Oscillations of glycolytic intermediates in yeast cells

Vol. 16, No. 2, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNlCATlONS

OSCU4TIOI'JS OF GLYCOLYTICINTERNEDIATESIlV YEAST CELLS A. Ghosh+ and B. Chance Johnson Research Foundation, University of Pennsylvania Philadelphia, Pennsylvania

Received

May

1, 1964

In previous papers we have described cyclic nucleotide

(RPN) in yeast cell

responses of reduced pyridine

suspensions which have been observed by direct

spectrophotometry at 340 nq~.(Chance, 1952, 199, methods (Chance, 1964a). servation lations

Considerable Interest

of a dampedsinusoidal train

1955), and by fluorometric has been aroused by the ob-

involving

over 12 consecutive oscil-

observed in Saccaromyces carlsbergensis (ATCC 4228)2

(Chance et al.,

1964a,b). In this paper, samples are removed periodically pension in which the reduced pyridine fluorometric

monitoring to oscillate

the concentrations tuations

sinusoidally,

of Rplr. In order to identify

tabolites

and the fluctuations

system.

to the fluc-

sites of glycolysis

chain (Chance et al.,

in-

In addition,

19%) has been applied

the phase relationships

of the me-

has been determined.

Growth of the cells is described elsewhere (Ch.arr:e et al., Metabolite

of

the crossover theorem derived for identifying

sites in the respiratory

to the glycolytic

the control

sus-

is observed by continuous

of the intermediates are recorded in relation

volved in this oscillation, control

nucleotide

from the yeast cell

19&b).

assays were madeaccording to the method of Maitra and Estabrook

(1964), based upon improvements on the methods of BC\cher as described by +

Information on these oscillations was communicated to Dr. F. Hommes of Rijmegen, who has confirmed the existence of the oscillations and the fluctuations of the mctabolite as deecribed here. + Calcutta, India. Present address: Saha Institute,

174

BIOCHEMICAL

Vol. 16, No. 2, 1964

Bergmeyer (l962).

t3 a larger reservoir

change fz

RESEARCH COMMUNICATIONS

The phase and amplitude 3f the %cillati3ns

m3nit3red c3ntinususly

at intervals

AND BIOPHYSICAL

the cuvette of which was connected

by means3f a pump. Samples were withdrawn periodically

precisely Fig. 1.

by a fluDrometer,

3f RPNwere

timed with the peaks and the ngdes 3f the fluorescence

Fez Figs. 2 and 3, samples were withdrawn every 10 sec.

Although glucose cD,uld n& be added simultaneously t3 the sampling reservoir and the flusrgmeter

cuvette,

a minute and a half elapsed between the addition

of glucose and the initiation transition.

Thus, the material

of oscillation

due t3 the aerobic-anaerobic

in the flu3rDmeter was well mixed with that

in the main sampling chamber.

U FDP

Gtuc. Add.

Time

In Minutes

After

Glucose

AddiYion

Figure 1. A dampedsinusoidal 3scillati3n of G-6-P and FDP measured simultaneously with fluorescence changes in a suspension of S. carlsbergensis (ATCC 4228). At the time of adding glucose (arrow) the cells are aerobic and spontaneously becDmeanaerobic at approximately 100 set after adding glucose.

(Expt.

8-8-63).

EXSERIMENTAL

Fructose diphDsphate. addition

of gluczie

A cyclic

FiEsuLTs

response Df pyridine

3r t3 the akwobic-anaerobic

175

transiti3n

nucle&ide

t3 the

3f Baker's yeast

Vol. 16, No. 2, 1964

BIOCHEMICAL

cells is identified

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

with the activation

of DPNreduction by glyceraldehyde-

j-phosphate dehydrogenase (GAPDH). Thus, the time wurse ose diphorphate (F'DP) concentration interest.

with respect to that of PPN is of greatest

Figure 1 shows dampedsinusoidal oscillations

close synchrony with the oscillations full

of changes of fruct-

cycles of oscillation.

It

phase of the dampedsinusoidal

of RPN, at least for one and one half

is apparent therefore, oscillations

those of PPK. This is consistent with

the phase is shifted

from

that the frequency and

of FDP are closely similar

the idea that the oscillations

reduction observed are due to oscillations Analyses were also carried

of FDP t,hat are in

in the level

to of PR

of FDP.

out for Glucose-j-phosphate (G-6-P).

Here,

that of ?DP and RPR durir.3 the period for the two

cycles in which RPN is oscillating

sinusoidally.

F-6-P was found to oscillate

in phase with G-S-P. Identification

of the crossover point. --

assay data on many of the glycolytic is determined by their oscillatory

the value of such data

the control

site of the

the portion of the enzymatic system that is caus-

For this purpose, we employ the crossover theorem

derived from the study of multisite prising

intermediates,

usefulness in identifying

mechanism, i.e.,

ing the oscillations.

I?rhile we are able to present

electron transport

Holmes and Connelly,

controls

and oxidative

19%).

in the multienzyme system cam-

phosphorylation

(Chance, Higgins,

A convenient graphical representation

plot the nameof the component in the linear

is to

sequence of enzymatic reactions

against the percentage change of the component with respect to its meanvalue in a particular

metabolic transition

--et al. (1964a)). glycolytic

Referring

flux

to Figure 1 we identify

with maximumpyridine

minimumglycolytic

(a related plot has been used by Lowry a condition

of maximum

nucleotide

reduction and one of

flux with minimumof pyridine

nucleotide reduction.

According to the crossover theorem, a control

site will

be identified

under

conditions of increasing flux by the point at which there is a crossover between a relative intermediates,

depletion of intermediates

and a relative

and vice versa with decreasing flux.

accumulation of

Therefore we plot as

Vol. 16, No. 2, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

a 100

0 -50 z E s ; 0.

,I / d’fiIL---f-% \ Crossover

Cl00

c,,,+

I

-100

c,,o-

Point

G6P

F6P

FDP

I D&P

2 GAP

RPN (Fluor)

aGP

100 x

I

I 3PGA I l,3PGA 2PGA

Figure 2. The crossover point for metabolite intermediates in dampedsinus3idalscillations of S. carlsbergensis (ATCC 4228). The abscissa represents the namesof the intermediates and the ordinates, the percentage change of the intermediate, with respect to the mean value of its concentration. The concentrations are compared at 150 seconds with that at 130 secmds, and at I-30 seconds with that at 100 seconds. A control site is identified by a depletion accumulation crossover point with increasing flux, and vvice versa with decreasing flux. (Expt. T-22-63)

values of the ordinate the change of concentration

of the intermediates

under conditions of increasing flux with reference to the average value of the concentration assays similar

of the intermediates

in the two states.

to those of Figure 1, a maximumof

In metabolite

I% reduction was found

at 150 set after

adding glucose and a minimumwas found at 130 set after

adding glucose.

We have therefore

plotted

percentage changes of the inter-

mediates against the nameof the intermediate It

is apparent that intermediates

in Figure 2 (dashed curve).

G-6-P and F-6-P are depleted and interme-

diates from FDP to 3-phosphoglycerate (3-PGA) are accumulated, a clear example of a depletion-accumulation

crossover point with increasing flux.

We can also compare the minimumof PN reduction at 130 set with the preceding maximumand thereby obtain the crossover point for conditions of diminishing 177

Vol. 16, No. 2, 1964

flux,

BIOCHEMICAL

as ?ldted

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

as a sol-id line in Figure 2.

Here, intermediates prior to

tie PFK step accumulate and those from F9? to 3-PGA are depleted.

\:ith

de-

crease 3f flux, an accumulation-depletion crossover point marks the cxtrol . slate. Thus, me find that the samepoint is operative in increases and decreases of flux

and conclude from this result

site in the oscillations

of gpycolytic

Phase plane plot. addition

to indicating

may also provide incisive The most appropriate

intermediates

The kinetics the control

of the glycolytic

information

graphical display of the oscillation

that of the other one at varims

a straight

characteristic

of one metabolite against

line at 45' slope indicates the two components to line at 135’

indicates that the

An open figure

indicates

of glucose until

possible to make incisive

the completion of the oscillation,

Of particular

it has been

interest

art the relations

of the

of G-6-P and FDP,

side of the crossover point of Fig. 2.

These

in Fig. 3.

are in millimicromolts

The values of the ordinate and the abscissa per 1010 cells. The graph starts at point 8, 80 stc

adding glucose, and then proceeds with points every 10 set through

to point 18, three min after

adding glucose.

nearly comprise a closed elliptical tical

from the

determinations of tht phase relationships

the two components on either

after

inter-

between zero and 180'.

By taking samplts every ten seconds throughout the interval

data are plotted

cycles.

of the phase plane plots can be recog-

in phase. Second, a straight

intermediates.

is

which in the terminology of biochemistry

phases of the two components are at 180'.

addition

in

mechanisms.

times throughout the oscillation

Three dominant characteristics

mediate phase shifts

intermediates,

on the type of oscillation

is represented by a graph of the concentration

be oscillating

is at PFK.

site according to the crossover theorem,

provided by the "phase plane plot",

nized; first,

that the metabolite control

figure.

Points 8 through 13 very A segment of a second ellip-

begins with point 13 and continues through 15.

a distortion

of the ellipticity

~-6-p and FDP are proportional

There is apparently

at point 16, and beyond that the changes of to each other. 178

Also at this point the oscil-

Vol. 16, No. 2, 1964

BIOCHEMICAL

1200

g IOOOs 0-o BOO5 d E

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

600-

1 4000 IL

1

200

Of-----200

400

G6Pmp

600

600

moles/ldO

1000

1200

Cells

Figure 3. Phase plot diagrams for G-6-P and F'DP. The concentration of G&-P and FDP of Fig. 2 are plotted against one another at times after addition of glucose from SO set to 3 min. (Expt. 7-22-63)

lations

observed fluorometrically

have lost considerable amplitude.

apparent, however, that the analytical

It is

data show an "open" phase plane plot

for F-6-P and FDP with a center that moves toward higher G-6-P concentrations and lower FDP concentrations

as the oscillations

of the plot disappears as the oscillations

proceed.

The "open" nature

subside.

DISCUSSION The crossover theorem which requires only metabolite measurementscorresponding to maxima and minima in the fluctuations identification the control

of control

sites in the respiratory

site of oscillations

in the glycolytic

A more incisive

evaluation

which indicated

a large phase shift

has been applied to the chain and shows Pm to be chain of S. carlsbergensis.

of the mechanismis provided by phase-plane diagrams between the substrate and the product at the

PFK site . While it is possible that other factors

contribute

to the crossover

phenomenainvolved,

the identification

of this site affords a basis for a chem-

ical interpretation

of U-E oscillation

mechanism. This identification

ported by tne characteristics Pressand Betz, 1964).

of the oscillations

ie suy

in a cell free system (Chance,

A minimum chemical mechanismfor dampedor continuous 179

Vol. 16, No. 2, 1964

sinusoidal

BIOCHEMICAL

oscillatiocs

b;r FDP, albeit rg64b ) .

This

zymatic

(Lard:.

reaction

nnd Per%,

cotioked

as a si,&

step

7 > octiva~led 3 prmuc-c

involves

el3713:

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

will

with

lF>‘i;), a first

step.

or by .;DP o&er

step

show damned or continuxs

P3X ma-’ be 3.ctivnted (Lmr;:

and Pass onnem,

as c! source

-nC an en-

oscillntixs

(9i&ns,

i.T&;

GLTJ -F-;-P F-6-P E At this enzyme to the

-Y-

+ E’-

. F&p

point

active

(1)

either

form

hDP or F'DPma;r convert an inactive

(ES).

T’ne activator

~a;~ reimin

bound

form E of the

to the

activated

enzpe E

FDP

I

Whichever

+

(4)

.?DP

is the

Ecti-vator,

it

case of FDP or by 1,3-diphospho~lycerate

must be exTended, kinnse

in the

‘02 sldolase

case of ADP.

in the In the

case of FDP

The dmped carlsbergensis

FDP + E*

-

E

-E2

.

2

sinusoidal

(LTCC 4221”;)

measured fluorometrically. pattern

FDP

E2 - FDP

(5)

+ G;:P

oscillation

shows a close

of &gcolytic synchrony

(6)

intermediates

of FLIP and RPE kinetics

A crossover point of the oscillatory

is found between G-6-P (F-6-P) and F'DPidentifying

metabolite

PFK as the control

site.

The phase relations

figure

in the phase plane plot is obtained during maximal oscillations;

figure

is more nearly closed as the oscillations

involving

activation

the basic oscillator

of G-6-P and FDP are of especial interest;

of PF'Kby one of its reaction mechanism. 180

of S.

subside.

an open the

A reaction mechanism

products is proposed as

Vol. 16, No. 2, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENT The support of the National Institutes Foundation for this research is gratefully

of Health and National Science acknowledged. The work was greatly

assisted by Miss Sigrid Elsaesser.

Bergmeyer, H.U., 1962. Methoden der enzymatichen Analyse, Verlag Chemie, Weinheim. Chance, B., 1952. Fed. Proc., 11, 196. Chance, B., 1952. Nature, 169,Tl5. Chance, B., lpT$.In!&e Mechxsm of EnzymeAction (W.D. McElroy and B. Glass, eds.), Baltimore, Johns Hopkins Press, p. 399. Chance, B., 1955. Harvey Lectures, 40, 145. Chance, B.,Hess, B., and Bets, A., BEchem. Biophys. Res. Commun.u 182. Chance, B., Higgins, J., Holmes, W., and Connelly, C. M., 1958. Nature, -'182 1190. Chance, B., Ghosh, A., Higgins, J., and Maitra, P. K., lp64a. N.Y. Acad. Sci. Meeting May 27-29, 1963 (in press). Chance, B., Estabrook, R., and Ghosh, A., 1964b. Proc. Natl. Acad. Sci.., 51 (in press). Higgins, J., 1964. Proc. Natl. Acad. Sci., 51 (in presb). Lardy, H., and Parks, R., 1956. In Enzymes: iji;its of Biological Structure and Function (O.H. Gaebler, ed.), New York, Academic Press, p. 584. Lowry, O.H., Passoneau, J.V., Hasselbergcr, F.X., and Schulz, D.W., 1964a. J. BLol. Chem., 9, 18. Lowry, O.H., and Passonneau, J.F., 196413. J. Biol. Chem., 39, 31. Maitra, P.K., and Estabrook, R.W., 1964. Anal. Biochem. (in press).

181