TJIE .JOURZ~AL OF RIOLOGIC~L CHE\IISTRY Vol. 240, No. 6, June 1965

Printd in C.S.A.

Rate-limiting Factors in Glycolysis and Inorganic Orthophos-

phate Transport in Rat Liver and Kidney Slices*

RAY Wu

From the Department of Biochemistry, The Public Health Research Institute of The City of New York, Inc., New Yod, New York 10009

(Received for publication, February 5, 1965)

It is generally recognized t’hat various types of cells or tissues differ in the mechanisms of metabolic control. Accordingly, the regulatory mechanisms for glgcolysis of each tissue must be determined individually. This paper is concerned with the rate- limiting factors of glycolysis of rat liver and rat kidney slices. The following fact’ors were analyzed, since one or several of them can control the over-all rate of glycolysis of the intact cells: (a) transport of glucose into the cells, e.g. limiting for glycolysis in muscles (l-4); (b) availability or transport of inorganic ortho- phosphate, e.g. limiting in ascites tumor cells (5) ; (c) availability of cofactors, e.g. adenine nucleotides was limiting in HeLa cells (6); (d) activity of glycolytic enzymes, limited either by the amount of enzyme or indirectly by the presence of enzyme in- hibitors in the cell, e.g. glucose G-phosphate inhibits hexokinase in the control of eryt,hrocyte g y 1 colysis (7), and ATP inhibits phosphofructokinase in the control of glycolysis of muscle (4, 8), brain (9), or tumors (10).

In addition to its role in the control of glycolysis, Pi seems to be important in other cellular activities, e.g. the control of respira- tion (11) and the balance of the cellular nucleotides (12). There- fore, Pi transport into the kidney cells was studied in greater det.ail. Preliminary reports on the energy requirement for Pi transport and on certain aspects of the control of glycolysis in these tissues have already appeared (10, 13).

EXPERIMENTAL PROCEDURE

Rat Liver and Kidney Slices--Albino male rats (Wistar strain) weighing between 200 and 250 g were used. The rats were either fed ad Zibitum or deprived of food for 50 hours before the experiment,. When perfusion was performed (14), the rat was killed by vertebrae dislocation, and the heart was perfused with Krebs-Ringer-bicarbonate buffer. When perfusion was not performed, the rat was decapit.ated and exsanguinated before the liver and kidneys were removed. Liver and kidneys were chilled and then sliced either with a McIlwain mechanical tissue chopper (Brinkmann Instruments, Inc., Great Neck, New York) to a thickness of 0.416 mm or with a Stadie-Riggs tissue slicer. The pooled slices were washed once with 15 ml of cold Krebs-Ringer- bicarbonate buffer, blotted, and weighed. Glycolysis and Pi transport were studied by incubating approximately 200 mg of slices in the presence of r4C-glucose, ‘*Pi, and 3H-inulin; after incubation, the slices and incubation mixture were treated as previously described (14). In addition to determining lactate

* This investigation was supported by Public Health Service Research Grant CA-05706 from the Xational Cancer Institute.

in the incubation medium, lactate in the slices was also deter- mined. The results in terms of lactat’e production represent the combined values of both these determinations. The determina- tions of total lactate (calorimetric method), l%Xactate, glucose, Pi, and other intracellular intermediates were performed as described previously (5, 14). DPN was determined fluorimetri- tally, in a system that contained 0.1 M Tris buffer (pH 9.5), 0.57, ethyl alcohol, and 20 pg of alcohol dehydrogenase. DPNH was determined fluorimetrically, in a system that contained 0.2 M t.riethanolamine buffer (pH 7.5), 0.002 M sodium pyruvate, and 10 pg of lactate dehydrogenase.

Intracellular Glucose and Lactate Concentration-The intra- cellular concentrations of glucose and lactate in kidney slices were determined as follows. Kidney cortex slices (200 mg per experiment) were incubated in a Warburg vessel with both a single and a double side arm (10) in the presence of 3H-inulin (200,000 cpm) and varying concentrations of r4C-glucose in 1.5 ml of Krebs-Ringer-bicarbonate buffer. After 10 min of gassing with the gas mixtures indicated in the tables, 14C-glucose and 3H-inulin were tipped from the double side arm into the main compartment. After 20 min of incubation at 37”, most of the incubation medium in the main compartment was tipped into the double side arm, and then 0.2 ml of 4 N perchloric acid was im- mediately tipped from the single side arm into the main compart- ment, which contained the slices and some remaining medium. After incubation for 2 more min t’o assure complete deproteiniza- tion, the vessel was opened to air and chilled. The incubation medium in the double side arm was removed with a capillary pipette and was used for the assay of glucose and 14C-lactate. After 1 ml of water had been added to the main compartment of the vessel, the slices were poured out with the perchloric acid into a graduated conical centrifuge tube, and the volume was noted. The slices were broken into fine pieces, the sample was cen- trifuged, and the supernatant solution was neutralized with KOH. After removal of KClO&, one part of the neutralized sample was used for the enzymatic determination of glucose with glucose-6-P dehydrogenase, another part was diluted 20-fold with water and passed through a Dowex l-acetate column (1 x 3 cm) and eluted with 15 ml of water to determine the 14C-glucose in this sample. A third part was used for the determination of intracellular 14C-lactate, and 0.05 ml was counted for 3H-inulin. The per- centage of 3H-inulin in the sample represents the amount of medium included in the cellular sample. This information was needed in order to correct for the 14C-glucose extracellularly trapped with the slices.

2373

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2374 Glycolysis and Pi Transport in Liver and Kidney Slices Vol. 240, No. 6

Glycolytic Enzymes-Glycolytic enzymes of the tissue homoge- dehydrogenase), except that EDTh (3 mM) was included to nates (600 X g supernatant solution) were assayed spectro- counteract metal inhibit,ion of this enzyme. Traces of Zn++

photometrically at pH 7.4 (5). Pyruvate kinase was measured were present in Tris buffer and KC1 solutions. Added Zn++, as before (coupled to DPNH oxidation in the presence of IacOate at 1 x low5 M, was found to inhibit pyruvate kinase activity by

TABLE I

Glycolysis and Pi uptake in rat liver slices Liver slices from fed rats were prepared by either a Stadie-Riggs slicer or a McIlwain chopper, and 220 mg of slices were used per

experiment. The slices were either suspended in 1.8 ml of Krebs-Ringer-bicarbonate buffer (Pi = 1.2 mM) or in serum where indi- cated. After incubation and gassing at 37” for 10 min, a mixture (0.05 ml) of l*C-glucose (8 pmoles = 100,000 cpm), 3H-inulin (0.2 mg = 300,000 cpm), and 32Pi (0.1 rmole = 20,000 cpm) was tipped into the main compartment from the side arm of the Warburg vessel. Results are expressed as micromoles per g of slices (based on the initial wet weight; values would be 40 to 70’% higher if based on the wet weight of slices at the end of incubation). -

) co2-02 COPN2 Slices prepared

by Perfusion Additions Amount Incubation time -

I 4C-Lactat

mi?z

30 60 30 60

2.4 1.2 4.2 1.7 2.4 1.0 4.8 1.6

60 4.2 60 7.4 60 8.7 60 6.0 60 5.5 60 3.3

1

- I: e : ‘2Pi uptake 4C-Lactate ‘2Pi uptake ‘otal acid”

1.6 2.4 2.0 3.6

10 15 12 18

1.8 12 2.8 24 3.2 23 2.5 17 2.5 17 1.9 13

i -

-

I-

1.6

1.4 2.0 1.4 1.4

0.2 0.2 0.3 0.3

0.3

0.4

0.5 0.3

Slicer Yes

Chopper Yes

Chopper NO None Rat serum 1.7 ml

1.7 ml 1.7 ml 1.7 ml 9 rnMd

Dialyzed rat serum6 Horse serum< Dialyzed horse serumb L-Lactate

a Measured manometrically. b Dialyzed for 60 hours against Krebs-Ringer-bicarbonate buffer; Pi at end of dialysis = 1.2 mM. ‘Pi = 1.5 mM.

d About 40yo of the added lactic acid was used up in 60 min.

TABLE II

Time course of glycolysis and glycogenolysis in rat liver slices

Liver from a perfused rat was used. Liver slices were prepared by the McIlwain tissue chopper, and 220 mg of slices were used per experiment. The slices, suspended in 1.8 ml of Krebs-Ringer-bicarbonate buffer, were incubated at 37” and gassed for 10 min; 0.05 ml of ‘*C-glucose (8 pmoles = 100,000 cpm) was then tipped in. Results are expressed as micromoles per g of slices. - -

Rat

coz-02 COPNz Incubation

time -7 - Glucose

producedC 4C-Lactat Total lactateb Total acidd Glucose”

produced

min

20 40 60 90

0.9 3.4 16 0.6 2.0 3.5 8

1.6 5.6 19 1.1 3.3 6.7 9

2.4 7.2 25 1.7 4.2 10.0 10 3.3 7.4 32 2.4 5.3 14.6 14

20 0.4 1.5 6 0.3 1.2 2.5 4 40 0.7 2.0 8 0.6 1.8 4.4 5 60 1.1 2.6 10 1.0 2.3 7.6 6 90 1.6 3.2 12 1.6 3.2 11.0 6

60 2.1 6.2 38 1.3 2.8 8.4 25 60 0 0 50 0 0 7.0 30 60 0 0 50 0 0 7.0 32

Addition Concentration

Iodoacetate 1.0 NaF 20

Fed

Fasted for 50 hours

Fed

i - a ‘%-Lactate calculation was based on the final glucose content instead of the amount of glucose initially introduced. b Total lactate values were corrected for lactate present in the slices at zero time (approximately 5 pmoles per g of liver slices). c Glucose produced = glucose at end of incubation minus glucose value after 10 min of gassing and preincubation. (Approximately

25 pmoles of glucose were produced in slices from fed rats during this 10 min of preincubation.) d Measured manometrically.

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June 1965 R. Wu

about 60 yO, but, this inhibition was completely reversed by EDTA (1 mM) or Pi (70 mM). Purified enzymes used for the various assays were purchased from Boehringer and Soehne, Germany.

RESULTS

Rat Liver Slices

Glycolysis and Pi Uptake-It has been customary to prepare liver slices with tissue slicers, but the McIlwain tissue chopper gave slices of more uniform thickness, and it was much more con- venient to use. Therefore, liver slices prepared by both methods were compared with regard to glycolysis and Pi uptake. The data in Table I indicate that after 30 and 60 min of incubation, slices prepared by the Stadie-Riggs slicer were quite comparable to slices prepared with the McIlwain chopper with respect to the accumulation of 1%~lactate and total acid, and the uptake of Pi. Therefore, the McIlwain tissue chopper was used for all other experiments.

Slices prepared from liver with and without perfusion of the animal were compared next. Again, the rates of glycolysis and 3zPi uptake were similar whether liver was taken from a perfused or nonperfused rat.

The effects of adding serum or lactate to the incubation me- dium are also shown in Table I. When rat serum was used in place of Krebs-Ringer-bicarbonate buffer, the accumulation of 14Clactate and total acid almost doubled; horse serum resulted in only a 4OC/, stimulation. Addition of L-lactate slightly lowered the aerobic accumulation of XLlactate. The effect of n-lactate may be due to its conversion to glycolytic intermediates by a reversal of glycolysis, thus diluting the labeled intermediates formed from ‘%-glucose.

Glycolysis of rat liver slices was measured in three ways, in order to distinguish it from glycogenolysis or acid formation from other endogenous sources. As shown in Table II, the accumula- tion of l*C-lact,ate was almost linear with time up to 90 min of incubation. Conversion of glycogen to lactate, calculated from the difference between total lactate and 14C-lactate, reached the maximum value at around 40 min. In slices of fed animals, both i4C-lactate and total lactate values were considerably higher aerobically than anaerobically, whereas in slices of fasted animals the differences were much less.

The classical method for measuring glycolyais is based on manomet,ric techniques. However, under anaerobic conditions, the values obtained by manometry were found to be 2 to 4 times higher than t#hose obtained by measuring the production of lactate calorimetrically (as will be shown in Table V, there is no such discrepancy with kidney slices). This discrepancy apparently represents the formation of volatile compounds or acids other than lactic acid (“extra acids”). Since both iodoacetate and NaF inhibited lactate production without affecting the formation of extra acids, the latter is therefore not, directly related to glycolysis (Table II). Formation of extra acids of a smaller magnitude has been reported by Elliott, Grieg, and Benoy (15), but the nature of the acids has not been determined. It seems likely that the acids may come from hydrolysis of lipid or protein.

Considerable amounts of glucose were produced from glycogen upon incubation of liver slices. Glucose production was higher in fed rats than in fasted animals, and was higher aerobically than anaerobically. Both iodoacetate and NaF slightly in- creased the accumulation of glucose.

The effects of inhibitors and high concentrations of Pi on glycolysis and Pi uptake are shown in Table III. The concentra- tion of Pi used in the medium (1.2 nnw) was much lower than that

in the cells (4 mM) in order to rule out simple diffusion and to facilitate the study of energy-dependent uptake of Pi. With the use of various inhibitors of respiration and glycolysis, the aerobic uptake of Pi was found to parallel only the rate of “oxy- gen uptake” but not the rate of glycolysis or the level of intracel- lular ATP. In fact, in the presence of sodium azide (2.5 mM), the intracellular ATP level was very low, yet Pi uptake was un- impaired. These data therefore indicate that Pi uptake depends mainly on energy derived from respiration. Under anaerobic conditions, the low rate of Pi uptake was probably due to ex- change, since it was unaffected by various inhibitors and it proceeded even in t,he absence of any energy supply. When the Pi levels in the medium became higher (8 to 20 mM) than that in the cells, diffusion appeared to be largely responsible for the marked increase in Pi uptake under both aerobic and anaerobic conditions. Aerobic 14C-lactate accumulation was found to be slightly stimulated by high levels of Pi.

Determinations of the intracellular concentrations of Pi, ADP, and ATP indicated that the ATP concentration was very low. Under aerobic conditions, the ATP concentration was approxi- mately 0.4 mM, whereas under anaerobic conditions it was as low as 0.03 mM (below the K, values for hexokinase and phospho-

TABLE III

Glycolysis and Pi uptake of liver slices in presence of inhibitors or high levels of Pi

Experimental conditions were the same as in Table I, except that liver slices from nonperfused rats were used. Krebs-Ringer- bicarbonate buffer (Pi = 1.2 mM) was used as the medium, and incubation time was 40 min. Results are expressed as in Table I.

Additions

None . . Dinitrophenol. Sodium azide Sodium azide

NaF............ 20 NaF 30 2-Deoxyglucose 10

None Pi . . . Pi . Pi.

coz-02 C02-N2

‘oncen ratior 32pp

lptake 1 xygen ptake’

Intra- cellular

ATPC

32pp lptake

0.2

3.5 2.5

1.6 1.6 10 0.43 0.8 0.5 1.4 0.8 1 0.06 1.7 1.2 5 0.05 0.5 1.9 1.5 8 0.08 0.5

0.2 1.7 9 0.18 0.5 0.1 1.9 9 0.15 0.9 1.6 0.40 0.5

3 8

20

0.9 0.9

0.1

0.4

1.0 0.9 1.0 1.0

2.0 1.6 10 0.5 2.2 2.4 10 1.0 2.4 4.2 12 2.4 2.8 8.6 5.2

a Expressed as micromoles of 3zPi uptake, and calculated from the counts that entered the cells (corrected for extracellularly trapped counts) divided by the specific activity of 32Pi in the incu- bation medium.

b Expressed as micromoles per g of liver slices per 40 min. Values for oxygen uptake were corrected for CO2 liberation due to glycolysis, but not for respiratory COZ production. They are not true oxygen-uptake values, but represent minimum values.

c Expressed as micromoles per ml of intracellular water.

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2376 Glycolysis and Pi TrampoTt in Liver and Kidney Xlices Vol. 240, S-o. 6

fructokinase). Therefore, ATP is likely to be rate-limiting for glycolysis of rat liver slices, at least under anaerobic conditions.

Glycolytic Enzyme Profile

The enzyme profiles of homogenates of rat liver and kidney are given in Table IV. In comparison with fasted animals, capacity for the formation of glucose-6-P from glucose in the liver of the fed rat was 2 to 3 t,imes higher; since the additional activity was detected only in the presence of high levels of glucose, it is presumably due to an increase in glucokinase (16). Hexokinase activit,y in the kidney was the same in fed or fasted rats. Al- though hexokinase activity was the lowest among glycolytic enzymes, it can support a rate of 14C-lactate formation at least 40 times higher than that observed in both liver and kidney slices.

Rat Kidney Slices

Glycolysis and Pi Uptake-Glycolysis was measured by three independent methods. Under anaerobic conditions, glycolysis in kidney slices as analyzed by the calorimetric determination of lactate formation gave values comparable to that of the mano- metric determination of total acid production (Table V). These measurements included lactate formation from X-glucose as

TABLE IV

Glycolytic enzyme projZes of homogenates of rat liver and kidney

Enzyme activities of tissue homogenates (600 X q supernatant solution) were measured at pH 7.4 and 26”. Results are expressed as micromoles of substrate turnover per min per 100 mg of homog- enate protein of the forward reaction, i.e. the direction of converting glucose to lactic acid. Enzyme activities measured in the presence of pyridine nucleotides have been corrected for DPNH oxidase or TPNH oxidase activity.

ElUyme

Hexokinase. .C

P-fructokinase.. Aldolase. Glyceraldehyde-

3-P dehydro- genase

3-Phosphoglyc- erate kinase .

S-Phosphoglyc- erate mutase

Enolase .

Pyruvate kinase Lactate dehy-

drogenase ATPase . . cu-Glycero-P de-

hydrogenase.

DPNH oxidase .

TPNH oxidase .

Rat liver Rat kidney

Fed animal I Tasted animal Fed animal Fasted animal

1.62 (0.6S)a

1.6 3.7

0 .28 (0. 24)”

1.3 3.1

.5 (2.2)a

5.8 4.3

.9 (1.8)”

5.7 4.1

23 18 28 30

250

2G 9

34

130 19

34

1.2

0.2

180

22 8

14

130 10

27

0.8

0.25

270 280

57 63 16 16

30 20

70 28

71 24

25 25

6

0.2

5

0.2

a Values in parentheses represent measurements made with glucose-6-P dehydrogenase and TPNH.

TABLE V

Time course of glycolysis and Pi uptake in kidney slices Kidney slices (170 mg of wet weight per experiment) from fed

rats were incubated in 1.9 ml of Krebs-Ringer-bicarbonate buffer, pH 7.5. A mixture (0.05 ml) of “Pi (0.1 Mmole = 26,000 cpm), ‘%-glucose (7 pmoles = 80,000 cpm), and 3H-inulin (0.2 mg = 300,000 cpm), was placed in the side arm and tipped into the main compartment after 10 min of gassing. After correction for the blank values of the control vessel, results are expressed as micro-

I min I I I coz-02 20 1.0 -0.9 3.1 1.7 6.8

40 1.8 -1.8 4.9 3.5 6.5 60 2.6 -2.3 G.2 4.8 6.5

--

rntrace11u1ar Pi concen-

tration

COz-Nz 20 2.8 8.0 7.3 1.4 9.0 40 6.2 15.0 14.6 1.4 8.6 60 11.0 22.0 22.0 1.4 8.6

Q Combined values of ‘%-lactate found in the slices and in the incubation medium.

b Corrected for lactate initially present in the slices. c Measured manometrically. d Aerobic minus anaerobic tissue 32Pi values. This calculation

is justified only when thereis no diffusion, i.e. whenthe Pi concen- tration in the medium is several times lower than the intracellular Pi concentration.

well as from endogenous substrates. However, neither method could be used for measuring lactate formation under aerobic con- ditions, since the rate of lactate utilization was greater than that of its production and a net loss of endogenous lactate was ob- served. Ry using ‘Y-glucose, however, 14C-lactate accumulation was found to increase linearly with time, up to 1 hour of both aerobic and anaerobic incubations. Anaerobic glycolysis (as 14C-lactate formation) was about 3 times faster than aerobic glycolysis, with a Pasteur effect close to 707,.

Aerobic Pi transport was linear with time up to 1 hour, whereas anaerobic Pi uptake (probably due to exchange) reached a con- stant value after 20 min. Pi transport will be discussed in greater detail lat.er.

It was found (not shown here) that kidney slices prepared with the Stadie-Riggs tissue slicer or with the McIlwain tissue chopper gave similar results in terms of glycolysis and Pi uptake. Kidney slices of fed rats gave somewhat higher rates of glycolysis than slices of fasted animals.

Comparison of glycolysis and Pi transport was made bet’ween whole kidney slices and their components, cortex and medulla. With values from whole kidney slices used as the control, cortex slices were found (Table VI) to have a lower rate of glycolysis but a higher rate of Pi transport and oxygen uptake. In con- trast, medulla slices had a higher rate of glycolysis but, a lower rate of Pi transport.

Effects of various activators and inhibitors of glycolysis and Pi uptake are shown in Table VII. Increasing the concentra- tions of Pi in the medium greatly stimulated Pi uptake but had no effect on glycolysis. n-Lactate increased the accumulation of 14C-lactate, presumably by diluting the %-lactate t,hat was formed and minimizing its oxidation and utilization. Rat, serum

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June 1965 R. Wu

contained appreciable amounts of lactate, and it stimulated i4C- lactate accumulation and Pi uptake. Dialyzed serum was much less effective. Inosine (4 mM) and imidazole (40 mM) both inhibited aerobic glycolysis slightly without any effect on Pi uptake. Under anaerobic conditions, all these effects were less

TABLE VIII

P, uptake in rut kidney cortex slices

Kidney cortex slices (220 mg of wet weight per experiment) were incubated at 37” in Warburg vessels in 1.3 ml of Krebs-Ringer-bicarbonate buffer (pH 7.5). A mixture (0.05 ml) of “Pi (0.1 pmole = 16,000 cpm), i4C-glucose (5.3 Mmoles = 40,000 cpm), and %-inulin (0.1 mg = 210,000 cpm), was placed in the side arm and tipped into the main compartment after 10 min of gassing. Results are expressed as micromoles per g of wet weight.

TABLE VI

Glycolysis and Pi uptake in whole kidney slices, cortex slices, and medulla slices

Kidney slices (120 mg per experiment from rats perfused with Krebs-Ringer-bicarbonate buffer) were incubated as in Table V, except that incubation was for 40 min.

Gas phase Final

:oncen- tration

ncuba- C-LX tion time

ttepro duced

0% ptake’

32pi

ptakt

coz-02 None None Dinitro-

phenol Sodium azidc Iodoacetate Iodoacetate NaF

COz-Nz None None Sodium azidc Iodoacetate

vu4

0.4

5 0.12 0.50

40

5 0.50

min

20 40 40

40 40 40 40

20 40 40 40

1.4 2.4 8.5

3.3 2.3 5.2 4.2 1.0 0

9.1 0.2 0 0.4

3.6 7.0 9.4 0

1.1 4.8 1.0 5.4

1.0 1.0 0.9 0.9

0.1 3.8 0 4.4

0 0 0 0

a Values for 02 uptake were corrected for CO* liberation due to glycolysis, but not for respiratory COZ production.

6 Aerobic minus anaerobic 32P values represent a2Pi transport.

Slices of kidney

Intra- cellular

32Pi

Intracellular Tr~sg:t Pi concen- 3 I tration

3.5 7.5 3.8 7.6 2.4 6.6

Gas phase La::& lactate uptakea Total Oxygen

coz-02 2.6 31 1.8 36 4.7 25

Whole* Cortex Medulla

3.9 4.1 2.7

Wholeb Cortex Medulla

COz-Ns 0.4 0.3 0.3

~ ~

8.9 7.5 7.0

a Values for “oxygen uptake” were corrected for COZ liberation due to glycolysis, but not for respiratory CO2 production. There-

fore, oxygen uptake represents minimum values. b Whole kidney slices, obtained after removing capsule and

materials in the kidney pelvis together with some medulla, were composed of, on protein basis, 75% of cortex and 25oj, of medulla.

TABLE VII

Effects of activators and inhibitors on glycolysis and Pi uptake

Kidney cortex slices (120 mg per experiment, from rats per- fused with Krebs-Ringer-bicarbonate buffer) were incubated as in Table T’. Experiments 1, 2, and 3 represent independent

pronounced. 2-Deoxyglucose strongly inhibited both the aerobic and anaerobic glycolysis without affecting Pi uptake.

Energy Requirement fcr Pi Transport-The uptake of Pi by kidney cortex slices was studied in greater detail. The data in Table VIII show that Pi uptake (with 1.2 mM Pi in the medium) was several times as high aerobically as anaerobically. Anaero- bic Pi uptake stopped after 20 min (see also Table V) and was unaffected by iodoacetate or azide. In contrast, aerobic Pi transport increased linearly with time and was susceptible to the following inhibitors. The transport was inhibited by dinitro- phenol and by high concentrations of aside or iodoacetat,e, all three of which strongly inhibited respiration.

There was only a slight inhibition of respiration by KaF or by low concentrations of iodoacetate (0.12 mM), both of which completely inhibited glycolysis without affecting Pi transport. The data indicate tha.t Pi transport depends predominantly on energy derived from oxidative phosphorylation. Since the intracellular concentration of Pi was about 4 times higher than that in the medium, and since the transport of Pi is dependent on an energy supply, it is regarded as active transport.

With the use of inhibitors, further experiments were carried out to explore the nature of the high energy compounds which supply energy for the transport of Pi. The selection of the inhibitors was based on the reports that azide and oligomycin

prevent the esterification of Pi in oxidative phosphorylation

(17-19). As shown in Table IX, at appropriate concentrations of azide, oligomycin, and dinitrophenol, respiration was inhibited

about 30% by all three compounds, and aerobic glycolysis was increased to levels similar to the anaerobic value. However, Pi transport was inhibited only by dinitrophenol, not by low con-

centrations of azide or oligomycin. These findings suggest that

experiments performed on different dr

CO%.01 C02-N2 Incuba- ion time MC. 32Pi

,actate ,ta.k& 32P;

ptak@ “ I

-

min

20 2.4 2.9 5.3 1.3 40 3.7 4.3 11.0 1.3 20 2.5 10.5 5.5 9.0 40 3.8 13.0 12.2 12.6

60 5.5 4.4 18.0 0.8 60 9.8 4.0 20.0 0.8 60 12.0 5.6 22.5 1.9 60 7.4 5.6 21.0 1.5

40 3.4 5.1 11.7 1.7 40 2.9 5.7 12.4 1.7 40 3.4 5.3 11.2 1.7 40 2.5 5.6 8.0 1.7 40 1.5 5.0 1.6 1.6 40 1.2 5.0 0.7 1.6

Final concen- tration

mill

20 20

9

4 9

40 3

10

Additions

Experiment 1 None............. None. Pi. . Pi. .

Experiment 2 None n-Lactate. Rat serumb. Dialyzed serumb

Experiment 3 None............. Inosine. Imidazole . Imidaeole 2.Deoxyglucose. 2-Deoxyglucose.

a Expressed as micromoles of “Pi uptake, and calculated as in Table III.

b Used in place of Krebs-Ringer-bicarbonate buffer during in- cubation. Pi content was 3 mM in rat serum and 1.2 mM in dialyzed rat serum. Lactate content was approximately 3 mM in rat serum.

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2378 Glycolysis and Pi Transport in Liver and Kidney Slices Vol. 240, No. 6

TABLE IX the energy-rich intermediates of oxidative phosphorylation, Pi uptake in presence and absence of inhibitors of respiration formed before the steps blocked by azide or oligomycin, can

Experimental conditions were the same as described in Table provide energy for the active transport of Pi. Even when the VIII, except that the concentrations of inhibitors were lower, as cellular ATP level was very low in the presence of azide, active indicated, and that the incubation time was 40 min for all experi- transport of Pi was not diminished; in contrast, if similarly low ments. Two identical sets of experiments were performed. The 1 evels first set was run as in Table VIII. In the second set, isotopic

of XTP were induced by dinitrophenol or by anaerobiosis,

compounds were placed in the main compartment, and 0.15 ml of transport of Pi was completely abolished.

10 N perchloric acid was placed in the side arm; at the end In order to correlate the extent of Pi transport and oxygen

of the incubation it was tipped in and the samples neutralized. uptake, the latter was measured more precisely by using Krebs-

After centrifugation, the supernatant solutions were used for the Ringer-phosphate buffer as medium with either 100% oxygen or

determination of intracellular ATP. Results are expressed as in air in the gas phase. It was found (Table X) that with 100% Table VIII. oxygen (or 95% in the case of COz-02), both Pi transport and

oxygen uptake were high, but aerobic glycolysis was low. With air as the gas phase, both Pi transport and oxygen uptake were lower but aerobic glycolysis was higher. The fact that the rate of oxygen uptake was doubled with 100% oxygen in place of air in the gas phase might explain why the Pi uptake was higher and why aerobic glycolysis was more strongly suppressed.

E$ect of Glucose Concentration on Glycolysis and on Intracellular Glucose Level-In testing of the possibility that the entrance of glucose might control the rate of glycolysis, 14C-lactate forma- tion was measured with varying levels of r4C-glucose in the in- cubation medium. Results in Table XI indicate that when the glucose concentration was increased from 0.5 to 6 mM, the rates

Intra- :ellular ATPb

1.30 0.10 0.05 0.60 0.40 0.10 0.05

Gas phase Additions azPi ‘ptakl

CO?-Or None 2.0 4.8 Dinitrophenol 0.13 mM 5.2 1.6 Dinitrophenol 0.25 mM 6.6 1.2 Oligomycin l.lrg/ml 7.2 5.0 Oligomycin 1.4pg/ml 7.6 4.8 Sodium azide 2.0 mM 8.2 4.9 Sodium azide 2.7 mM 8.4 4.5

COTN2 None I / G.8 1.0

“Pi tram port

02 UP- takea

3.8 0.6 0.2 4.0 3.8 3.9 3.5

0

18 14 11 15 14 15 14

0.10

a Values for 02 uptake were corrected for CO2 liberation due to of both aerobic and anaerobic 14C-lactate accumulation in kidney

glycolysis, but not for respiratory COZ production. cortex slices were more than tripled. Not shown in the table is * Expressed as micromoles per ml of intracellular water. the finding t.hat the rate of 1%.glucose uptake paralleled that of

TABLE X

Effect of varying the percentage of oxygen in gas phase on glycolysis and Pi uptake in kidney cortex slices

Kidney cortex slices (80 mg per experiment) were incubated in Warburg vessels in 2 ml of medium at 37” for 40 min. n-Lactate (2.7 mM) was added to eachvessel to ensure sufficient substrate for respiration. A mixture of “Pi, i4C-glucose, and 3H-inulin was placed in the side arm as in Table T’. Results are expressed as micromoles per g of wet weight.

Medium” Addition inal concentra tion

KOH in center well Gas phase F ‘“C-Lactate

produced 3xygen uptake 3”Pi uptake !Pi tK+llSpOrtb

2.0 44c 6.0 10.1 32c 2.1

8.0 57 4.1 11.0 49 2.4 6.2 25c 3.8

2.8 126 6.3 9.0 54 2.1 2.0 45c 5.6

7.5 0 2.1

2.6 49c 6.5 9.8 39” 2.5

7.0 10.0

28” 3.7 2.0

3.9

0

1.7 0 1.4

4.2 0 3.5

0

4.0 0

1.7 0

?m.f

0.17

0.17

0.17

0.17

KRB-2

KRP-2

None 2,4-Dinitrophenol

None 2,4-Dinitrophenol None

None 2,4-Dinitrophenol None

coz-02

Air

- -

+ + -

+ + -

-

- -

- -

KRP-2 02

KRB-2

KRB-2d

KRP-2d

COrNz

CO?02

Air

None 2,4-Dinitrophenol

None 2,4-Dinitrophenol

Q KRB, Krebs-Ringer-bicarbonate. KRB-2 was the same as KRB buffer, except that Pi concentration was increased to 2 mM. KRP-2, Krebs-Ringer-phosphate buffer, except that Pi concentration was lowered to 2 mM.

* 32Pi transport represents the difference of “Pi uptake in the presence and absence of 2,4-dinitrophenol. c Oxygen uptake in this experiment was corrected for CO2 liberation due to glycolysis, but not for liberation due to respiration. d Imidazole (9 mM) was included in these experiments to provide additional buffering capacity. At 9 mM, imidazole had no effect

on glycolysis and “Pi uptake (see Table VII).

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June 1965 R. Wu 2379

TABLE XI 61 Rate of glycolysis in kidney slices with varying

concentrations of glucose in the medium Kidney cortex slices (55 mg per experiment) were incubated in

2 ml of Krebs-Ringer-bicarbonate buffer with varying amounts of ‘Gglucose (14,000 cpm per pmole) for 20 min. ‘%-Lactate values were calculated and corrected for blank values obtained in the presence of ‘%-glucose but without kidney slices. At least 70% of the initial glucose was present at the end of incubation. There- fore, glucose concentration during incubation was essentially constant. Results are expressed as micromoles per g of wet weight.

V

:I ,‘-:..\, 12345678

s (mM) “C-Glucose concentra-

tion in medium

‘*C-Lactate formation

CO%.02 COYiv2

Pasteur effect

FIG. 1. Apparent K, values of intracellular glucose for intra- cellular hexokinase in kidney cortex slices. The results presented in Table XII are plotted according to the method of Hofstee (20). The K, values are determined from the slope of such a plot. The values obtained are not necessarily the true K, values because the rate of glycolysis was measured over a 20.min period which may not represent initial rates.

% 58 61 61 59 58 GO

0.5 0.37 0.87 1 0.60 1.53 2 0.86 2.20 4 1.16 2.57 6 1.30 3.10 8 1.30 3.30 r4C-lactate accumulation. Interestingly enough, the percentage

of the Pasteur effect remained essentially the same. The intracellular concentration of glucose was found to in.

crease with increasing levels of glucose in the medium (Table

XII) ; however, the intracellular concentrations were always much

lower than that in the medium. This indicates that the trans-

port of glucose was too slow to achieve equilibration between the

medium and the cells. Furthermore, as can be seen in Fig. 1,

hexokinase in the kidney slices was found to have a high apparent

K, value for glucose (above 0.4 mM under both aerobic and anaerobic conditions). In comparison, the K, for hexokinase in

kidney homogenates was 0.03 1nM.l Similar differences between

the K, values for muscle hexokinase in the tissues and in the homogenates have been reported (21, 22).

It can be concluded that the entrance of glucose (at 6 mM or

below) into kidney slices is a rate-limiting factor both for aerobic and anaerobic glycolysis. However, the limitation of glucose

transport appears to be similar under aerobic and anaerobic

conditions, as reflected by the constancy in the percentage of

Pasteur effect (Table XI) at various levels of glucose. Factors responsible for the Pasteur effect are then explored.

Control of Aerobic Glycolysis by P-fructokinase-Among the

probable factors analyzed, the inhibition of P-fructokinase activity appeared to be the most important factor in the control

of kidney glycolysis. This conclusion was based on the deter- mination of the levels of intracellular glycolytic intermediates.

Although the rate of glucose phosphorylation is slower aerobically

than anaerobically, the level of glucose-6-P was higher but

fructose-di-P was much lower under aerobic conditions (Table XIII). The lower act,ivity of I’-fructokinase under aerobic

conditions is most likely due to its inhibition by a high int,racel-

lular ATP level (23). Mansour and Mansour (24) as well as

1 The K, value for hexokinase in the kidney homogenates was determined by measuring the activity of hexokinase spectro- photometrically in a system which contained : triethanolamine buffer, 50 mM, pH 7.4; TPN, 0.15 mM; ATP, 2 mM; MgCl?, 2 mrvr; glucose-6-P dehydrogenase, 0.5 unit; and varying concentrations of glucose (0.02 to 1.0 mM). Density readings at 340 m/l were made at 30.set intervals for 3 min. The results are plotted as in Fig. 1, and the K, value calculated from the slope of such a plot.

TABLE XII

Intracellular concentration of glucose in kidney slices

The procedure for measuring intracellular glucose was given in “Experimental Procedure.” The results of three independent experiments, each with duplicate samples for every 14C-glucose level, were averaged and expressed as intracellular glucose con- centration.

ntracellularn glucose concentration

Ii

‘C-Glucose 3

in the tr H-Inuli apped

slices

C-Lactate produced

= I/b

14 n in Gas phase v:s

Total &ICOX

SC

?I%&- % 1 10.2 2 12.1 4 12.1 8 11.1

0.3 0.2 1.3 4.3 0.8 0.6 1.8 2.3 1.2 0.9 2.2 1.8 2.0 1.5 2.5 1.3

1 12.6 0.6 0.4 2.5 2 12.6 1.2 0.6 3.3 4 11.8 2.0 1.2 4.1

8 14.0 2.6 1.7 4.4

COY02

coa-Ns 4.2 2.8 2.0 1.7

me were

I ICC a Since corrections for extracellularly trapped glu

large, the corrected values for intracellular glucose were not com-

pletely accurate. 6 14C-Lactate production represents the total 14C-lactateformed.

About 709& of ‘Glactate was found in the medium and 3Oye found in the slices. Results are expressed as micromoles per g of wet weight per 20 min.

c Determined with glucose-6-P dehydrogenase and TPN, and

corrected for glucose trapped extracellularly. Results are ex- pressed as mM of intracellular glucose.

d Determined after passing neutralized cellular sample through Dowex l-acetate column, and corrected for glucose trapped extra- cellularly.

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2380 Glycolysis and Pi Transport in Liver and Kidney Xlices Vol. 240, K’o. 6

Passonneau and Lowry (25) have shown that in cell-free systems, the inhibition of P-fructokinase by ATP can be relieved by

AMP, cyclic AMP, ADP, Pi, etc. In kidney slices, however,

the intracellular levels of Pi and ADP were similar under aerobic and anaerobic conditions, but, AMP levels were much lower

TABLE XIII Concenlration of intracellular intermediates in rat kidney slices

Kidney cortex slices (220 mg of wet weight) were incubated at 37” in Warburg vessels. The final fluid volume was 1.5 ml, which contained 1.3 ml of Krebs-Ringer-bicarbonate buffer (pH 7.5), 1.8 Mmoles of Pi, 8 rmoles of 14C-glucose (60,000 cpm), and 0.1 mg of 3H-inulin (210,000 cpm). Results represent averages from four experiments. Concentrations of intracellular compounds were corrected for the amounts present in the medium which was included in the cellular sample, and results are expressed as micro- moles per ml of intracellular water (14).

I lace Intracellular coynceJiea.tion (corrected

Gas phase Incuba- Lactate Glucose 1 t. mn time pro- uptake

I duced Glgu-cpoase- Fyy- ATP AMP Pi

-I- fnin

coz-02 20 1.4 0.072 0.018 1.1 0.15 6.2 40 2.4 3 0.074 0.016 1.2 0.14 6.1

COs-N2 20 5.6 0.029 0.065 0.20 0.45 8.3 / 40 10.8 12 0.028 0.066 0.16 0.42 8.0

a The level of fructose-6-P was approximately 25% that of glu- cose-6-P.

b These samples also included triose phosphates.

TABLE XIV

Concentration of intracellular sugar phosphates and pyridine nucleotides

Experimental conditions were the same as in Table XIII, ex- cept that incubation time was 20 min. For the determination of DPNH, additional duplicate experiments were performed, in which perchloric acid in the single side arm was omitted. At the end of incubation, the medium was tipped into the double side arm, the vessel was then removed from the manometer, and 1 ml of 0.5 N KOH was quickly introduced into the main compartment of the vessel to inactivate the kidney slices. After centrifugation, the KOH-containing supernatant solution was neutralized with Tris-Pi buffer (0.5 M-0.5 M, pH 5.8) to pH 8.0. The neutralized sample was then centrifuged in a Spinco ultracentrifuge at 60,000 X 9 for 20 min. The clear supernatant solution was used for DPNH determination.

Gas phase

co*-02

COs-Nz

-

, 3ucose con- ‘?C- centration Lacta, in medium produc

NZM

1.2 0.7 2.4 1.1 4.8 1.6 9.6 1.6

1.2 1.1 2.4 1.5 4.8 2.2 9.6 2.3

kc l- &l (

6

Intracellular concentration

;lUCOSE? 6-P

0.062 0.012 0.56 0.064 0.012 0.080 0.011 0.54 0.085 0.011

0.031 0.028 0.030 0.030

-

0.030 0.15 0.045 0.050 0.14 0.052

DPNH

0.08

0.08

0.17

0.17

I )PNH ~- DPN

0.14

0.15

1.1

1.2

aerobically. Therefore, the low AMP level in conjunction with high ATP was responsible for the low rate of P-fructokinase under aerobic conditions, and appears to be responsible for the Pasteur effect .

In the experiments just described, sat,urating amounts of

glucose were used. Rate-limiting fact,ors for aerobic glycolysis in the presence of limiting amounts of glucose were next explored. As shown in Table XIV, under aerobic conditions, with increas- ing glucose concentration, the glucose-6-P level increased with an increasing rate of glycolysis, but fructose-di-P levels remained constant,. This may be interpreted as follows. An increase in glucose supply resulted in a higher rate of glucose phosphoryla- tion, and an increased fructose-6-P level in turn stimulated P- fruct’okinase activity (25), resulting in a 2-fold increase in aerobic 14C-lactate accumulation. It must be realized that even though P-fructokinase activity is stimulated, it is still a major rate- limiting enzyme for aerobic glycolysis. This is indicated by the increase in the glucose-6-P level without a parallel increase in the fructose-di-P level. Thus, P-fructokinase appeared to be inhibited under aerobic conditions at both low and high levels of glucose. This conclusion is consistent with the fact that, at any given level of glucose, aerobic glucose-6-P concentration was always higher than that under anaerobic conditions, whereas fructose-di-P levels are always lower aerobically.

Control of Anaerobic Glgcolysis in Rat Kidney Slices-Anaerobic glycolysis was not mainly limited by P-fructokinase, since with increasing glucose levels, the glucose-6-P level remained constant, but the fructose-di-1’ level almost doubled. Therefore, an enzyme after the P-fructokinase step also became rate-limiting for anaerobic glycolysis. This step is presumably catalyzed by glyceraldehyde-3-P dehydrogenase, an enzyme known to be inhibited when the ratio of DPNH to DPN is relatively high (26). Assays for DPNH and DPN (Table XIV) showed that the DPNH:DPN ratio was 8 times higher anaerobically than aero- bically. Therefore, the relatively high DPNH and low DPN levels under anaerobic conditions are likely to cause partial inhibition of glyceraldehyde-3-P dehydrogenase. In addition, anaerobic glycolysis was also controlled coordinately by hexo- kinase and P-fructokinase. However, owing to the lower levels of glucose-6-P and ATP, the inhibition of hexokinase and P- fructokinase, respectively, was less severe under anaerobic condi-

t,ions than under aerobic conditions.

DISCUSSION

With the classical manometric method for the determination of glycolysis (together with glycogenolysis) in liver slices, Qi&

values of 1 to 10 have been reported (15, 27-34), but the exact reason for this large variation was not clear. In this paper, anaerobic acid production gave an average value of 1.4. How-

ever, when lactate was determined calorimetrically, much lower

values (between 0.3 and 0.5) were obtained. The calorimetric lactate values can be regarded as valid measurements of gly- colysis, especially since its production is susceptible to inhibition by iodoacetate or NaF. On the other hand, production of total acid and volatile compounds, as measured by manometric meth-

ods indirectly, is not significantly inhibited by these poisons;

therefore, these measurements grossly overestimate the true rate

of glycolysis. The rate of entrance of glucose into rat liver was very rapid

(35). The rate-limiting factor for glycolysis in liver slices was

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June 1965 R. Wu 2381

mainly the supply of ATP for hexokinase and P-fructokinase. Not only was the intracellular level of ATP very low, but both hexokinase and P-fructokinase had rather low capacities, about one-quarter that of the kidney. Therefore, it is not surprising that the rate-limiting factors for glycolysis in liver and kidney slices are entirely different.

The observation that Pi transport in kidney slices depends on high energy intermediates of oxidative phosphorylation partly resembles the findings of Van Rossum (36, 37) for cation trans- port in rat liver slices which u-as 50% inhibited by oligomycin. However, the findings on Pi transport presented here indicate that nearly all the energy may be derived from energy-rich intermediates formed even in the presence of oligomycin or sodium azide, since Pi transport, was not inhibited by sodium azide under conditions in which the intracellular ATP level was drastically lowered, whereas complete inhibition was observed if a comparable diminution of the intracellular concentration of ATP was induced by dinitrophenol or anaerobiosis. In addition, since the effects of oligomycin and sodium azide on transport were similar, it further strengthened the conclusion that energy- rich intermediates can support Pi transport.

It was recently reported that transport of Pi and Mg++ into beef heart mitochondria was inhibited by dinitrophenol but not by oligomycin (38) ; similar results were reported on the respira- tion-dependent accumulation of Pi and Ca++ by rat liver mito- chondria (39). However, transport of Pi into intact cells may be more complicated, since additional steps are probably in- volved in transporting Pi through the cell membrane, and its relationship to Pi t,ransport in mitochondria has yet to be ex- plored.

SUMMARY

1. The rate-limiting factors for glycolysis and inorganic orthophosphate (Pi) transport in rat liver and kidney slices have been analyzed.

2. In liver slices, the rate of aerobic glycolysis appeared to be partly limited by ATP and Pi, whereas anaerobic glycolysis was limited mainly by ATP. Transport of Pi required energy, and occurred only under aerobic conditions.

3. In kidney slices, the rates of both aerobic and anaerobic glycolysis were limited by glucose when its concentration was less than 6 mM. Aerobic glycolysis appeared to be further limited by a severe inhibition of the activities of I’-fructokinase and hexokinase, which in turn was associated with the Pasteur effect.

4. Active transport of Pi into kidney slices was maximal under conditions of rapid respiration. It appeared that high energy intermediates of oxidative phosphorylation can provide energy

for the transport process.

.-2cknowZedgment-The excellent technical assistance of Mrs. Helen Power is gratefully acknowledged.

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Ray Wuin Rat Liver and Kidney Slices

Rate-limiting Factors in Glycolysis and Inorganic Orthophosphate Transport

1965, 240:2373-2381.J. Biol. Chem. 

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