimulation by the addition of monoiodoacetate, together with the marked stimulation of lactate production on raising pH, indicated that the glycolytic pathway was activated on raising the pH of the medium. Similar results were reported by GEVERS AN[) DAWDLE9. Induction of anaerobiosis abolished the stimulation of glycolysis by pH, suggesting that events somewhat related to the oxidative process are also involved in the pH-stimulated glucose uptake and lactate production by muscle tissue. TABLE I GLUCOSE PHRAGM
UPTAKE
AND
LACTATE
PRODUCTION
BY THE
INTACT-CELL
PREPARATION
FROM
RAT
DIA-
INCUBATED AT p H 7"4 OR 8.O
Q u a r t e r - d i a p h r a g m s from fasted r a t s were i n c u b a t e d in i ml of K r e b s R i n g e r b i c a r b o n a t e s o l u t i o n a t 37 ° for 9o m i n in an a t m o s p h e r e of 93 % O 2 - 7 % COe (pH 7.4) or 99.5 o//o 02 o.5 %. CO.2 (pH 8.o). O~ was r e p l a c e d b y N~ in the a n a e r o b i c p a r t of E x p t . 2. T he c o n c e n t r a t i o n of glucose w a s t25 nlg %. M P D : m e a n p a i r difference ~: s t a n d a r d error of t h e m e a n w i t h t h e n u m b e r of o b s e r v a t i o n s in p a r e n t h e s e s . N.S., n o t significant.
Expt.
Incubated at
MPD
P
pH 7.4
pill 8.0
2.83 2.23 2-65 2.7 °
4. i S 2.54 5.oo 2.65
+I.35 @°'31 +2-35 ....0.05
(5) (5) (6) (6)
o.oI N.S. < o.ol N.S.
5.45 6,30
+ 2 . 5 4 • o. I99 (0) + 0 . 2 3 :L o.231 (6)
< o.ol N.S.
Glucose uptake (mg/g tissue per 9° rain) i 2
Monoiodoacetate (o.I mM) Aerobic Anaerobic
+
d ~ ~
0.225 0"209 0.248 o. IT8
•
Lactate production (mg/g tissue per 90 rain) 2
Aerobic Anaerobic
2.91 6.07
pH dependency of the glycolytic activity of the cell-free mltscle extract The glycolytic activity of the cell-free muscle extract fortified with glucose and cofactors was examined at two different p H values. Fig. I shows that a majol factor ]3iochim. Biophys. Acta, 124 (1966) 3 i o - 3 2 2
pH-DEPENDENT
313
GLYCOLYSIS
determining the apparent effectiveness of pH change on glycolytic rate was the initial concentration of ATP in the reaction mixture. At higher pH, acceleration of glycolysis was observed in the presence of higher concentrations of ATP, while at lower pH neither lactate production nor glucose utilization by the extract was markedly stimulated by increasing the concentration of ATP. As a result, a marked difference of glycolytic rate was observed between the lower and higher pH as the concentration of ATP was raised, whereas no significant dependency of the glycolytic activity on pH was evident at the lowest concentration of ATP. Such observations for the cell-free preparation may be consistent with the above findings with the intact-cell preparation of rat diaphragm, that anaerobiosis renders glycolytic rate unaffected by a change in the pH of the medium (Table I), because incubation of isolated rat diaphragm under anaerobic conditions resulted in a marked reduction of ATP content in the tissue 2. It can be seen in Fig. 2 that the extract aged for a period of 30 min or longer in the absence of substrate failed to respond to change in the pH of the medium even at a higher concentration of ATP. The results shown in Fig. I indicate that the apparent pH optimum of the glycolytic activity is dependent upon the level of ATP, whereas Fig. 2 suggests that this apparent pH optimum is lost on aging of the enzyme system. The marked increase of glycolysis at higher pH as compared with that at lower pH will henceforth be referred to, for the sake of brevity, as "pH effect".
c -~1o00o.~ L O- E
_~ 5oo-
00
~ 2~5 5
7;5 ~b
[ATP] (raM}
c 500"
~ 1ooo.o "~
~o
c
L
"o o .
~E -~ 350.
~6 -~ 5oc ~o
o uO E !:L
.E 5 0 0
o~
oE
0
2.5
5 Z5 10 [ATP] (raM)
30
Aging
60
t i m e (rain)
o
3'0 Aglng t l m e
go (rnin)
Fig. I. Effect of A T P c o n c e n t r a t i o n on glycolytic a c t i v i t y of t h e cell-free m u s c l e e x t r a c t i n c u b a t e d a t p H 7.2 or 7.6. Cell-free e x t r a c t p r e p a r e d from r a t d i a p h r a g m was i n c u b a t e d a t 37 ° for 9o m i n in a n a t m o s p h e r e of 9 3 % 0 2 - 7 % CO2 (pH 7.2) or lOO% 02 (pH 7.6) w i t h t h e a d d i t i o n of 5 m M glucose. T h e reaction m i x t u r e c o n t a i n i n g 0. 3 ml of t h e cell-free e x t r a c t w a s a d d e d as described in t h e t e x t in a t o t a l v o l u m e of 0.35 ml. O - - O , i n c u b a t e d at p H 7.2; 0 - - 0 , i n c u b a t e d a t p H 7.6. Fig. 2. Effect of a g i n g on giycolytic a c t i v i t y of cell-free m u s c l e e x t r a c t i n c u b a t e d at p H 7.2 or 7.6. Cell-free e x t r a c t from r a t d i a p h r a g m was aged at 37 ° in a n a t m o s p h e r e of 9 3 % 0 2 - 7 % COs (pH 7.2) for t h e period i n d i c a t e d on t h e abscissa with t h e a d d i t i o n of 1.2 m M N A D +, 5.4 m M n i c o t i n a m i d e a n d 2 m M ATP. Aged e x t r a c t was t h e n i n c u b a t e d with f u r t h e r a d d i t i o n of 8 m M A T P a n d 5 m M glucose for 3 ° m i n in a n a t m o s p h e r e of 9 3 % 0 2 - 7 % COs or lOO% 02 . O - - O , i n c u b a t e d a t p H 7.2; 0 - - 0 , i n c u b a t e d a t p H 7.6.
Activity of individual enzymes at different pH levels Table II shows that the glycolytic enzymes from ketose-I-phosphate aldolase to lactate dehydrogenase were not concerned with the "pH effect", since lactate production from Fru-i,6-P 2 was not markedly stimulated by raising pH at a higher concentration of ATP (IO mM). Lactate production was strikingly accelerated when glucose, Glu-6-P or Fru-6-P was employed as substrate. The activity of hexokinase, glucosephosphate isomerase and phosphofructokinase was then followed at different Biochim. B$ophys. Acta, 124 (1966) 31o-322
314
51. u i
TABLE
II
GLYCOLYTIC ACTIVITY OF THE C E L L - F R E E
MUSCLE
EXTRACT INCUBATED AT p U
7.2 I)i,~ 7.13
C e l l - f r e e m u s c l e e x t r a c t w a s i n c u b a t e d in a n a t m o s p h e r e of 93 % O 2 - 7 % ('()~ ( p H 7.2) n r I o o % ()2 ( p H 7.6). E a c h t u b e c o n t a i n e d o.2 m l of e x t r a c t , 5 m M s u b s t r a t e a n d o t h e r a d d i t i o n s a s d e s c r i b e d in t h e t e x t in a t o t a l v o l u n i e of o . 2 5 m l . W h e n g l u c o s e w a s a d d e d , i n c u b a t i o n w a s c o n d u c t e d a t 37 ° f o r 6 o n i i n : w h e n h e x o s e p h o s p h a t e w a s u s e d , a t 22 ° for 15 r a i n . T h e c o n c e n t r a t i o n of A T P w a s ~o r a M .
Subslrate
Lacla& prodztction (mlOnole/mg protein) pH 7.2
Glucose Glu-6-P Fru-6- P Fru-I,6-P~
TABLE
455 657 055 IO22
pH 7.6 144 ° lO22 i 134 ~I 5 0
II1
ttEXOKINASE ACTIVITY OF THE CELL-FREE MUSCLE EXTRACT INCUBATED AT p H
7.2 OR 7.0
(;ell-free e x t r a c t f r o m r a t d i a p h r a g m w a s i n c u b a t e d a t 3 o~' f o r 3 ° r a i n in a n a t m o s p h e r e of (~3 % ()'.,-7 % CO2 ( p H 7.2) o r I o o % (),, ( p H 7.6). T h e c o n c e n t r a t i o n of 2 - d e o x y g l u c o s e w a s t r a M . A TP
added
2-l)eox;vghtcose utili.:,aliou (mluuole/mg prolei~)
(roB1)
15H 7.2 2. 5 lo
pH 7.6
I40
i07
1 (~.t
2 o9
~_hosphofructokinase ph o spho~lucoisomerase
:0.2 E
O O.2 "6 g o.1
P o 0.1 o c m
g c_
o )
°o
5
0
Time (rain)
5
Fig. 3. G l u c o s e p h o s p h a t e i s o m c r a s c a n d p h o s p h o f r u c t o k i n a s e a c t i v i t i e s ot tile c c l l - f r c c ll/tt>,clt e x t r a c t a t p f t 7.2 o r 7.o. E a c h c u v e t t e c o n t a i n e d 2. 5 m l o . l M T r i s b u f f e r ( p H 7.2 o r 7.()) a n d o. 5 m l e x t r a c t . In g l u c o s e p h o s p h a t e i s o m e r a s e a s s a y 0 . 2 5 t i m o l e N A 1 ) I > , o.2 u n i t glucose-(~ p h o s p h a t e d e h y d r o g e n a s e a n d 0 / ~ m o l e s M g C l i w e r e a ( i d e d (final \ o l m n e 3.2 ml) a n d a h s o r b a n c e a t 3 4 o m]~ w a s f o l l o w e d a f t e r t h e a d d i t i o n of o . a 5 , u m o l e F r u - 0 P . P'or t h e p h o s p h o f r u c t o k i n a s e a s s a y 0 . 2 5 p m o l c N A D H , to F g g l y c c r ° l - 3 - p h ° s p h a t e d e h y d r o g e n a s e , 5 ° [qL k e t o s e I - p h o s p h a t e a l d o l a s e , o. 3 t t g t r i o s e p h o s p h a t e i s o m e r a s e a n d A T P (I o r 3 r a M ) w e r e a d d e d . P'inal v o l u m e 3.-' ml. R e d u c t i o n in a b s o r b a n c e a t 34 o m p w a s f o l l o w e d , a f t e r t h e a d d i t i o n of t~'ru-0 P (o. J r a M ) . O O, incubated at pH 7.2; O--O, i n c u b a t e d a t p H 7.0: / , 2,, i n c u b a t e d a t p H 7.-' ~ i t h h i g h e r c o n c e n t r a t i o n of A T P ; A " A , i n c u b a t e d a t p H 7.(} w i t h h i g h e r c o n c e n t r a t i o n of ,VI'IL
Hiochim. t~io~]t~,s ,qcla, 124 (~0(~) 3 ~ ° 322
p H - D E P E N D E N T GLYCOLYSIS
315
pH levels. Hexokinase activity, expressed as the rate of disappearance of 2-deoxyglucose, was not significantly accelerated when the pH was raised from 7.2 to 7.6 (Table III). The activity of glucosephosphate isomerase was also virtually indifferent to the change of pH of the buffer (Fig. 3). In contrast, Fig. 3 also shows the importance of phosphofructokinase actixdty in the "pH effect", in which the inhibition of phosphofructokinase activity 1° was exaggerated at a lower pH level according to spectrophotometric assay in the diluted state. Consequently, an apparent stimulation of phosphofructokinase by shifting the pH to a higher level was only markedly evident in the presence of a higher concentration of ATP. The results obtained for the concentrated extract (Table IV) lend support to the significance of phosphofructokinase. When glucose was employed as substrate, glycolytic activity of the extract was limited by hexokinase in the presence of ATP at 2.5 raM, and thus neither Glu-6-P nor Fru-6-P was detected after incubation (Expt. 2 in Table IV). In the presence of a much higher concentration of ATP, as a result of phosphofructokinase inhibition, measurable amounts of Glu-6-P and Fru-6-P existed after incubation, suggesting that phosphofructokinase rather than hexokinase acts as a rate-limiting step in this case. The interconversion rate between Glu-6-P and Fru-6-P was found to be extremely high in the extract (cf. refs. II, 12). When glucose was either combined with yeast hexokinase or replaced by Glu-6-P to avoid the involvement of hexokinase, the residual level of Glu-6-P and Fru-6-P after incubation could represent the phosphofructokinase activity, because the reaction catalyzed by phosphofructokinase is rate-limiting, as verified by the absence of Fru-I,6-P 2 after incubation. The stimulation of phosphofructokinase on raising pH T A B L E 1V LEVEL OF GLYCOLYTIC INTERMEDIATES AFTER INCUBATION AT p H 7.2 OR p H 7.6 Cell-free e x t r a c t from r a t d i a p h r a g m was i n c u b a t e d a t 22 ° for 15 m i n in a n a t m o s p h e r e of 93 % 0 3 7 % COz (p H 7.2) or lOO% O a (pH 7.6). E a c h t u b e c o n t a i n e d t h e e x t r a c t (0.2 ml) a n d o t h e r c o f a c t o r s in a final v o l u m e of 0.25 ml. The r e a c t i o n was s t o p p e d b y t h e a d d i t i o n of HC104. The s u p e r n a t a n t n e u t r a l i z e d w i t h 2 M K O H was s u b m i t t e d to e n z y m i c a n a l y s i s as d e s c r i b e d in t e x t . The c o n c e n t r a t i o n of s u b s t r a t e was 5 mM. W h e r e i n d i c a t e d , y e a s t h e x o k i n a s e , 5 ° / z g pe r t ube , was a d d e d .
Expt. No.
I*
Substrate
Intermediate analyzed
Levels after incubation (m#mole/mg protein) 2.5 m M A T P
zo m M A T P
p H 7.2 p H 7.6
p H 7.2 p H 7.6
Glu-6-P
Glu-6-P Fru-6-P Fru-I,6-P
223 l°9 o
26 44 o
319 128 o
IOO 66 20
Glucose
Glu-6-P Fru-6-P Glu-6-P Fru-6-P
o o 314 15I
o o 155 7°
82 42 487 220
31 25 215 94
Glu-6-P Fru-6-P
622 218
239 III
748 257
337 131
Glucose plus y e a s t h e x o k i n a s e Glucose plus y e a s t h e x o k i n a s e
* E D T A - M g ~+ (0. 5 mM) was added.
Biochim. Biophys. Acta, 124 (1966) 31o-322
316
M. UI
and the inhibition on adding a larger a m o u n t of A T P were manifested by a fall and rise, respectively, in the residual levels of both Glu-6-P and F r u - 6 - P (Table IV). It is a well-known fact t h a t Glu-6-P is a potent inhibitor of hexokinase. Since the inhibition or activation of phosphofructokinase is effective in raising or lowering the Glu-6-P level, respectivdy, (due to the rapid equilibrium between Glu-0-P and F r u - 6 - P as a result of high activities of glucosephosphate isomerase) it m a y be assumed t h a t modification of t)hosphofructokinase activity can exert a profound influence on hexokinase activity. Such a conclusion was verified by the d a t a in Table V, in which the rate of inhibition of hexokinase in the presence of F r u - 6 - P was reduced to some extent b y activating phosphofructokinase at a higher pH level. The significance of phosphofructokinase in the regulation of hexokinase activity via the (ilu-6-P level was shown in erythrocytes b y ROSE AND O'CONNEL11 and in brain b y AISENB E R G 13.
Disappearance of "pH effect" in the aged extract As is evident in Fig. 2, aging of the cell-free muscle extract without addition of substrate diminished the overall glyeolytic activity with a concomitant disappearance of the "pH effect". Table VI shows that hexokinase (in the absence of TABLE
V
HEXOKINASG
A C T I V I T Y OF T H E
CELL-FREE
MUSCLE
EXTRACT
WITH
OR W'ITHOUT ADDED
Fru-6-P
C e l l - f r e e e x t r a c t f r o m r a t d i a p h r a g m w a s i n c u b a t e d a t 22 ° f o r 15 m i n in t h e p r e s e n c e of l o m M A T P in 93 o/ /o 0 . ) - 7 o, 7o C O 2 ( p H 7.2) or IOO°'o 02 { p H 7.6). F i n a l v o l u m e , 0 . 2 5 m l . T h e c o n c e n t r a t i o n s of 2 - d e o x y g l u c o s e a n d F r u - 6 - P w e r e e a c h r m M .
Fru-6- P
-
2- Deoxyglucose utilizatio~l (ml~mole/mg protein)
-
+
TABLE
pH 7.2
p H 7.6
i2i t7
138 54
VI
ACTIVITIES
OF
GLYCOLYTIC ENZYMES
BEFORE
AND
AFTER
AGING OF T H E
EXTRACT
A g i n g of c e l l - f r e e e x t r a c t w a s c o n d u c t e d a s in F i g . 2. H e x o k i n a s e a n d g l u c o s e p h o s p h a t c i s o m e r a s e a c t i v i t i e s w e r e a s s a y e d a s d e s c r i b e d i n T a b l e IX: a n d F i g . 3, r e s p e c t i v e l y . P r o d u c t i o n of l a c t a t e w a s m e a s u r e d a f t e r i n c u b a t i o n a t 22 c' f o r 15 m i n w i t h t h e a d d i t i o n of F r u - r , 6 - P 2 (2 r a M ) .
Before aging pH 7.2
drier agiug pH 7.6
pH 7.2
pft 7.0
Hexohinase activity (2-deoxyghtcose utilization, mltmole/mg protein) "Without Glu-6-P With Glu-6-P
157 12
182 73
171 2o
~SS 33
Glucosephosphate isomerase activity (Fru-6-P utilized, ml*mole/mg protein) 895
Io88
895
1o53
Lactate production with the addition of Fra-z,6-P~ as substrate (mt*mole/mg protei~z) 453
Biochim. Biophys. Acta, 124 ( i 9 6 0 ) 3 I O - 3 2 2
585
317
p H - D E P E N D E N T GLYCOLYSIS
GIu-6-P), glucosephosphate isomerase and complex enzyme systems below ketosei-phosphate aldolase survived the aging process without loss of activity. It can be seen in Fig. 4 that phosphofructokinase activity was markedly depressed by aging with striking reduction in the degree of pH dependency. This finding is further supported by tile data in Table VII, in which the level of Glu-6-P and Fru-6-P after incubation, in the presence of 2.5 mM of ATP, was elevated in the aged extract.
~J
c
~
Time (mln)
5
Fig. 4. Phosphofructokinase activity before and after aging of the cell-free muscle extract. Phosphofructokinase was assayed with the addition of 3 mM ATP as described in Fig. 3- O - - © , non-aged e x t r a c t incubated at p H 7.2; 0 - - 0 , non-aged e x t r a c t at p H 7.6; A - - A , aged e x t r a c t at p H 7.2; ~k--Jk, aged e x t r a c t at p H 7.6. TABLE VII LEVELS
OF
GLYCOLYTIC
INTERMEDIATES
AFTI~R
INCUBATION
OF
NON-AGED
AND
AGED
EXTRACT
The extract was incubated before or after aging and s u b m i t t e d to enzymic analysis as described in Table IV.
Expt. No.
Substrate
Glucose
Intermediate analyzed
Glu-6-P Fru-6-P
Glucose plus yeast hexokinase
Glu-6-P Fru-6-P
2
GIu-6-P
Glu-6-P Fru-6-P
A TP (raM)
Levelsafter incubation (m#moles/mg protein) Non-aged
Aged
pH 7.2 pH 7.6
pH 7 2
pH 7.6
2. 5 io 2. 5 IO 2. 5 IO 2. 5 IO
o 82 o 42 314 487 151 220
23 68 27 46 480 525 221 249
17 51 21 38 450 514 193 220
2.5 io 2.5 IO
39 273 32 115
o 31 o 25 155 215 7° 94
292 316 138 139
Hexokinase activity in the glycolytic system assayed in the presence of Glu-6-P at higher pH was markedly inhibited upon aging the extract (Table VI). In view of the above proposition that Glu-6-P-induced inhibition of hexokinase in the non-aged extract is apparently reduced at higher pH as a result of increased phosphofructokinase activity (Table V), the apparent inhibition of hexokinase in the presence of Biochim. Biophys. Acta, 124 (1966) 31o-322
318
M. uI
Glu-6-P observed at higher pH in the aged extract m a y be reasonably attributed to the failure of phosphofructokinase activation under such conditions. Minor responsiveness of phosphofructokinase in the aged extract (as compared with that in the non-aged extract) to the change of p H is also noted in all the experiments presented in Table VII. Moreover, the magnitude of the inhibition of phosphofructokinase b y high concentrations of ATP was often reduced when aging was conducted prior to incubation. Thus, the aging process m a y depress the enzymic activity and reduce the response to changes of p H and ATP concentrations. Such modification of phosphofructokinase activity m a y lze due to changes in the enzyme itself rather than the involvement of non-protein factors, since, as shown in Table V I I I , the phosphofructokinase activity of the extract prepared by combining aged extract with non-aged extract in equal amounts was practically identical to the value calculated from the separate enzyme activities of the aged and non-aged extracts. Furthermore, the supernatants of the boiled extracts of aged and non-aged preparations did not differ in their effect on phosphofructokinase activity when added to the extract incubated with Fru-6-P as substrate. TABLE VIII PHOSPHOFRUCTOKINASE
ACTIVITY
OF
NON-AGED
AND
AGED
EXTRACT
Cell free e x t r a c t from r a t d i a p h r a g m , before or after a g i n g as d e s c r i b e d in Fig. 2, was i n c u b a t e d w i t h t h e a d d i t i o n of 5 mM F r u - 6 - P . E a c h t u b e c o n t a i n e d o.2 ml of " n o n - a g e d " e x t r a c t , o.2 ml of " a g e d " , or o.I ml of each ( " n o n - a g e d plus a g e d " ) : final vol ume , o.25 nil. Boiled s u p e r n a t a n t was p r e p a r e d b y i m m e r s i n g " n o n - a g e d " or " a g e d " e x t r a c t in b o i l i n g w a t e r for i5o sec a n d t h e n c e n t r i f u g i n g off t h e p r e c i p i t a t e d protein, o. t ml of each w a s a d d e d to t h e r e a c t i o n m i x t u r e before i n c u b a t i o n , t h e final x o l u m e b e i n g o.35 ml in such cases.
Addition
None* Boiled s u p e r n a t a n t of n o n - a g e d e x t r a c t B o i l e d s u p e r n a t a n t of aged e x t r a c t
Fret 6-1) level after incubation (mktmole/mg protein) Non aged
Aged
Non aged ph*s aged
211 3oo 3oo
443 455 4'io
314 (327)
* The figure in p a r e n t h e s e s is the m e a n of t h e v a l u e s o b t a i n e d for t h e " n o n - a g e d " a n d " a g e d " e x t r a c t s alone. S t a n d a r d d e v i a t i o n s c a l c u l a t e d were 3.5, lO.8 a n d 9.4 fur " n o n - a g e d " , " a g e d " a n d " n o n - a g e d plus aged", r e s p e c t i v e l y .
A TP-induced inhibition of muscle phosphofruclokinase at various pH levels Results presented above pointed to the significance of ATP concentration in the apparent stimulation of glycolysis by pH in which phosphofructokinase activity acted as a limiting factor. The rate of the reaction catalyzed by tile phosphofruetokinase isolated from rabbit skeletal muscle was then tested at various concentrations of ATP and hydrogen ions. Enzyme assay was not done at pH values below 6.7,~ because of the loss of buffering capacity of the Tris buffer below pH 6.6. Fig. 5 shows t h a t the p H optimum of phosphofructokinase was shifted to the alkaline side on increasing ATP concentration. The reaction did not proceed at all when assayed at lower p H in the presence of excess ATP. In Fig. 6, initial velocities were plotted against ATP concentrations in the "inhibitory phase" at various pH levels. It can be seen t h a t precipitous inhibition occurred at lower pH (below 7.6) as the ATP Biochim. Biophys..4cla, 124 (x960) 31o-322
pH-DEPENDENT
319
GLYCOLYSIS
concentration was raised to 3 raM. Increase in the p H of the medium resulted in a striking restoration of phosphofructokinase activity in the presence of excess ATP. A similar recovery from ATP-induced inhibition was found to take place when the amount of Fru-6-P was increased as shown in Fig. 7. :3,. E
~-- 0,2!
0
O
~c
,J E o.2-
Ja5 L;5
~ .c_
.C "E-
g~ c
~.~
o
J
o.1-
~-
8.o
7.0
9.o
pH
16,0
kJ
0
~A
v [.AMP] (raM)
Fig. 5. p H - p h o s p h o f r u c t o k i n a s e a c t i v i t y c u r v e s at h i g h e r c o n c e n t r a t i o n s of ATP. P h o s p h o f r u c t o kinase purified f r o m r a b b i t skeletal m u s c l e according to GATT AND RACKER7 a n d stored b y susp e n d i n g in 5 ° % s a t u r a t e d (NH4)2SO 4 at o ° was diluted 2•-fold with o.I M Tris buffer (pH 7.6). E a c h c u v e t t e c o n t a i n e d I. 5 ml o.i M Tris buffer c o n t a i n i n g MgC12 (final concentration, 3 mM), o.oi m l diluted p h o s p h o f r u c t o k i n a s e , o.i ml ATP, o.o 3 ml of e n z y m e m i x t u r e ( k e t o s e - I - p h o s p h a t e aldolase, t r i o s e p h o s p h a t e isomerase a n d glycerol-3-phosphate d e h y d r o g e n a s e , each e q u i v a l e n t to t h e a m o u n t capable of c o n v e r t i n g i / * m o l e s u b s t r a t e p e r rain) a n d o. 4 # m o l e N A D H . T h e reaction was initiated b y t h e a d d i t i o n of o.05 m M F r u - 6 - P . Decrease in a b s o r b a n c e a t 34 ° m # d u r i n g t h e initial 2 rain was p l o t t e d as initial velocity on t h e ordinate. T e m p e r a t u r e , 22 °. O - - O , o.I m M ATP; A--&,o.4mMATP; El--[2, ImMATP; • O, 3mMATP;A--A,5mMATP; re__m, 8 m M ATP. Fig. 6. Effect of A T P c o n c e n t r a t i o n s on t h e kinetics of p h o s p h o f r u c t o k i n a s e . T h e e x p e r i m e n t w a s carried o u t as described in legend to Fig. 5- O - - O , p H 6.78; A - - & , p H 7.1; [ ] - - E l , p H 7.3; ~--~,pH7.6; O--Q, pH8.o;A--~k, pH8.3; .--I, p H 8.8; Q - - Q , p H 9 . 3 ; × - - × , p H g . 6 . :::L.
E O
0.5"6.c_
~~ o.3 o
~0.2
._c .c
~
ol
c u
o
2
4
6
8
10
12
14
[AT P.] (raM)
Fig. 7. Effect of F r u - 6 - P c o n c e n t r a t i o n s on t h e kinetics of p h o s p h o f r u c t o k i n a s e . T h e e x p e r i m e n t was carried o u t as described in legend to Fig. 5. O - - O , o.o25 m M F r u - 6 - P ; & - - A , o.o 5 m M F r u - 6 - P ; [ ] - - E l , 0.075 m M F r u - 6 - P ; 0 - - 0 , o.i m M F r u - 6 - P ; A - - , , 0.25 m M F r u - 6 - P ; I - - I , 0. 5 m M F r u - 6 - P .
It m a y be reasonably assumed that the "pH effect" of muscle glycolysis is due to the pH-dependent inhibition of phosphofructokinase b y ATP as illustrated in Figs. 5 and 6.
Biochim. Biophys. Mcta, 124 (i966) 31o-322
320
M. UI
DISCUSSION
In the preceding papers 1,2, it was shown that induction of alkalosis in the organism as well as incubation of nmscle slices in the alkaline medium markedly affected the action of hormones such as epinephrine on carbohydrate metabolism. Marked hyperlactacidemia observed during alkalosis and the promotion of lactate formation i~ vitro by the muscle slices in a medium at higher pH was suggestive of accelerated glycolysis in such a case. Studies with the cell-free glycelytic systems reported here have indicated that glycolysis is actually stimulated by raising p t t and t h a t the major factor responsible for the " p H effect" is phosphofructokinase which operates as one of the rate-limiting factors in gtycolysis. The significance of phosphofructokinase, together with hexokinase, in the regulation of glycolysis has repeatedly been pointed out by m a n y investigators~°, 12-16. The supposition that the " p H effect" observed in the overall glycolytic reaction is solely due to the alteration of phosphofructokinase activity appears to be disfavored only b y the finding that, while the overall glycolytic rate was not affected by pH change at the lower ATP concentration (Fig. I), the phosphofructokinase activity (expressed in terms of residual levels of Glu-6-P and Fru-6-P after incubation) was stimulated by raising the p H of the medium even at the lower concentration of ATP (Table IV). This apparent discrepancy m a y be explained as follows. In the experiments shown in Fig. I, muscle extract was incubated at 37 ° for a long period of time (9 ° min) in contrast to the mild incubation employed for experiments shown in Table IV (22 ° for IO min). During incubation of whole extract at a higher temperature, added ATP was found to undergo rapid hydrolysis through activation of particulate ATPase activity. It m a y be deduced, therefore, that the effective doses of ATP in Fig. I were far less than those given on the abscissa. Moreover, tile experiment presented in Fig. 3 demonstrates that at a lower concentration of ATP phosphofructokinase activity was not essentially affected by a change in the pH of the medium. A closer exainination {}f rabbit skeletal nmscle phosphofructokinase, as shown in Figs. 5-7, revealed the dependence of ATP-induced inhibition upon the pH level, confirming the direct relationship between "pH effect" and ATP concentration. As shown in Table V, the modification of t)hosphofructokinase activity is reflected in the hexokinase reaction through the mediation of change in (;lu-6-1' levels. On the other hand, the hexokinase reaction could control the phosphofructokinase activity, because Fru-6-P, rapidly formed by glucosephosphate isomerase from Glu-6-P, exerts a profound influence on the inhibitory action of ATP on phost)hofructokinase as shown in Fig. 7 (see also refs. i 7 --I0). Thus, interdependence of the possible rate-limiting steps, hexoldnase and phosphofructokinase, would contribute to the regulatory mechanism involved in cellular glycolytic rates. Since (ilu-6-I) also functions as an activator of UDPglucose-glycogen glucosyl transferase (EC z.4.I.I I ) and as a substrate for glucose-6-phosphate dehydrogenase, which supplies the reduced form of NADP + for lipogenesis or other reduction steps in metabolism, it would appear t h a t the regulation of phosphofructokinase activity exerts a broad influence on cellular metabolic activity. As is evident in Fig. 6, muscle phosphofructokinase activity is profoundly influenced b y a change of ATP concentration from o.5 to 2 mM, depending on p H wtlues ranging from 7.I to 7.6 when Fru-6-P was maintained at o.o 5 raM. In view <~f the Biocki.z. Biopkys. Acta, I24 (I9()()) 31o 3 ; 2
pH-DEPENDENT
GLYCOLYSIS
321
fact that these concentrations of ATP, Fru-6-P and H ÷ never involve a marked departure from cellular concentrations (cf. ref. I7), it is possible that subtle change of hydrogen ion concentration in the cellular fluid plays a physiological role in the regulation of cellular glycolytic activity. For example, a drop in cellular hydrogen ion concentration that is assumed to take place during active oxidative phosphoryiation in the mitochondria could act as a physiological feed-back mechanism protecting the glycolytic activity from excessive blocking via the Pasteur effect. PASSONNEAU AND LOWRY10 postulated that in muscle phosphofructokinase there are two ATP sites, one catalytic and one allosteric, and the ATP-induced inhibition m a y be--according to their postulation--due to the binding of ATP on the allosteric site. A similar observation has been reported for yeast is and E s c h e r i c h i a coli 19 phosphofructokinase. The pattern shown in Figs. 6 and 7 illustrates the velocity of phosphofructokinase reaction after ATP began to occupy the inhibitory site. The fact that sigmoid, rather than hyperboloid, curves were obtained for the plots of velocity v e r s u s ATP concentration m a y be suggestive of the existence of two (or more) inhibitory sites for ATP in addition to one catalytic site. As the pH was raised, the slope of the inhibition curve became less. This m a y be a manifestation of the reduced affinity of ATP for the inhibitory site(s). It m a y be proposed, then, that a drop in hydrogen ion concentration causes a conformational change at the allosteric site probably by spatial changes in hydrogen bonding or in other molecular linkages, thus preventing ATP from occupying the allosteric site(s). Detailed discussion on the inhibitory site for ATP in the phosphofructokinase protein, together with other kinetic data, will be published elsewhere. The aging of cell-free muscle extract in the presence of ATP led to a diminution of phosphofructokinase activity as well as a disappearance of the inhibition due to increased ATP concentration and of the stimulation by increased pH (Fig. 4 and Table VII). The activities of the aged and non-aged extracts were additive when assayed together for the enzyme activity, as shown in Table V I I I . This, together with the similarity of the effect of boiled extracts on phosphofructokinase, seemed to rule out changes, during aging, in the level of certain heat-stable metabolites or cofactors that could affect the activity of the enzyme, and might suggest that changes in the enzyme protein itself were involved. VINI~ELA et al. ~° reported that aging of crude yeast extract in the presence of N a F rendered the phosphofructokinase unresponsive to the inhibition by ATP. Thus, loss of susceptibility to the inhibition by ATP after aging is common to yeast and muscle phosphofructokinase. In view of recent reports that phosphofructokinase undergoes a reversible conversion from an inactive to active form~°,21 and is a dissociable protein22, 23 characteristic of an allosteric enzyme 24, modification of reactivity after aging of the extract m a y be explained on the basis of "desensitization" of the allosteric protein. This would be as a result of dissociation of the enzyme molecule into subgroups or of other drastic conformational changes due to exposure to higher temperature without substrate. Further studies are now in progress on the kinetic behavior of muscle phosphofructokinase after a variety of "desensitization processes". NEFERENCES I M. UI, Am. J. Physiol., 2o9 (1965) 353. 2 M. UI, Am. J. Physiol., 209 (1965) 359.
Biochim. Biophys. Acta, 124 (1966) 31o-322
322
M. u i
3 V. S. VVTARAVDEKAR AND L. D. SASLA~V, J . Biol. Chem., 234 (1959) 1945. 4 S. B. BARKER AND W. H. SUMMERSON, J. Biol. Chem., 138 (1941) 535. 5 F. TAYLOR, in S. P. COLO~VICK AND N. O. I(APLAN, Methods in Enzymology, Vol. 1, \ c a d e m i c Press, New Yo rk , 1955, p. 31o. 6 M. W. SLEIN, in S. P. COLOWICK AND •. O. I"~APLAN, Methods in. Enzymology, Vol. 7, A c a d e m i c Press, N e w Yo rk , 1955, p. 3o4 . 7 S. GATT ANn E. RACKER, J. Biol. Chem., 234 (I959) l o l 5. 8 0 . I-I. LOWRY, N. J. ROSEBROISGH, A. L. FARR AND ]{. J. RANDALL, J. 13iol. Che~z., 193 (i~)5J) 265 . 9 W. QEVERS AND E. DAWDLE, Clin. 5"ci., 25 (1963) 344" IO J. ¥*. PASSONNEAU AND O. H. LOWRY, Bioehem. Biophys. Res. Commun., 7 (1962) lo. 11 I. A. R o s e AND E. L. O'CONNEL, J. Biol. Chem., 239 (1964) 12. 12 P. (-')ZAND AND H. T. NARAHARA, J. Biol. Chem., 239 (1964) 3146. 13 A. C. AISENBERG, J. Biol. Chem., 234 (1959) 441 . 14 R. WtJ AND E. RACKER, J. Biol. Chem., 234 (1965) lO29. 15 O. H. LOVCRY, J. V. PASSONNEAU, F. X. HASSELBERGI~2R AND D. ~V. SCHULZ, ./. Biol. Uhem, 239 (1964) 18. 16 D. M. REGEN, W . W. DAvis, H. E. MORGAN AND C. l(. PARK, J. Biol. Chem., 239 (1964) 43. 17 T. E. MANSOUR, J. Biol. Chem., 238 (1963) 2285. 18 A. RAMAIAH, J. A. HATHAWAY AND D. E. ATKINSON, J. Biol. Chem., 239 (1964) 361(). 19 D. E. ATKINSON AND G. M. WALTON, J. Biol. Chem., 24o (1965) 757. 2o E. VINI3ELA, M. L. SALAS, M. SALAS AND A. SOLS, Biochem. Biophys. Res. Commun., 15 (~904) 243. 21 T. E . MANSOUR, J. Biol. Chem., 24o (1965) 2165. 22 T. E. MANSOUR, N. \V. WAKID AND H. M. SPROUSE, Biochem. Biophys. Res. CommaJz., ~0 (1965) 72~. 23 A. PARMEGGIANI AND E. G. b2REBS, Federation Proc., 24 (1965) 284. 24 J. MONOD, J. WYMAN .aND J.-P. CHANGEUX, J. Mol. Biol., 12 (1965) 88.
Bioehim. Biophys. Aeta, 124 (1966) 31o 322
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
25580
A ROLE OF P H O S P H O F R U C T O K I N A S E IN p H - I ) E P E N D E N T REGULATION OF GLYCOLYSIS MICHIO uI Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido Univcrsi/3:, Sapporo (Japan)
(Received January 4th, t966
SUMMARY
i. Glycolysis in a cell-free extract from rat diaphragm was stimulated at elevated pH in the presence of higher concentrations of ATP. A similar situation was also observed in the case of intact-cell preparations of rat diaphragm. 2. It was found that the rate of inhibition of phosphofructokinase (ATP:Dfructose 6-phosphate I-phosphotransferase, EC 2.7.I.11) activity by excess NFP was strictly dependent upon the pH; at pH 7.3 an increase in ATP concentration from I to 1. 7 mM resulted in a sudden inhibition of phosphofructokinase activity, whereas at pH 7.6 such an effect was not observed until the ATP level was raised to 2 3 mM (o.o5 mM fructose 6-phosphate was employed as substrate). Consequently phosphofructokinase activity was profoundly influenced by quite a minute change in pH when larger amounts of ATP were present in the glycolytic system. 3-Since the glucose 6-phosphate which accumulated during phosphofructokinase inhibition was inhibitory to hexokinase (ATP : D-hexose 6-phosphotransferase, EC 2.7.1.1), the pH-induced stimulation of phosphofructokinase caused an exaggerated acceleration of overall glycolysis. 4. Preincubation of the cell-free extract without substrates (aging) rendered phosphofructokinase protein unresponsive both to the stimulation on raising pH and to the inhibition by ATP, It is tentatively proposed that the affinity of the inhibitory site(s) of phosphofructokinase for ATP is affected by hydrogen ion concentration and that aging causes a change of conformation of the enzyme, thus blocking the control mechanism of phosphofructokinase.
INTRODUCTION
Recently the present author has reported that the hyperglycemic action of epinephrine was abolished during alkalosis 1. This abolition was due to tile blockage of the epinephrine-induced inhibition of glucose utilization by muscle at higher wdues of body fluid pH (refs. I, 2). During the course of these studies, it was found that an increase of pH in the incubation medium resulted in a marked increase in glucose uptake by isolated rat diaphragm. It may be expected that enzymes in the metabolic chain responding to changes of environmental pH would also play a significant role in the mechanisms of hormonal control of carbohydrate metabolism. The purpose of Biochbm Biophys. Acta, ~z4 (lO0()) 3Io-3zz
pH-DEPENDENT GLYCOLYSIS
3II
the present study is to establish which enzymes or enzyme systems m a y be responsible for the apparent p H dependency of muscle glucose utilization. As will be seen, the results obtained indicate the significance of phosphofructokinase (ATP: D-fructose 6-phosphate I-phosphotransferase, EC 2.7.1.11) in the regulatory mechanisms of glycolysis. MATERIALS AND METHODS
The experiments employing the rat diaphragm preparations containing intact cells (Table I) were conducted as already descri~edl, 2. All of the data included in the present paper, other than those in Table I, were obtained in experiments with cell-free preparations of muscle glycolytic systems. These experiments were carried out as described below. Cell-free muscle extract was prepared by mincing the rat diaphragm in a m o r t a r at o ° in a medium containing 15o mM K +, 5 mM Mg2+, 12o mM CI-, 24 mM HC03-, 2 mM PO43-. The ratio of the amount of tissue to medium used was i : IO. The mince was centrifuged at 700 × g for IO min to remove the residual intact cells and nuclei. Substrate was added in a final concentration of 5 mM unless otherwise specified; NAD + (1.2 mM), nicotinamide (5.4 mM) and ATP at the concentration indicated were also added. When required, the extract was aged (preincubated without substrate) at 37 ° for a period indicated prior to the incubation. Incubation was carried out in an atmosphere of either 93 % 0 3 - 7 To COs or ioo To 03 at 22 ° or 37 ° for the period indicated. The p H of the cell-free preparation after incubation was 7.15-7.25 for an incubation in 93 % 0 3 - 7 To CO2 and 7.55-7.65 for IOO % 0 2. (As already reported, the pH of the medium buffered with 24 mM HCO 8was 7.3-7.4 in an atmosphere of 93 % 03--7 % COs (refs. I, 2), and introduction of Oo. into incubation flasks rendered the pH as high as p H 8.6. The presence of IOO o~ /o protein in the cell-free extract, however, prevents the increase of pH of the medium in the oxygenated flask.) The reaction was stopped by the addition of HC104 to a final concentration of 5 %. In order to determine glucose or 2-deoxyglucose, the deproteinized aliquot, after being neutralized, was further treated with Ba(OH)2 and Z n S Q to remove phosphorylated sugars and glycogen. Glucose was analyzed with an anthrone reagent. Colorimetric procedures were applied for the determination of 2-deoxyglucose a and lactate 4. Intermediates of the glycolytic pathway were enzymically assayed by following the change in absorbance at 340 m~ as described below: Glu-6-P, by the reduction of NADP + in the presence of glucose-6-phosphate dehydrogenase (EC 1.1.1.49); Fru-6-P, by the reduction of NADP + in the presence of glucosephosphate isomerase (EC 5.3.1,9) and glucose-6-phosphate dehydrogenase; Fru-I,6-P 2 by the oxidation of NADH in the presence of ketose-I-phosphate aldolase (EC 4.i.2.7), triosephosphate isomerase (EC 5.3.1.1) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8); triosephosphate, by the oxidation of NADH in the presence of triosephosphate isomerase and glycerol-3-phosphate dehydrogenase. The amount of the intermediates to be assayed was calculated according to the molar extinction of N A D H or N A D P H as well as from the standard curves obtained with known amounts of substrates. The following chemicals were obtained from Sigma Chemical Co.: ATP, ADP, NAD +, NADP+, 2-deoxyglucose, Glu-6-P, Fru-6-P, Fru-i,6-P2, yeast hexokinase Biochim. Biophys. Acta, 124 (1966) 31o- 322
312
~I. uI
(EC 2.7.I.~), glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, triosephosphate isomerase. Ketose-I-phosphate aldolase and glucosephosphate isomerase were prepared in this laboratory from rat skeletal muscle according to TAYLOR5 and SLEIX6. Phosphofructokinase was also prepared from rabbit skeletal muscle according to G.~'rT aND RAC~;ERv. The protein concentration in the reaction mixture was determined according to LOWRY et al. s and the metabolic activity of the cell-free extract was expressed, on the basis of mg protein, as the mean value of three or four observations. Standard deviations were calculated to substantiate the significance of the differences (or agreements) ; these are given, however, only when rather subtle differences (or agreements) are mentioned for the discussion of the results. RESULTS
Experiments with isolated rat diaphragm preparations containing intact cells In agreement with the previous report 2, glucose uptake by isolated rat quarterdiaphragm was markedly stimulated when the incubation was conducted at a higher p H (Table I). Prevention of this stimulation by the addition of monoiodoacetate, together with the marked stimulation of lactate production on raising pH, indicated that the glycolytic pathway was activated on raising the pH of the medium. Similar results were reported by GEVERS AN[) DAWDLE9. Induction of anaerobiosis abolished the stimulation of glycolysis by pH, suggesting that events somewhat related to the oxidative process are also involved in the pH-stimulated glucose uptake and lactate production by muscle tissue. TABLE I GLUCOSE PHRAGM
UPTAKE
AND
LACTATE
PRODUCTION
BY THE
INTACT-CELL
PREPARATION
FROM
RAT
DIA-
INCUBATED AT p H 7"4 OR 8.O
Q u a r t e r - d i a p h r a g m s from fasted r a t s were i n c u b a t e d in i ml of K r e b s R i n g e r b i c a r b o n a t e s o l u t i o n a t 37 ° for 9o m i n in an a t m o s p h e r e of 93 % O 2 - 7 % COe (pH 7.4) or 99.5 o//o 02 o.5 %. CO.2 (pH 8.o). O~ was r e p l a c e d b y N~ in the a n a e r o b i c p a r t of E x p t . 2. T he c o n c e n t r a t i o n of glucose w a s t25 nlg %. M P D : m e a n p a i r difference ~: s t a n d a r d error of t h e m e a n w i t h t h e n u m b e r of o b s e r v a t i o n s in p a r e n t h e s e s . N.S., n o t significant.
Expt.
Incubated at
MPD
P
pH 7.4
pill 8.0
2.83 2.23 2-65 2.7 °
4. i S 2.54 5.oo 2.65
+I.35 @°'31 +2-35 ....0.05
(5) (5) (6) (6)
o.oI N.S. < o.ol N.S.
5.45 6,30
+ 2 . 5 4 • o. I99 (0) + 0 . 2 3 :L o.231 (6)
< o.ol N.S.
Glucose uptake (mg/g tissue per 9° rain) i 2
Monoiodoacetate (o.I mM) Aerobic Anaerobic
+
d ~ ~
0.225 0"209 0.248 o. IT8
•
Lactate production (mg/g tissue per 90 rain) 2
Aerobic Anaerobic
2.91 6.07
pH dependency of the glycolytic activity of the cell-free mltscle extract The glycolytic activity of the cell-free muscle extract fortified with glucose and cofactors was examined at two different p H values. Fig. I shows that a majol factor ]3iochim. Biophys. Acta, 124 (1966) 3 i o - 3 2 2
pH-DEPENDENT
313
GLYCOLYSIS
determining the apparent effectiveness of pH change on glycolytic rate was the initial concentration of ATP in the reaction mixture. At higher pH, acceleration of glycolysis was observed in the presence of higher concentrations of ATP, while at lower pH neither lactate production nor glucose utilization by the extract was markedly stimulated by increasing the concentration of ATP. As a result, a marked difference of glycolytic rate was observed between the lower and higher pH as the concentration of ATP was raised, whereas no significant dependency of the glycolytic activity on pH was evident at the lowest concentration of ATP. Such observations for the cell-free preparation may be consistent with the above findings with the intact-cell preparation of rat diaphragm, that anaerobiosis renders glycolytic rate unaffected by a change in the pH of the medium (Table I), because incubation of isolated rat diaphragm under anaerobic conditions resulted in a marked reduction of ATP content in the tissue 2. It can be seen in Fig. 2 that the extract aged for a period of 30 min or longer in the absence of substrate failed to respond to change in the pH of the medium even at a higher concentration of ATP. The results shown in Fig. I indicate that the apparent pH optimum of the glycolytic activity is dependent upon the level of ATP, whereas Fig. 2 suggests that this apparent pH optimum is lost on aging of the enzyme system. The marked increase of glycolysis at higher pH as compared with that at lower pH will henceforth be referred to, for the sake of brevity, as "pH effect".
c -~1o00o.~ L O- E
_~ 5oo-
00
~ 2~5 5
7;5 ~b
[ATP] (raM}
c 500"
~ 1ooo.o "~
~o
c
L
"o o .
~E -~ 350.
~6 -~ 5oc ~o
o uO E !:L
.E 5 0 0
o~
oE
0
2.5
5 Z5 10 [ATP] (raM)
30
Aging
60
t i m e (rain)
o
3'0 Aglng t l m e
go (rnin)
Fig. I. Effect of A T P c o n c e n t r a t i o n on glycolytic a c t i v i t y of t h e cell-free m u s c l e e x t r a c t i n c u b a t e d a t p H 7.2 or 7.6. Cell-free e x t r a c t p r e p a r e d from r a t d i a p h r a g m was i n c u b a t e d a t 37 ° for 9o m i n in a n a t m o s p h e r e of 9 3 % 0 2 - 7 % CO2 (pH 7.2) or lOO% 02 (pH 7.6) w i t h t h e a d d i t i o n of 5 m M glucose. T h e reaction m i x t u r e c o n t a i n i n g 0. 3 ml of t h e cell-free e x t r a c t w a s a d d e d as described in t h e t e x t in a t o t a l v o l u m e of 0.35 ml. O - - O , i n c u b a t e d at p H 7.2; 0 - - 0 , i n c u b a t e d a t p H 7.6. Fig. 2. Effect of a g i n g on giycolytic a c t i v i t y of cell-free m u s c l e e x t r a c t i n c u b a t e d at p H 7.2 or 7.6. Cell-free e x t r a c t from r a t d i a p h r a g m was aged at 37 ° in a n a t m o s p h e r e of 9 3 % 0 2 - 7 % COs (pH 7.2) for t h e period i n d i c a t e d on t h e abscissa with t h e a d d i t i o n of 1.2 m M N A D +, 5.4 m M n i c o t i n a m i d e a n d 2 m M ATP. Aged e x t r a c t was t h e n i n c u b a t e d with f u r t h e r a d d i t i o n of 8 m M A T P a n d 5 m M glucose for 3 ° m i n in a n a t m o s p h e r e of 9 3 % 0 2 - 7 % COs or lOO% 02 . O - - O , i n c u b a t e d a t p H 7.2; 0 - - 0 , i n c u b a t e d a t p H 7.6.
Activity of individual enzymes at different pH levels Table II shows that the glycolytic enzymes from ketose-I-phosphate aldolase to lactate dehydrogenase were not concerned with the "pH effect", since lactate production from Fru-i,6-P 2 was not markedly stimulated by raising pH at a higher concentration of ATP (IO mM). Lactate production was strikingly accelerated when glucose, Glu-6-P or Fru-6-P was employed as substrate. The activity of hexokinase, glucosephosphate isomerase and phosphofructokinase was then followed at different Biochim. B$ophys. Acta, 124 (1966) 31o-322
314
51. u i
TABLE
II
GLYCOLYTIC ACTIVITY OF THE C E L L - F R E E
MUSCLE
EXTRACT INCUBATED AT p U
7.2 I)i,~ 7.13
C e l l - f r e e m u s c l e e x t r a c t w a s i n c u b a t e d in a n a t m o s p h e r e of 93 % O 2 - 7 % ('()~ ( p H 7.2) n r I o o % ()2 ( p H 7.6). E a c h t u b e c o n t a i n e d o.2 m l of e x t r a c t , 5 m M s u b s t r a t e a n d o t h e r a d d i t i o n s a s d e s c r i b e d in t h e t e x t in a t o t a l v o l u n i e of o . 2 5 m l . W h e n g l u c o s e w a s a d d e d , i n c u b a t i o n w a s c o n d u c t e d a t 37 ° f o r 6 o n i i n : w h e n h e x o s e p h o s p h a t e w a s u s e d , a t 22 ° for 15 r a i n . T h e c o n c e n t r a t i o n of A T P w a s ~o r a M .
Subslrate
Lacla& prodztction (mlOnole/mg protein) pH 7.2
Glucose Glu-6-P Fru-6- P Fru-I,6-P~
TABLE
455 657 055 IO22
pH 7.6 144 ° lO22 i 134 ~I 5 0
II1
ttEXOKINASE ACTIVITY OF THE CELL-FREE MUSCLE EXTRACT INCUBATED AT p H
7.2 OR 7.0
(;ell-free e x t r a c t f r o m r a t d i a p h r a g m w a s i n c u b a t e d a t 3 o~' f o r 3 ° r a i n in a n a t m o s p h e r e of (~3 % ()'.,-7 % CO2 ( p H 7.2) o r I o o % (),, ( p H 7.6). T h e c o n c e n t r a t i o n of 2 - d e o x y g l u c o s e w a s t r a M . A TP
added
2-l)eox;vghtcose utili.:,aliou (mluuole/mg prolei~)
(roB1)
15H 7.2 2. 5 lo
pH 7.6
I40
i07
1 (~.t
2 o9
~_hosphofructokinase ph o spho~lucoisomerase
:0.2 E
O O.2 "6 g o.1
P o 0.1 o c m
g c_
o )
°o
5
0
Time (rain)
5
Fig. 3. G l u c o s e p h o s p h a t e i s o m c r a s c a n d p h o s p h o f r u c t o k i n a s e a c t i v i t i e s ot tile c c l l - f r c c ll/tt>,clt e x t r a c t a t p f t 7.2 o r 7.o. E a c h c u v e t t e c o n t a i n e d 2. 5 m l o . l M T r i s b u f f e r ( p H 7.2 o r 7.()) a n d o. 5 m l e x t r a c t . In g l u c o s e p h o s p h a t e i s o m e r a s e a s s a y 0 . 2 5 t i m o l e N A 1 ) I > , o.2 u n i t glucose-(~ p h o s p h a t e d e h y d r o g e n a s e a n d 0 / ~ m o l e s M g C l i w e r e a ( i d e d (final \ o l m n e 3.2 ml) a n d a h s o r b a n c e a t 3 4 o m]~ w a s f o l l o w e d a f t e r t h e a d d i t i o n of o . a 5 , u m o l e F r u - 0 P . P'or t h e p h o s p h o f r u c t o k i n a s e a s s a y 0 . 2 5 p m o l c N A D H , to F g g l y c c r ° l - 3 - p h ° s p h a t e d e h y d r o g e n a s e , 5 ° [qL k e t o s e I - p h o s p h a t e a l d o l a s e , o. 3 t t g t r i o s e p h o s p h a t e i s o m e r a s e a n d A T P (I o r 3 r a M ) w e r e a d d e d . P'inal v o l u m e 3.-' ml. R e d u c t i o n in a b s o r b a n c e a t 34 o m p w a s f o l l o w e d , a f t e r t h e a d d i t i o n of t~'ru-0 P (o. J r a M ) . O O, incubated at pH 7.2; O--O, i n c u b a t e d a t p H 7.0: / , 2,, i n c u b a t e d a t p H 7.-' ~ i t h h i g h e r c o n c e n t r a t i o n of A T P ; A " A , i n c u b a t e d a t p H 7.(} w i t h h i g h e r c o n c e n t r a t i o n of ,VI'IL
Hiochim. t~io~]t~,s ,qcla, 124 (~0(~) 3 ~ ° 322
p H - D E P E N D E N T GLYCOLYSIS
315
pH levels. Hexokinase activity, expressed as the rate of disappearance of 2-deoxyglucose, was not significantly accelerated when the pH was raised from 7.2 to 7.6 (Table III). The activity of glucosephosphate isomerase was also virtually indifferent to the change of pH of the buffer (Fig. 3). In contrast, Fig. 3 also shows the importance of phosphofructokinase actixdty in the "pH effect", in which the inhibition of phosphofructokinase activity 1° was exaggerated at a lower pH level according to spectrophotometric assay in the diluted state. Consequently, an apparent stimulation of phosphofructokinase by shifting the pH to a higher level was only markedly evident in the presence of a higher concentration of ATP. The results obtained for the concentrated extract (Table IV) lend support to the significance of phosphofructokinase. When glucose was employed as substrate, glycolytic activity of the extract was limited by hexokinase in the presence of ATP at 2.5 raM, and thus neither Glu-6-P nor Fru-6-P was detected after incubation (Expt. 2 in Table IV). In the presence of a much higher concentration of ATP, as a result of phosphofructokinase inhibition, measurable amounts of Glu-6-P and Fru-6-P existed after incubation, suggesting that phosphofructokinase rather than hexokinase acts as a rate-limiting step in this case. The interconversion rate between Glu-6-P and Fru-6-P was found to be extremely high in the extract (cf. refs. II, 12). When glucose was either combined with yeast hexokinase or replaced by Glu-6-P to avoid the involvement of hexokinase, the residual level of Glu-6-P and Fru-6-P after incubation could represent the phosphofructokinase activity, because the reaction catalyzed by phosphofructokinase is rate-limiting, as verified by the absence of Fru-I,6-P 2 after incubation. The stimulation of phosphofructokinase on raising pH T A B L E 1V LEVEL OF GLYCOLYTIC INTERMEDIATES AFTER INCUBATION AT p H 7.2 OR p H 7.6 Cell-free e x t r a c t from r a t d i a p h r a g m was i n c u b a t e d a t 22 ° for 15 m i n in a n a t m o s p h e r e of 93 % 0 3 7 % COz (p H 7.2) or lOO% O a (pH 7.6). E a c h t u b e c o n t a i n e d t h e e x t r a c t (0.2 ml) a n d o t h e r c o f a c t o r s in a final v o l u m e of 0.25 ml. The r e a c t i o n was s t o p p e d b y t h e a d d i t i o n of HC104. The s u p e r n a t a n t n e u t r a l i z e d w i t h 2 M K O H was s u b m i t t e d to e n z y m i c a n a l y s i s as d e s c r i b e d in t e x t . The c o n c e n t r a t i o n of s u b s t r a t e was 5 mM. W h e r e i n d i c a t e d , y e a s t h e x o k i n a s e , 5 ° / z g pe r t ube , was a d d e d .
Expt. No.
I*
Substrate
Intermediate analyzed
Levels after incubation (m#mole/mg protein) 2.5 m M A T P
zo m M A T P
p H 7.2 p H 7.6
p H 7.2 p H 7.6
Glu-6-P
Glu-6-P Fru-6-P Fru-I,6-P
223 l°9 o
26 44 o
319 128 o
IOO 66 20
Glucose
Glu-6-P Fru-6-P Glu-6-P Fru-6-P
o o 314 15I
o o 155 7°
82 42 487 220
31 25 215 94
Glu-6-P Fru-6-P
622 218
239 III
748 257
337 131
Glucose plus y e a s t h e x o k i n a s e Glucose plus y e a s t h e x o k i n a s e
* E D T A - M g ~+ (0. 5 mM) was added.
Biochim. Biophys. Acta, 124 (1966) 31o-322
316
M. UI
and the inhibition on adding a larger a m o u n t of A T P were manifested by a fall and rise, respectively, in the residual levels of both Glu-6-P and F r u - 6 - P (Table IV). It is a well-known fact t h a t Glu-6-P is a potent inhibitor of hexokinase. Since the inhibition or activation of phosphofructokinase is effective in raising or lowering the Glu-6-P level, respectivdy, (due to the rapid equilibrium between Glu-0-P and F r u - 6 - P as a result of high activities of glucosephosphate isomerase) it m a y be assumed t h a t modification of t)hosphofructokinase activity can exert a profound influence on hexokinase activity. Such a conclusion was verified by the d a t a in Table V, in which the rate of inhibition of hexokinase in the presence of F r u - 6 - P was reduced to some extent b y activating phosphofructokinase at a higher pH level. The significance of phosphofructokinase in the regulation of hexokinase activity via the (ilu-6-P level was shown in erythrocytes b y ROSE AND O'CONNEL11 and in brain b y AISENB E R G 13.
Disappearance of "pH effect" in the aged extract As is evident in Fig. 2, aging of the cell-free muscle extract without addition of substrate diminished the overall glyeolytic activity with a concomitant disappearance of the "pH effect". Table VI shows that hexokinase (in the absence of TABLE
V
HEXOKINASG
A C T I V I T Y OF T H E
CELL-FREE
MUSCLE
EXTRACT
WITH
OR W'ITHOUT ADDED
Fru-6-P
C e l l - f r e e e x t r a c t f r o m r a t d i a p h r a g m w a s i n c u b a t e d a t 22 ° f o r 15 m i n in t h e p r e s e n c e of l o m M A T P in 93 o/ /o 0 . ) - 7 o, 7o C O 2 ( p H 7.2) or IOO°'o 02 { p H 7.6). F i n a l v o l u m e , 0 . 2 5 m l . T h e c o n c e n t r a t i o n s of 2 - d e o x y g l u c o s e a n d F r u - 6 - P w e r e e a c h r m M .
Fru-6- P
-
2- Deoxyglucose utilizatio~l (ml~mole/mg protein)
-
+
TABLE
pH 7.2
p H 7.6
i2i t7
138 54
VI
ACTIVITIES
OF
GLYCOLYTIC ENZYMES
BEFORE
AND
AFTER
AGING OF T H E
EXTRACT
A g i n g of c e l l - f r e e e x t r a c t w a s c o n d u c t e d a s in F i g . 2. H e x o k i n a s e a n d g l u c o s e p h o s p h a t c i s o m e r a s e a c t i v i t i e s w e r e a s s a y e d a s d e s c r i b e d i n T a b l e IX: a n d F i g . 3, r e s p e c t i v e l y . P r o d u c t i o n of l a c t a t e w a s m e a s u r e d a f t e r i n c u b a t i o n a t 22 c' f o r 15 m i n w i t h t h e a d d i t i o n of F r u - r , 6 - P 2 (2 r a M ) .
Before aging pH 7.2
drier agiug pH 7.6
pH 7.2
pft 7.0
Hexohinase activity (2-deoxyghtcose utilization, mltmole/mg protein) "Without Glu-6-P With Glu-6-P
157 12
182 73
171 2o
~SS 33
Glucosephosphate isomerase activity (Fru-6-P utilized, ml*mole/mg protein) 895
Io88
895
1o53
Lactate production with the addition of Fra-z,6-P~ as substrate (mt*mole/mg protei~z) 453
Biochim. Biophys. Acta, 124 ( i 9 6 0 ) 3 I O - 3 2 2
585
317
p H - D E P E N D E N T GLYCOLYSIS
GIu-6-P), glucosephosphate isomerase and complex enzyme systems below ketosei-phosphate aldolase survived the aging process without loss of activity. It can be seen in Fig. 4 that phosphofructokinase activity was markedly depressed by aging with striking reduction in the degree of pH dependency. This finding is further supported by tile data in Table VII, in which the level of Glu-6-P and Fru-6-P after incubation, in the presence of 2.5 mM of ATP, was elevated in the aged extract.
~J
c
~
Time (mln)
5
Fig. 4. Phosphofructokinase activity before and after aging of the cell-free muscle extract. Phosphofructokinase was assayed with the addition of 3 mM ATP as described in Fig. 3- O - - © , non-aged e x t r a c t incubated at p H 7.2; 0 - - 0 , non-aged e x t r a c t at p H 7.6; A - - A , aged e x t r a c t at p H 7.2; ~k--Jk, aged e x t r a c t at p H 7.6. TABLE VII LEVELS
OF
GLYCOLYTIC
INTERMEDIATES
AFTI~R
INCUBATION
OF
NON-AGED
AND
AGED
EXTRACT
The extract was incubated before or after aging and s u b m i t t e d to enzymic analysis as described in Table IV.
Expt. No.
Substrate
Glucose
Intermediate analyzed
Glu-6-P Fru-6-P
Glucose plus yeast hexokinase
Glu-6-P Fru-6-P
2
GIu-6-P
Glu-6-P Fru-6-P
A TP (raM)
Levelsafter incubation (m#moles/mg protein) Non-aged
Aged
pH 7.2 pH 7.6
pH 7 2
pH 7.6
2. 5 io 2. 5 IO 2. 5 IO 2. 5 IO
o 82 o 42 314 487 151 220
23 68 27 46 480 525 221 249
17 51 21 38 450 514 193 220
2.5 io 2.5 IO
39 273 32 115
o 31 o 25 155 215 7° 94
292 316 138 139
Hexokinase activity in the glycolytic system assayed in the presence of Glu-6-P at higher pH was markedly inhibited upon aging the extract (Table VI). In view of the above proposition that Glu-6-P-induced inhibition of hexokinase in the non-aged extract is apparently reduced at higher pH as a result of increased phosphofructokinase activity (Table V), the apparent inhibition of hexokinase in the presence of Biochim. Biophys. Acta, 124 (1966) 31o-322
318
M. uI
Glu-6-P observed at higher pH in the aged extract m a y be reasonably attributed to the failure of phosphofructokinase activation under such conditions. Minor responsiveness of phosphofructokinase in the aged extract (as compared with that in the non-aged extract) to the change of p H is also noted in all the experiments presented in Table VII. Moreover, the magnitude of the inhibition of phosphofructokinase b y high concentrations of ATP was often reduced when aging was conducted prior to incubation. Thus, the aging process m a y depress the enzymic activity and reduce the response to changes of p H and ATP concentrations. Such modification of phosphofructokinase activity m a y lze due to changes in the enzyme itself rather than the involvement of non-protein factors, since, as shown in Table V I I I , the phosphofructokinase activity of the extract prepared by combining aged extract with non-aged extract in equal amounts was practically identical to the value calculated from the separate enzyme activities of the aged and non-aged extracts. Furthermore, the supernatants of the boiled extracts of aged and non-aged preparations did not differ in their effect on phosphofructokinase activity when added to the extract incubated with Fru-6-P as substrate. TABLE VIII PHOSPHOFRUCTOKINASE
ACTIVITY
OF
NON-AGED
AND
AGED
EXTRACT
Cell free e x t r a c t from r a t d i a p h r a g m , before or after a g i n g as d e s c r i b e d in Fig. 2, was i n c u b a t e d w i t h t h e a d d i t i o n of 5 mM F r u - 6 - P . E a c h t u b e c o n t a i n e d o.2 ml of " n o n - a g e d " e x t r a c t , o.2 ml of " a g e d " , or o.I ml of each ( " n o n - a g e d plus a g e d " ) : final vol ume , o.25 nil. Boiled s u p e r n a t a n t was p r e p a r e d b y i m m e r s i n g " n o n - a g e d " or " a g e d " e x t r a c t in b o i l i n g w a t e r for i5o sec a n d t h e n c e n t r i f u g i n g off t h e p r e c i p i t a t e d protein, o. t ml of each w a s a d d e d to t h e r e a c t i o n m i x t u r e before i n c u b a t i o n , t h e final x o l u m e b e i n g o.35 ml in such cases.
Addition
None* Boiled s u p e r n a t a n t of n o n - a g e d e x t r a c t B o i l e d s u p e r n a t a n t of aged e x t r a c t
Fret 6-1) level after incubation (mktmole/mg protein) Non aged
Aged
Non aged ph*s aged
211 3oo 3oo
443 455 4'io
314 (327)
* The figure in p a r e n t h e s e s is the m e a n of t h e v a l u e s o b t a i n e d for t h e " n o n - a g e d " a n d " a g e d " e x t r a c t s alone. S t a n d a r d d e v i a t i o n s c a l c u l a t e d were 3.5, lO.8 a n d 9.4 fur " n o n - a g e d " , " a g e d " a n d " n o n - a g e d plus aged", r e s p e c t i v e l y .
A TP-induced inhibition of muscle phosphofruclokinase at various pH levels Results presented above pointed to the significance of ATP concentration in the apparent stimulation of glycolysis by pH in which phosphofructokinase activity acted as a limiting factor. The rate of the reaction catalyzed by tile phosphofruetokinase isolated from rabbit skeletal muscle was then tested at various concentrations of ATP and hydrogen ions. Enzyme assay was not done at pH values below 6.7,~ because of the loss of buffering capacity of the Tris buffer below pH 6.6. Fig. 5 shows t h a t the p H optimum of phosphofructokinase was shifted to the alkaline side on increasing ATP concentration. The reaction did not proceed at all when assayed at lower p H in the presence of excess ATP. In Fig. 6, initial velocities were plotted against ATP concentrations in the "inhibitory phase" at various pH levels. It can be seen t h a t precipitous inhibition occurred at lower pH (below 7.6) as the ATP Biochim. Biophys..4cla, 124 (x960) 31o-322
pH-DEPENDENT
319
GLYCOLYSIS
concentration was raised to 3 raM. Increase in the p H of the medium resulted in a striking restoration of phosphofructokinase activity in the presence of excess ATP. A similar recovery from ATP-induced inhibition was found to take place when the amount of Fru-6-P was increased as shown in Fig. 7. :3,. E
~-- 0,2!
0
O
~c
,J E o.2-
Ja5 L;5
~ .c_
.C "E-
g~ c
~.~
o
J
o.1-
~-
8.o
7.0
9.o
pH
16,0
kJ
0
~A
v [.AMP] (raM)
Fig. 5. p H - p h o s p h o f r u c t o k i n a s e a c t i v i t y c u r v e s at h i g h e r c o n c e n t r a t i o n s of ATP. P h o s p h o f r u c t o kinase purified f r o m r a b b i t skeletal m u s c l e according to GATT AND RACKER7 a n d stored b y susp e n d i n g in 5 ° % s a t u r a t e d (NH4)2SO 4 at o ° was diluted 2•-fold with o.I M Tris buffer (pH 7.6). E a c h c u v e t t e c o n t a i n e d I. 5 ml o.i M Tris buffer c o n t a i n i n g MgC12 (final concentration, 3 mM), o.oi m l diluted p h o s p h o f r u c t o k i n a s e , o.i ml ATP, o.o 3 ml of e n z y m e m i x t u r e ( k e t o s e - I - p h o s p h a t e aldolase, t r i o s e p h o s p h a t e isomerase a n d glycerol-3-phosphate d e h y d r o g e n a s e , each e q u i v a l e n t to t h e a m o u n t capable of c o n v e r t i n g i / * m o l e s u b s t r a t e p e r rain) a n d o. 4 # m o l e N A D H . T h e reaction was initiated b y t h e a d d i t i o n of o.05 m M F r u - 6 - P . Decrease in a b s o r b a n c e a t 34 ° m # d u r i n g t h e initial 2 rain was p l o t t e d as initial velocity on t h e ordinate. T e m p e r a t u r e , 22 °. O - - O , o.I m M ATP; A--&,o.4mMATP; El--[2, ImMATP; • O, 3mMATP;A--A,5mMATP; re__m, 8 m M ATP. Fig. 6. Effect of A T P c o n c e n t r a t i o n s on t h e kinetics of p h o s p h o f r u c t o k i n a s e . T h e e x p e r i m e n t w a s carried o u t as described in legend to Fig. 5- O - - O , p H 6.78; A - - & , p H 7.1; [ ] - - E l , p H 7.3; ~--~,pH7.6; O--Q, pH8.o;A--~k, pH8.3; .--I, p H 8.8; Q - - Q , p H 9 . 3 ; × - - × , p H g . 6 . :::L.
E O
0.5"6.c_
~~ o.3 o
~0.2
._c .c
~
ol
c u
o
2
4
6
8
10
12
14
[AT P.] (raM)
Fig. 7. Effect of F r u - 6 - P c o n c e n t r a t i o n s on t h e kinetics of p h o s p h o f r u c t o k i n a s e . T h e e x p e r i m e n t was carried o u t as described in legend to Fig. 5. O - - O , o.o25 m M F r u - 6 - P ; & - - A , o.o 5 m M F r u - 6 - P ; [ ] - - E l , 0.075 m M F r u - 6 - P ; 0 - - 0 , o.i m M F r u - 6 - P ; A - - , , 0.25 m M F r u - 6 - P ; I - - I , 0. 5 m M F r u - 6 - P .
It m a y be reasonably assumed that the "pH effect" of muscle glycolysis is due to the pH-dependent inhibition of phosphofructokinase b y ATP as illustrated in Figs. 5 and 6.
Biochim. Biophys. Mcta, 124 (i966) 31o-322
320
M. UI
DISCUSSION
In the preceding papers 1,2, it was shown that induction of alkalosis in the organism as well as incubation of nmscle slices in the alkaline medium markedly affected the action of hormones such as epinephrine on carbohydrate metabolism. Marked hyperlactacidemia observed during alkalosis and the promotion of lactate formation i~ vitro by the muscle slices in a medium at higher pH was suggestive of accelerated glycolysis in such a case. Studies with the cell-free glycelytic systems reported here have indicated that glycolysis is actually stimulated by raising p t t and t h a t the major factor responsible for the " p H effect" is phosphofructokinase which operates as one of the rate-limiting factors in gtycolysis. The significance of phosphofructokinase, together with hexokinase, in the regulation of glycolysis has repeatedly been pointed out by m a n y investigators~°, 12-16. The supposition that the " p H effect" observed in the overall glycolytic reaction is solely due to the alteration of phosphofructokinase activity appears to be disfavored only b y the finding that, while the overall glycolytic rate was not affected by pH change at the lower ATP concentration (Fig. I), the phosphofructokinase activity (expressed in terms of residual levels of Glu-6-P and Fru-6-P after incubation) was stimulated by raising the p H of the medium even at the lower concentration of ATP (Table IV). This apparent discrepancy m a y be explained as follows. In the experiments shown in Fig. I, muscle extract was incubated at 37 ° for a long period of time (9 ° min) in contrast to the mild incubation employed for experiments shown in Table IV (22 ° for IO min). During incubation of whole extract at a higher temperature, added ATP was found to undergo rapid hydrolysis through activation of particulate ATPase activity. It m a y be deduced, therefore, that the effective doses of ATP in Fig. I were far less than those given on the abscissa. Moreover, tile experiment presented in Fig. 3 demonstrates that at a lower concentration of ATP phosphofructokinase activity was not essentially affected by a change in the pH of the medium. A closer exainination {}f rabbit skeletal nmscle phosphofructokinase, as shown in Figs. 5-7, revealed the dependence of ATP-induced inhibition upon the pH level, confirming the direct relationship between "pH effect" and ATP concentration. As shown in Table V, the modification of t)hosphofructokinase activity is reflected in the hexokinase reaction through the mediation of change in (;lu-6-1' levels. On the other hand, the hexokinase reaction could control the phosphofructokinase activity, because Fru-6-P, rapidly formed by glucosephosphate isomerase from Glu-6-P, exerts a profound influence on the inhibitory action of ATP on phost)hofructokinase as shown in Fig. 7 (see also refs. i 7 --I0). Thus, interdependence of the possible rate-limiting steps, hexoldnase and phosphofructokinase, would contribute to the regulatory mechanism involved in cellular glycolytic rates. Since (ilu-6-I) also functions as an activator of UDPglucose-glycogen glucosyl transferase (EC z.4.I.I I ) and as a substrate for glucose-6-phosphate dehydrogenase, which supplies the reduced form of NADP + for lipogenesis or other reduction steps in metabolism, it would appear t h a t the regulation of phosphofructokinase activity exerts a broad influence on cellular metabolic activity. As is evident in Fig. 6, muscle phosphofructokinase activity is profoundly influenced b y a change of ATP concentration from o.5 to 2 mM, depending on p H wtlues ranging from 7.I to 7.6 when Fru-6-P was maintained at o.o 5 raM. In view <~f the Biocki.z. Biopkys. Acta, I24 (I9()()) 31o 3 ; 2
pH-DEPENDENT
GLYCOLYSIS
321
fact that these concentrations of ATP, Fru-6-P and H ÷ never involve a marked departure from cellular concentrations (cf. ref. I7), it is possible that subtle change of hydrogen ion concentration in the cellular fluid plays a physiological role in the regulation of cellular glycolytic activity. For example, a drop in cellular hydrogen ion concentration that is assumed to take place during active oxidative phosphoryiation in the mitochondria could act as a physiological feed-back mechanism protecting the glycolytic activity from excessive blocking via the Pasteur effect. PASSONNEAU AND LOWRY10 postulated that in muscle phosphofructokinase there are two ATP sites, one catalytic and one allosteric, and the ATP-induced inhibition m a y be--according to their postulation--due to the binding of ATP on the allosteric site. A similar observation has been reported for yeast is and E s c h e r i c h i a coli 19 phosphofructokinase. The pattern shown in Figs. 6 and 7 illustrates the velocity of phosphofructokinase reaction after ATP began to occupy the inhibitory site. The fact that sigmoid, rather than hyperboloid, curves were obtained for the plots of velocity v e r s u s ATP concentration m a y be suggestive of the existence of two (or more) inhibitory sites for ATP in addition to one catalytic site. As the pH was raised, the slope of the inhibition curve became less. This m a y be a manifestation of the reduced affinity of ATP for the inhibitory site(s). It m a y be proposed, then, that a drop in hydrogen ion concentration causes a conformational change at the allosteric site probably by spatial changes in hydrogen bonding or in other molecular linkages, thus preventing ATP from occupying the allosteric site(s). Detailed discussion on the inhibitory site for ATP in the phosphofructokinase protein, together with other kinetic data, will be published elsewhere. The aging of cell-free muscle extract in the presence of ATP led to a diminution of phosphofructokinase activity as well as a disappearance of the inhibition due to increased ATP concentration and of the stimulation by increased pH (Fig. 4 and Table VII). The activities of the aged and non-aged extracts were additive when assayed together for the enzyme activity, as shown in Table V I I I . This, together with the similarity of the effect of boiled extracts on phosphofructokinase, seemed to rule out changes, during aging, in the level of certain heat-stable metabolites or cofactors that could affect the activity of the enzyme, and might suggest that changes in the enzyme protein itself were involved. VINI~ELA et al. ~° reported that aging of crude yeast extract in the presence of N a F rendered the phosphofructokinase unresponsive to the inhibition by ATP. Thus, loss of susceptibility to the inhibition by ATP after aging is common to yeast and muscle phosphofructokinase. In view of recent reports that phosphofructokinase undergoes a reversible conversion from an inactive to active form~°,21 and is a dissociable protein22, 23 characteristic of an allosteric enzyme 24, modification of reactivity after aging of the extract m a y be explained on the basis of "desensitization" of the allosteric protein. This would be as a result of dissociation of the enzyme molecule into subgroups or of other drastic conformational changes due to exposure to higher temperature without substrate. Further studies are now in progress on the kinetic behavior of muscle phosphofructokinase after a variety of "desensitization processes". NEFERENCES I M. UI, Am. J. Physiol., 2o9 (1965) 353. 2 M. UI, Am. J. Physiol., 209 (1965) 359.
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M. u i
3 V. S. VVTARAVDEKAR AND L. D. SASLA~V, J . Biol. Chem., 234 (1959) 1945. 4 S. B. BARKER AND W. H. SUMMERSON, J. Biol. Chem., 138 (1941) 535. 5 F. TAYLOR, in S. P. COLO~VICK AND N. O. I(APLAN, Methods in Enzymology, Vol. 1, \ c a d e m i c Press, New Yo rk , 1955, p. 31o. 6 M. W. SLEIN, in S. P. COLOWICK AND •. O. I"~APLAN, Methods in. Enzymology, Vol. 7, A c a d e m i c Press, N e w Yo rk , 1955, p. 3o4 . 7 S. GATT ANn E. RACKER, J. Biol. Chem., 234 (I959) l o l 5. 8 0 . I-I. LOWRY, N. J. ROSEBROISGH, A. L. FARR AND ]{. J. RANDALL, J. 13iol. Che~z., 193 (i~)5J) 265 . 9 W. QEVERS AND E. DAWDLE, Clin. 5"ci., 25 (1963) 344" IO J. ¥*. PASSONNEAU AND O. H. LOWRY, Bioehem. Biophys. Res. Commun., 7 (1962) lo. 11 I. A. R o s e AND E. L. O'CONNEL, J. Biol. Chem., 239 (1964) 12. 12 P. (-')ZAND AND H. T. NARAHARA, J. Biol. Chem., 239 (1964) 3146. 13 A. C. AISENBERG, J. Biol. Chem., 234 (1959) 441 . 14 R. WtJ AND E. RACKER, J. Biol. Chem., 234 (1965) lO29. 15 O. H. LOVCRY, J. V. PASSONNEAU, F. X. HASSELBERGI~2R AND D. ~V. SCHULZ, ./. Biol. Uhem, 239 (1964) 18. 16 D. M. REGEN, W . W. DAvis, H. E. MORGAN AND C. l(. PARK, J. Biol. Chem., 239 (1964) 43. 17 T. E. MANSOUR, J. Biol. Chem., 238 (1963) 2285. 18 A. RAMAIAH, J. A. HATHAWAY AND D. E. ATKINSON, J. Biol. Chem., 239 (1964) 361(). 19 D. E. ATKINSON AND G. M. WALTON, J. Biol. Chem., 24o (1965) 757. 2o E. VINI3ELA, M. L. SALAS, M. SALAS AND A. SOLS, Biochem. Biophys. Res. Commun., 15 (~904) 243. 21 T. E . MANSOUR, J. Biol. Chem., 24o (1965) 2165. 22 T. E. MANSOUR, N. \V. WAKID AND H. M. SPROUSE, Biochem. Biophys. Res. CommaJz., ~0 (1965) 72~. 23 A. PARMEGGIANI AND E. G. b2REBS, Federation Proc., 24 (1965) 284. 24 J. MONOD, J. WYMAN .aND J.-P. CHANGEUX, J. Mol. Biol., 12 (1965) 88.
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