Eur. J. Biochem.

61, 181 190 (1976)

~

Self-Association of Human Erythrocyte Phosphofructokinase

Kinetic Behaviour in Dependence on Enzyme Concentration and Mode of Association
Klaus-Wolfgang WENZEL, Boris I. KURGANOV, Gerolf ZIMMERMANN, Victor A. YAKOVLEV,
Wolfgang SCHELLE,NBERGER, and Eberhard HOFMANN
Institute of Physiological Chemistry, Karl-Marx-University, Leipzig
and Institute of Vitamin Rcsearch, Moscow

(Received April 16/July 30, 1975)

The kinetic behaviour of human erythrocyte phosphofructokinase has been analyzed over a re-
lative wide range of enzyme concentration (0.02 - 1.7 pg/ml). The kinetic cooperativity which becomes
apparent when the enzymic reaction rate is plotted versus the fructose 6-phosphate concentration
decreases with increasing enzyme concentration. Simultaneously, a decrease of the half-saturation
concentration for fructose 6-phosphate [S],., is observed. Maximum velocity passes through a maxi-
mum at increasing enzyme concentrations.
Sets of curves representing specific enzymic activity of phosphofructokinase versus enzyme con-
centration obtained at various fixed concentrations of fructose 6-phosphate and ATP are analyzed.
The shapes of these curves are interpreted in terms of an association model of human erythrocyte
phosphofructokinase, in which an inactive dimer ( M , 190000) and active multimers of the dimeric
form are involved.
The conclusion is drawn that the sigmoidal shape of the plots of the enzymic reaction rate versus
fructose 6-phosphate concentration is partially caused by a displacement of the equilibrium between
different states of association of phosphofructokinase to multimers by this substrate. On the other
hand, the inhibition of the enzyme by high concentrations of ATP may be partially caused by a shift
of this equilibrium to the state of the inactive dimer.

Various allosteric enzymes may undergo associa-
tion-dissociation reactions and the state of equilibrium
between different oligomeric forms may be deter-
mined by substrates, coenzymes and allosteric effec-
tors. The displacement of equilibrium between them
by metabolites may be one of the mechanisms of
enzyme regulation in vivo [l - 71.
When the individual oligomeric forms of an
enzyme differ in their kinetic parameters, the displace-
ment of the equilibrium between them should result
in deviations from simple kinetic behaviour and may
give rise to the appearance of a sigmoidal shape of
the substrate or effector concentration vs velocity
curves. The idea that in the case of certain enzymcs
the displacement of equilibrium between various
The results of this investigation were partially reported by
B. I. Kurganov at the FEBS Advanced Course No. 27 on Muihe-
tnaiical Models of Metuholic Regulation in Dobogoko (Hungary)
1-5 September, 1974.
Ahbrcviafion. a , the specific activity of phosphofructokinase in
U/mg protein where 1 U = conversion of 1 pmol fructosc 6-phos-
phate per min.
E n q w m . Phosphofructokinase (EC 2.7.1.11); fructose bisphos-
phate aldolase (EC 4.1.2.13); triosephosphate isomerase (EC 5.3.
1 . l ) ; glycerol-3-phosphate dehydrogenase (EC 1.3.1.8).
oligomeric forms might be one of the molecular
reasons of substrate or effector kinetic cooperativity
has been independently expressed by three groups
of investigators [ 10- 151.
The phosphofructokinases from heart, skeletal
muscle, and erythrocytes have been demonstrated as
self-associating enzymes, the kinetic and regulatory
characteristics of which depend on the protein con-
centration [8,9,17 - 281. Phosphofructokinase from
human erythrocytes occurs in various associated
forms, which seem to be multimers of an inactive
dimer having a molecular weight of 190000 [18,19].
By means of density gradient centrifugation and frontal
gel chromatography [16,18,19] it could be established
that the reversible association of the enzyme takes
place at the protein concentrations generally used in
the kinetic experiments. Therefore, the self-association
of erythrocyte phosphofructokinase may be taken
as a rational basis for the interpretation of the changes
of the kinetic pattern of the enzyme with enzyme
concentration.
In this paper, the kinetic behaviour of human
erythrocyte phosphofructokinase will be analyzed at
various concentrations of the enzyme. Especially,
182
the effect of enzyme concentration on catalytic activity,
on fructose 6-phosphate affinity and on ATP inhibi-
tion, as well as on kinetic cooperativity will be de-
scribed. The results will be interpreted in terms of the
association behaviour of human erythrocyte phos-
phofructokinase and an appropriate model will be
proposed.
Recently, the kinetics of erythrocyte phospho-
fructokinase has been interpreted in terms of the
allosteric model of Monod et al. [29].
MATERIALS AND METHODS
Human erythrocyte phosphofructokinase, checked
regularly for homogeneity in sodium dodecylsulphate
electrophoresis, was prepared as described previously
[30]. The enzyme used in this study had a specific activ-
ity of 135 U/mg protein at pH 8.0 and 25 "C. The other
enzymes as well as their substrates were purchased from
C. F. Boehringer Mannheim GmbH, FRG.
Phosphofructokinase activities have been measured
in a photometer Eppendorf at 334 nm, pH 7.1 and
25 "C by coupling the enzyme with aldolase, triose-
phosphate isomerase and glycerol-3-phosphate de-
hydrogenase. Phosphofructokinase as well as the
auxiliary enzymes were dialyzed free from ammonium
sulphate. Care was taken that the auxiliary enzymes
were always in excess. The assay mixture contained
in 1-ml volume : 100 mM Tris-HC1,lO mM KC1,l mM
EDTA, 5 mM 2-mercaptoethanol, 0.15 mM NADH,
10 pg aldolase, 10 pg triosephosphate isomerase,
10 pg glycerol-3-phosphate dehydrogenase. The actual
concentrations of fructose 6-phosphate and of ATP
are given in the text. In all cases the ratio [Mg'] : [ATP]
was kept constant to 1 : 1. The reaction was started
by the addition of 10 p1 of phosphofructokinase.
Before adding to the assay mixture the phosphofructo-
kinase was properly diluted and kept in a buffer con-
taining 100 mM potassium phosphate, pH 7.1, 5 mM
2-mercaptoethanol, 1 mM EDTA, and the fixed
substrate in the same concentration as used in the
corresponding set of experiments. The enzyme has
been found to be stable over the period required to
perform an adequate set of experiments. For calcula-
tion of the reaction rates only the linear sections of
the recorded curves were used.
Protein concentrations were determined by the
method of Janatovd et al. [31] using human serum
albumin as a standard. Specific activity (symbolized
as a) has the dimension ofU/mg protein where 1 U rep-
resents the conversion of 1 pmol fructose 6-phosphate
per min.
RESULTS
The kinetics of human erythrocyte phosphofructo-
kinase has been investigated at different enzyme
Cooperative Association of Erythrocyte Phosphofructokinase
concentrations in the range of 0.02- 1.7 pg/ml. At
first, the dependence of the specific enzymic activity,
a, on the concentration of fructose 6-phosphate was
recorded at various fixed concentrations of ATP
(1,2, 3,4, and 5 mM). The curves obtained at 2 and
5 mM ATP are shown in Fig. 1 . With increasing
enzyme concentrations the [S], value for fructose
6-phosphate decreases. The maximum specific activity
amaxpasses through a maximum with increasing
enzyme concentration. The curves are sigmoid, the
shape of which is dependent on the concentration of
enzyme and of ATP (Fig. 1A, C). In Fig. 1 B and D
the Hill plots of the curves are presented. The
maximum activities used for construction of the Hill
plots were obtained directly from the experimental
points at high substrate concentrations. The slopes
of the curves (nH)at half-saturation of the enzyme with
fructose 6-phosphate decrease significantly with in-
creasing phosphofructokinase concentration. A sum-
mary of these relationships is given in Fig. 2. From the
dependence of the maximum specific activity amaxon
enzyme concentration it follows that an increase of the
ATP concentration causes a displacement of curve
maximum towards higher enzyme concentrations.
Fig. 3 represents the dependence of the half-
saturation concentration of the enzyme for fructose
6-phosphate, [S], 5 , on the concentration of ATP at
various enzyme concentrations. Between 2 and 3 mM
ATP a steep increase of the [S],,, value occurs. The
slope of the curves increases with decreasing enzyme
concentration.
In Fig.4 the dependence of specific enzymic
activity on enzyme concentration at various fixed
concentrations of fructose 6-phosphate is demon-
strated in logarithmic coordinates. Fig. 4A, B, C, and
D refer to 1, 2, 4 and 5 mM ATP, respectively. At
1 mM ATP the specific enzymic activity decreases
regularly with decreasing enzyme concentration at
sufficientlylow concentrations of fructose 6-phosphate
(0.01, 0.02 and 0.05 mM) (Fig.4A). The slope of the
curve at 0.01 mM fructose 6-phosphate is near unity
at sufficiently low concentrations of the enzyme ;
with increasing enzyme concentrations the slope
increases and then it decreases again. The extremum
value of the slope exceeds unity. A similar behaviour,
but less pronounced, is also observed at 0.02 and
0.05 mM fructose 6-phosphate. At increasing fructose
6-phosphate concentration a mximum appears in the
shape of the curves. At 2,4 and 5 mM ATP respectively,
the shapes of the log a lrewus log [El, plots change
analogously with increasing fructose 6-phosphate
concentrations (Fig. 4B, C, D).
The dependence of the specific enzymic activity
on the enzyme concentration produces a sigmoidal
curve (Fig. 5).
In Fig.6 the dependences of the limiting values
of the specific enzymic activity at [E],+Go on the
K.-W. Wenzel, €3. 1. Kurganov, G. Zimmermann, V. A. Yakovlev, W. Schellenberger, and E. Hofmann 183

0 1 2 3 -2 -1 0
[Fructose 6- phosphate] (mM) log [Fructose 6- phosphate]

0 .c45
X-
/ X Y X Y 0.1 "

0
0 5 10 15 -2 -1 0
[Fructose 6 - phosphate] (mM ) log [Fructose 6 - phosphate]
Fig. 1. Dependence o j the specific enzymic activity, a, of human erythrocyte phosphojructokinase on the concentration 0f:fructose 6-phosphate
of various enzyme concentrations, [El,. (A, B) 2 mM ATP; (C, D) 5 mM ATP. amaxis the limiting value of a at [ S l - z

fructose 6-phosphate concentration are shown. The
respective values of activity were taken from experi-
ments performed at high enzyme concentrations as
presented in Fig. 4. The maximum specific activities
at [El,-+ 00 are equal at sufficiently high concentra-
tions of ATP and of fructose 6-phosphate. However,
the fructose 6-phosphate concentration necessary
to yield maximum velocity depends on the ATP
concentration : the sigmoidal curve is shifted to the
right when the ATP concentration is increased.
In Fig.7 the dependence of specific activity on
the enzyme concentration at various fixed concen-
trations of ATP is presented in logarithmic coordi-
nates. Fig.7A, B, C, and D refer to 0.01, 0.02, 0.05
and 0.1 mM fructose 6-phosphate, respectively. The
experimental points were obtained from measuring
the dependence of enzyme activity on ATP concen-
tration, which resulted in a series of biphasic curves
caused by the action of ATP as substrate and inhibitor
(not shown). The position of each curve maximum as
well as the strength of inhibition depend significantly
on fructose 6-phosphate and enzyme concentration.
With increasing fructose 6-phosphate and enzyme con-
centration the curve maximum shifts to the right and
184
10 4
-2 -1 0 present knowledge about human erythrocyte phos-
log [ d o
Fig. 2. Dependence of the Hill coefficient nHfor fructose 6-phosphate
at the halflsaturationpoint qjthis substrate ( A ) and of the muximum
specific enzymic activity amox( B ) of human erythrocyte phospho-
jructokinase on the enzyme concentration [ E l , at various fixed
concentrations qf ATP. Dimensions of [EL are pg/ml
0 1 2 3 4 5 6
[ATPI (mM)
Fig. 3. Dependence of the halflsaturation concentralion [S]o.5 of
human erythrocyte phosphofmctokinase .for ,fructose 6-phosphate
on the concentration of A T P at various j k e d enzyme concentru-
tions [ E l o
ATP inhibition decreases. As shown in Fig.7, where
these experiments are presented as a log a versus log
[El, plot, the slope of the curves approaches unity
at sufficiently low concentrations of the enzyme.
At 0.1 mM fructose 6-phosphate a maximum occurs
Cooperative Association of Erythrocyte Phosphofructokinase
at low ATP concentration and this maximum disap-
pears when the concentration is increased.
DISCUSSION
Erythrocyte phosphofructokinase undergoes an
indefinite reversible association reaction which in-
volves a catalytically inactive ‘monomer” M with a
molecular weight of about 190000 and active poly-
meric forms with molecular weights up to several
millions [18,19]. The association equilibria are strongly
influenced by the enzyme concentration as well as
by substrates and effectors [19]. Regarding the mode
of association several models may be considered. The
most straightforward models compatible with our
phofructokinase and its association behaviour are
represented in Schemes 1 and 2. In Scheme 3 the
enzyme associates by association of the monomeric
unit M to the polymeric forms, while in Scheme 2
the association is divided into the formation of the
dimers from the monomers M and into further associa-
tion of the dimers.
inactive ~ active polymers
monomer ~
Scheme I
M%Mz
7 $M4 c“’,...+ “ r M ~ i+ hl,
I
inactive active polymers
monomer I
,
Scheme 2
Experiments show that at pH 7.1 fructose 6-phos-
phate displaces the equilibria in the direction of
polymeric forms, whereas ATP favours dissocia-
tion [19].
I t is principally assumed that the alterations in the
kinetic behaviour of the enzyme induced by changing
the protein and the substrate concentrations are due
to changes in the association state of the enzyme.
The dependence of the specific enzymic activity
on the enzyme concentration exhibits the following
qualitative characteristics : (a) a decrease of specific
enzymic activity with decreasing enzyme concentra-
tion at sufficiently low concentrations of the enzyme,
(b) the occurrence of a plateau in the specific enzymic
activity at high enzyme concentrations, and (c) the
~-
‘ In the context of the modcl, the term ‘monomer’ is used I’or
the basic associating unit of‘ the enzyme, which is itself a dirncr
composed of two subunits with M , N 95000 [18,19].
K.-W. Wenzel, B. I. Kurganov, G. Zimrnennann, V. A. Ydkovlev, W. Schellenberger, and E. Hofrnann 185

ru-6-PI (mM)

1.5 1.5

1 .o 1 .o

m m
m
9 -
g,

0.5 0.5

/ ' /
0
y1 i
0.02*7
0

@/'

0.01
-0.: -0.5

r u - 6 - P ] (mM)

1.5 1.5

1 .o 1 .o

0 rn
-m -B
0.5 0.5

0 0

1.5
-0.5 - .-
-0.5
-2 -1 0
log [Elo

Fig. 4. Drpendence of' the specifk enzjmir activity of' human erytizwcyte phospllofruc.tokinase on the enzj'nie concentrution, Logarithmic co-
ordinates; the concentrations of fructose 6-phosphate (Fru-6-P) are given in the figure. ATP concentrations: (A) 1 mM, (B) 2 mM, (C) 4 mM
and (D) 5 mM. Dimensions of [E,], are pg/ml, of a U/mg protein
I86 Cooperative Association of Exythrocyte Phosphofructokinase

Fig. 5 . Sigmoidal dependence of the specific uctivity vf humun erythrocyte phosphojructokinase on the enzyme concentration. Fructose
6-phosphate: 0.01 mM (0)and 0.02 mM (A); 1 m M ATP

20

-0
n
5 10
iFructose 6 - phosphate] (mM)

Fig. 6. Dependence of’ the limiting specific uctivity qf’phosphqf~uctokinuseon fructose 6-phosphate. Concentration of ATP : 4 mM (A) and
5 mM (0).The values were obtained by extrapolation of the respective curves shown in Fig. 4 to infinitely high enzyme concentrations

appearance of a maximum in the log a versus log
[El, plots under certain conditions. The decrease
of specific enzymic activity with decreasing enzyme
concentration indicates that the monomer is en-
zymically inactive, whereas the existence of the plateau
demonstrates that at high degree of association the
kinetic properties do not depend on the association
state of the enzyme. The occurrence of a maximum in
the plots indicates that the dimeric form M, (char-
acterizing in both association schemes the enzyme
entity with a molecular weight of 380000) differs
kinetically from the higher associated forms, reflecting
the plateau region.
From Fig. 1 and Fig.4 it may be concluded that
thc dimer has a higher maximum specific activity
but a lower affinity to fructose 6-phosphate than the
higher polymeric species. Therefore, it has to be as-
sumed that the sigmoidal shape of the fructose 6-phos-
phate velocity curve as well as ATP inhibition arise
at least partially from the displacement of the associa-
tion equilibria between different association states
of the enzyme.
A tentative model of the association behaviour
of human erythrocyte phosphofructokinase as a first
approach is now proposed. The following semi-
quantitative considerations are based on association
by Scheme 1 in which the free monomer is supposed
to be catalytically inactive. The specific activity of the
dimer is denoted by a,. As a first approximation,
it is assumed that all polymeric forms Mi at i 2 3
should have the same specific enzymic activity u3.
From this the following expression for the specific
activity is derived :
For calculation of the specific enzymic activity, the
distribution of the enzyme between the different
association states must be known. In order to obtain
a quantitative description the different association
steps have to be characterized by the association
constants. It is assumed that the association starts
with the nucleation step
M+M&M2 (2)
K.-W. Wenzel, B. 1. Kurganov. G. Zimmermann, V. A. Yakovlev, W. Schellenberger, and E. Hofmann 187

1.5 A B
1.5

1 .o 1 .o

.5

0
.M s
-8 0.5 -8'0.5

.o 1

0 0

-0.5 -0.5
-1 0 -1 0
log (€10 log IElo

,.
L D
1.5 1.5

1 .o 1 .o

m m
-g0.5 -g 0.5

0 0

-0.5 -0.9

Fig. 7 . Dependence of the ~ p ~ ~ uctivity

i f i c ofphosphojiwtokinase on the enzyme concentration. Logarithmic coordinates; concentrations of ATP
as indicated in the figure, Fructose &phosphate concentration: 0.01 mM (A), 0.02 mM (B), 0.05 mM (C) and 0.1 mM (D). Dimensions of
[El, are pg/ml and of a U!mg protein

which is characterized by the association constant KN for Scheme 1 or

[MI2
KN = P421
~

(3) M2 ( i - j ) - M,,
+ M2, 5 i 2 2; 1 I .j < i (4b)
for Scheme 2. For simplicity of the mathematical
and proceeds with the propagation steps
formulation, the following considerations are restricted
Mi-j + Mj$ Mi i 2 3 ; 1 ~j < i (4a) to the special case of Scheme 1, in which j = 1,
188 Cooperative Association of Erythrocyte Phosphofructokinase

1.
0

01

-2 -1 0 1 2 3 4
log K N (€10
Fig.8. Dependence qf a/a2 on K,. f E l 0 in logarithmic coordinates.
Computer output generated from E,qns (6) and (9) for a non-
cooperative associating enzyme system for various values of the
ratio a , p 2

meaning that only monomers may associate with

the multimeric forms. Hence, the association constant
K p is defined as
CM i l
Kp = M i - l ] [MI .
From the conservation equation of the enzyme the
following implicit equation for the fraction of mo-
nomerf; = [M]/[E]" is obtained

From the fraction of monomer the amounts of

dimeric and polymeric forms are obtained according
to Eqns (3) and (5) as:
log K"do
Fig. 9. ( A ) Dipndcwcc o/a/a, O I I K,. . [ E l o lit lognrirhmic coordiirutes
und ( B ) influence of the degree of'cooperutivity Kp& on tltc .S/ILII)P
qf ihe curves. Computer output based on Eqns(6) and (9) for
(A) various values of the ratio a,/a2 at K , / K , = 10 (positive co-
By insertion of Eqn (8) into Eqn (1) one obtains operative association) and (B) for various values of K p / K Nat a,~u,
a = Q2L + a?& = 0.1

= [E1O,f: + a3
(l - fl - KN [E1O,fi)* (9)
In this model, the dependence of specific enzymic
activity u on enzyme concentration [El, is determined
by the following four parameters: u2, u3, K , and Kp.
All of them depend on the actual concentrations of
fructose 6-phosphate and of ATP.
The association process is non-cooperative if
KN = K p . If the propagation steps are favoured in
comparison to the nucleation step, i.e. if KN < K p ,
the association process may be described as positive
cooperative [32].
The kinetic pattern generated by the model is
demonstrated in Fig. 8 and 9. As in the experimental
curves, the double logarithmic plot of the dependence
of activity on enzyme concentration has been used.
Due to the fact that the shape of the curves generated
from Eqn (9) depends only on the ratios of a3/a2and
KP/KN,the enzyme activity was normalized on the
activity a, of the enzyme form M, and the enzyme
concentration on the association constant KN. These
normalized curves may be compared directly with
the experimental ones as presented in Fig.4 and 7
after simple displacement of the coordinates by
-log KN and +log a,.
In Fig. 8 non-cooperative association is simulated.
With variation of the ratio u3/u2,maxima at aJa,
< 1 occur. The slopes of the increasing parts of the
K.-W. Wcnzcl, B. 1. Kurganov, G. Zimmermann, V. A. Yakovlev, W Schellcnberger, and E. Hofmann
1.5
I log a3
B; 0.01 rnM Fru-6-P
1rnM ATP
1 i'
-0.5 4 by independent physical methods are in progress.
-2 -1 0 1
log [El0
Fig. 10. C'oniparison q j t h e u.~periinentalcurve obtained ar 0.01 mM
fructose 6-phosphate and I mM ATP (taken .from Fig.4A) with
the model based on Eqns ( 6 ) and ( 9 ) by non-linear regression
analysis. The full line represents a cooperatively associating system,
the dotted line the non-cooperative case. For the cooperative case,
the parameters have been obtained as: K N = 0.059 ml . pg-',
K , = 4.85 ml pg-', u2 = 159.6 U . mg-', and u3 = 20.7 U . mg-'.
For the non-cooperative case: KN = K, = 1.3ml.pg-', a, = 6.8 U
. mg-' and a3 = 37.5 U . mg-'
curves do not exceed unity. This is in contrast to the
shape of the experimental curves shown in Fig. 4 and 7.
Therefore, the association process of the enzyme
cannot be described by assuming one single associa-
tion constant.
On the other hand, the curves in Fig.9 were gen-
erated by assuming cooperative association. In Fig. 9A
the dependence of the shape of the curves on a& is
shown. Their slopes reach unity at [El,+ 0.
In contrast to non-cooperative association, the
slope may exceed unity at certain enzyme concentra-
tions. The higher the cooperativity, the greater the
slope (Fig.9B). The occurrence of the curve maximum
does not only depend on aJa,, but also on K p / K N .
A maximum may appear when a,/a, < 1, but is
abolished at increasing cooperativity (Fig. 9 B).
The more general case of association of Scheme 1,
where,j 2 1, and of Scheme 2 give qualitatively similar
results.
In Fig. 10 the model based on Eqn (9) is fitted
to the experimental curve for 0.01 mM fructose
6-phosphate and 1 mM ATP (taken from Fig.4) by
non-linear regression analysis and assuming non-
cooperative as well as cooperative association. It may
be seen that the curve based on the model of non-
cooperative association gives systematic deviations,
189
whereas the curve calculated by assuming two different
association constants reflects sufficiently the experi-
mental results.
Qualitatively, the action of ATP may be explained
by assuming that it only decreases KN but is without
effect on uJa2 and on K p .
The effects of fructose 6-phosphate appear to be
more complex. In terms of this model it seems to
influence all four parameters. The appearance of
curve maxima with increasing fructose 6-phosphate
concentration may result either from a weakening of
cooperativity in the association process and/or from
decreasing u3/a2.
A quantitative check of these qualitative statements
needs the integration of the action of ATP and fructose
6-phosphate into this model. Such an analysis in
combination with investigations of the association
pattern of human erythrocyte phosphofructokinase
REFERENCES
1. Datta, P., Gest, H. & Segal, H. L. (1964) Proc. Natl Acad.
Sci. U.S.A. 51, 125-133.
2. Frieden, C. (1968) in Regulation of Enzyme Activity and
Allosteric Inferucfions,pp. 59- 86, Univcrsitetsforlaget,Oslo.
3. Frieden, C. (1971) Annu. Rev. Biochem. 40, 653-696.
4. Bonsignore, A., de Flora, A., Mangiarotti, M . A,, Lorenzoni,
I., Cancedda, R. & Dina, D. (1968) Ifal. J . Biochem. 17,
90 -97.
5. Le John, H. B., Mc Crea, B. E., Suzuki, I. &Jackson, S . (1969)
J. Biol. Chem. 244, 2484-2493.
6. Stancel, G. M. & Deal, W. C., Jr (1969) Biochemistry, 8,4005-
4011.
7. Constantinidcs, S. M. & Deal, W. C., Jr (1969) J . B i d . Chem.
244, 5695 - 5702.
8. Hulme, E. C . & Tipton, K. F. (1971) FEBS Left. 12, 197-200.
9. Hofer, H. W. (1971) Hoppe-Seyler's Z . Physiol. Chem. 352,
997 - 1004.
10. Kurganov, B. I. (1967) Chemistry und Technology of Polymers.
USSR, 11, 140- 163.
11. Kurganov, B. I. (1968) Mu/. Biol. 2, 430-438.
12. Nichol, L. W., Jackson, W. J . H. & Winzor, D J. (1967) Bio-
chemistry, 6, 2449 - 2456.
13. Frieden, C. (1967) J . B i d . Chem. 242, 4045-4052.
14. Kurganov, B. I. (1973) Acta B i d . Med. Ger. 31, 181 -201.
15. Kurganov, B. I. (1974) Mol. Biol. 8,525-535.
16. Hofmann, E., Kurganov, B. I., Schellenberger, W.. Schulz, J.,
Sparmann. G., Wenzel, K.-W. & Zimmermann. G. (1975)
Adv. Enzyme Re@. 13, 247 - 277.
17 Layzer, R. B., Rowland, L. P. & Bank, W. J. (1969) J. Bid.
Chem. 244,3823-3831.
18 Wenzel, K.-W., Zimmermann, G., Gauer, J . , Diezel, W., Liebe,
St. & Hofmann, E. (1972) FEBS Lett. IY, 285-289.
19 Zimmermann, G . , Wenzel, K.-W., Gauer, J. & Hofmann, E.
(1973) Eur. J . Biochem. 40, 501 - 505.
20. Tarui, S., Kono, N. & Uyeda, K. (1972) J . Biol. Chem. 247,
1138- 1145.
21. Staal, G. E.J., Koster, J . F., van Dijck, P. & van der Loo, A.
(1973) Int. J . Biochem. 4, 74-78.
22. Parmeggiani, A ,, Luft, J. K., Love, D. S . & Krebs, J. (1966)
J. Biol. Chem. 241, 4625-4637.
190 K.-W. W e n d rt al. : Cooperative Association of Erythrocyte Phosphofructokinase

23. Paetkau, V. & Lardy, H . A. (1967) J . Biol. Chem. 242, 2035- 28. Brennan, S.O., Davis, P. F. & Midwinter, G. G. (1974) Eur.
2042. J . Biochem. 42,489 -494.
24. Mansour, T. E. & Ahlfors, C. E. (1968) J . Biol. Chem. 243, 29. Otto, M., Heinrich, R., Kiihn, B. & Jacobasch, G. (1974)
2523- 2533. Eur. J . Biochem. 4Y, 169- 178.
25. Leonard, K. R. & Walker, I. 0. (1972) Eur. J . Biochem. 26, 30. Wenzel, K.-W., Gauer, J., Zimmermann, G. & Hofmann, E.
442 - 448. (1972) FEBS Lett. 19,281 -284.
26. Aaronson, R. P. & Frieden, C. (1972) J. Biol. Chem. 247, 31. Janatova, J., Fuller, J . K. & Hunter, M. J. (1968) J . Bid.
7502- 7509. Chem. 243,3612-3622.
27. Pavelich, M. J. & Hammes, G. G. (1973) Biochemistry, 12, 32. Winklmair, D. (1971) Arch. Biochem. Biophys. 147, 509-514.
1408- 1414.

K.-W. Wenzel, G. Zimmermann, W. Schellenberger, and E. Hofmann,

Physiologisch-ChemischesTnstitut, Bereich Medizin der Karl-Marx-Universitat Leipzig,
DDR-701 Leipzig, LiebigstraDe 16, German Democratic Republic

B. I. Kurganov and V. A. Yakovlev, Institute of Vitamin Research, Nauchny pr. 14A, Moskva, U.S.S.R. 117246