Vol.1, No.4, 121-131 (2013) Advances in Enzyme Research
http://dx.doi.org/10.4236/aer.2013.14013
Spinach aldolase interactions with rabbit, chicken,
and fish muscle phosphofructokinase-1*
Anita Williams, Ami Abbott, Jessica Chadwick, Alicia Thomas, Nathalia Cruz,
Alice Deng, Leah Ordinanza, John Tat, Percy Russell#
Department of Medical Education, School of Medicine, University of California, Division of Biology, San Diego, USA;
#Corresponding Author:
Received 15 July 2013; revised 15 August 2013; accepted 19 August 2013
Copyright © 2013 Anita Williams et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Previous studies showed that rabbit muscle
phosphofructokinase-1 (PFK-1) activity losses
due to dilution, due to inhibition by ascorbate,
and due to some lithium salt s were prevented by
rabbit muscle aldolase. Chicken PFK-1 and fish
PFK-1 interacted w ith ascorbate and were inhib-
ited, consistent with a previously proposed
function that ascorbate facilitates glycogen in
resting muscle by inhibiting glycolysis. This
report shows that a plant enzyme, spinach al-
dolase, has the same ability to prevent rabbit
muscle PFK-1 activity loses as rabbit muscle
aldolase and in some instances it was a better
protector from activity losses than rabbit aldo-
lase. Spinach aldolase also protected chicken
and fish PFK-1s from inhibitions by ascorbate
and from activity losses due to dilution. Preven-
tion of losses PFK-1 activities from animal spe-
cies by a plant protein, spinach aldolase, sug-
gests an evolutionary conservative relationship
between PFK-1s and aldolases.
Keywords: Phosphofructokina se -1; Spi nach
Aldolase Interactions; Carbonate Inhibitions; Rabbit
Aldolase; Evolutionary Conservative Relationships;
Ascorbate Inhibition
1. INTRODUCTION
Influenced by several metabolic intermediates, phos-
phofructokinase-1 (PFK-1) is the putative controlling
enzyme of glycolysis. Compounds of potential therapeu-
tic value were examined to determine their influence on
PFK-1 activity. A report on the anti-metastatic effect of
an ascorbic (AA) fatty acid derivative [1] suggested a
study of other AA fatty acid derivatives. The effects of
AA on muscle PFK-1 from three animal species, mam-
mal, bird, and fish, were studied. A purpose of this study
was to further demonstrate or not the role of AA as a
facilitator of glycogen synthesis in resting muscle [2,3].
Because brain depends on glycolysis as an energy source
and because lithium salts are used therapeutically in
manic-depressive syndrome [4-9], another purpose was
to study the ability of Li2CO3 to inhibit rabbit muscle (rm)
PFK-1. Because spinach (sp) aldolase and rm aldolases
share several characteristics, comparison of rm aldo-
lases and sp aldolase interactions with mammalian
PFK-1s was undertaken; the abilities of rm aldolase and
sp aldolase were compared for their abilities to prevent
inhibitions of rm PFK-1.
2. MATERIALS
Biologicals and Chemicals
Sigma-Aldrich Co was the commercial source of
chemicals and enzymes in these experiments unless
stated otherwise. Rabbit muscle (rm) aldolase (A 8811)
was free of phosphofructokinase-1 (PFK-1), adenylate
kinase (AK) and lactate dehydrogenase (LDH) under our
conditions. Spinach (sp) aldolase preparation is described
below.
3. METHODS
The PFK-1 buffers were 10 mM Tris-phosphate, pH
8.0 (TP8) and all other solutions were pH 8.0 unless
stated otherwise. Temperatures were 25˚C.
3.1. Statistics
Each experiment (n) was done in duplicate by other
hands; the minimum number of experiments was n = 6.
*Declaration of interest statement: The authors report no declarations
of interes
t
.

A. Williams et al. / Advances in Enzyme Research 1 (2013) 121- 131
122
Data were acceptable when the standard error of the
mean (SEM) was less than ±10%. Error bars represent
±10% of the average SEM.
3.2. PFK-1 Preparations
Rabbit muscle (Oryctolagus cuniculus), chicken mus-
cle (cm, Gallus gallus) and red snapper muscle (fm, Lut-
janus peru) phosphofructokinase-1s (PFK-1, EC 2.7.1.56)
were prepared by our laboratory from frozen tissue after
a method by Kemp [10]. After determining that a PFK-1
preparation was void of aldolase activity, PFK-1 prepara-
tions were stored at 4˚C as 60% saturated ammonium
sulfate precipitates. Precipitates of rm PFK-1 were useful
in experiments for several weeks; cm PFK-1 and the fm
PFK-1 ammonium sulfate precipitates were not so stable
—cm PFK-1 was used up to 2 weeks after preparation
and fm PFK-1 was used up to a few days after prepara-
tion. A criterion for experimental use was an activity of
50% or greater than the original preparation. It was de-
termined that under these conditions there was no differ-
ence from a fresh preparation.
At 3.1 ± 0.1 eu/mL, stock PFK-1 solutions were con-
sidered 3 µM PFK-1 in 10 mM TP8. Dilutions were
made from 3 µM PFK-1 stock solutions to final concen-
trations given in individual experiments.
3.3. Criteria for PFK-1 Purity
Primary criteria for PFK-1 preparations were the ab-
sence of aldolase, LDH, and AK activities. The purity of
PFK-1 was also determined using enzyme activities and
SDS polyacrylamide gel electrophoresis (Figure 1) of rm
PFK-1 preparations. SDS PAGE used 12 percent cross-
linked gels and the Mini-Protean II Cell assembly as
given previously [11]. Bio-Rad gels were silver stained
for proteins using the procedure of Morrissey [12].
3.4. Standard PFK-1 Assay
PFK-1 activities, F 6-P (fructose 6-phosphate) + ATP =
F 1,6-BP (fructose 1,6 -bisphosphate), were measured
with a modification of the method by Anderson et al.
[13]. A 1.0 assay mixture contained 2 mM F 6-P; 1 mM
ATP (A 7699); 3 mM MgCl2; 0.13 mM NADH (N 1161);
1.7 eu/mL glyceraldehyde 3-phosphate dehydrogenase
(G 0763); 18 eu/mL triose phosphate isomerase (G1881);
1.3 eu/mL aldolase (A 8811); and 10 mM TP8 buffer as
final concentrations. A molar absorptivity value of 6220
converted NADH absorbance changes to µmoles of
product formed. One PFK-1 enzyme unit (eu) of activity
is defined as formation of 1 µmole of NAD+/min.
3.5. Dilute PFK-1 Assays
When PFK-1 activity rates were below 0.05 eu/min in
a 100 µL sample, assay system components in the Stan-
dard PFK-1 assay above were concentrated to 10 times
final assay concentrations into 0.1 mL. This allowed
PFK-1 test samples up to 0.9 mL in the assay for more
accurate rate activity measurements.
3.6. Aldolase Assay
Reagents for measurement of aldolase activity were
the same as the Standard PFK-1 assay above, except that
Figure 1. Polyacrylamide gel electrophoresis of a typical PFK-1 preparation. On
the right and left is a polyacrylamide gel electrophoresis with protein standards
and their molecular weights. In the center is a sample of a typical PFK-1 prepara-
tion.

A. Williams et al. / Advances in Enzyme Research 1 (2013) 121- 131 123
2 mM F 6-P and 1 mM ATP were omitted and replaced
by 2 mM F 1,6-BP. One eu of aldolase activity was de-
fined as formation of 1 µM NAD+/min.
3.7. LDH Assay
Measurement of LDH activity was according to Vas-
sault [14]. One eu of LDH activity was defined as forma-
tion of 1 µmole of NAD+/min.
3.8. Rabbit Muscle Aldolase
Rabbit muscle aldolase was obtained from Sigma-Al-
drich Co and was void of any PFK-1 activity. The per-
cent contamination of other enzymes, based on eu/mL
were as follows: LDH, 0.03 eu/mL; enolase, 0.1 eu/mL;
and adenylate kinase, 0.2 eu/mL, none of which inter-
fered with losses or preventions of losses of PFK-1 ac-
tivities.
3.9. Spinach Aldolase Preparation
The preparation of sp aldolase used was reported pre-
viously [15]. Figure 2 shows a polyacrylamide gel elec-
trophoresis (PAGE) of a typical sp aldolase preparation.
Purities of sp aldolase was determined by enzymatic de-
terminations of other enzyme contaminants and by SDS
polyacrylamide gel PAGE were run with 12 percent
cross-linked gels and silver stained for proteins accord-
ing to Morrissey [12]. The PAGE shows a contamination
of AK at 20 - 25 kD that was difficult to remove without
significant losses of sp aldolase. It was determined that
AK had no effect on either inhibitions of PFK-1 or loss
of activity due to dilution. When there was an occasional,
small contamination of sp PFK-1 activity in sp aldolase,
the appropriate PFK-1 activity was subtracted from sam-
ples containing sp aldolase.
3.10. Addition to Samples
The enzyme to be tested was added last from a 3 µM
stock solution. The order of reagent additions to final
samples concentrations were as follows: buffer, 10 mM
TP8; PFK-1; aldolase; inhibitors; and test-enzyme. A 45
min incubation allowed activities losses due to dilution
to stabilize, followed by an 1 hr incubation after inhibitor
additions, when activities were determined.
3.1 1. Ascorbate-Fatty Acid D erivatives
The L-ascorbate (AA) fatty acid derivatives were ob-
tained from TCI and Alfa Aesar.
Structures of the AA-fatty acid derivatives are shown
in Figure 3.
3.12. Measurements of Protein
Concentrations
PFK-1 protein concentrations during purification pro-
cedures were determined using the following formula:
mg protein/mL = 1.55 A280 – 0.76 A260, where A280 and
A260 are absorbencies at 280 nm and 260 nm, respec-
tively [16].
Figure 2. Polyacrylamide gel electrophoresis of a typical sp aldolase
preparation; On the right is a polyacrylamide gel electrophoresis with pro-
tein standards and their molecular weights. On the left is a sample of a sp
aldolase preparation. The major band on the left is associated with sp aldo-
lase and the minor band is associated adenylate kinase (AK, 21 kD), which
had no effect on activity losses due to dilution, activity losses due to AA
inhibition, or prevention of activity losses due to the presence rm aldolase.

A. Williams et al. / Advances in Enzyme Research 1 (2013) 121- 131
124
Figure 3. Structure of ascorbate fatty acid derivatives.
4. RESULTS
Activities relative to 0-time controls are shown in Fig-
ures 4 and 5 for cm PFK-1 and fm PFK-1 because of the
large differences in activities at various enzyme concen-
trations: 30 nM PFK-1, 100 nM PFK-1, and 300 nM
PFK-1. Figures 4(a) and (b) show characteristic differ-
ences between cm PFK-1 and fm PFK-1 dilutions with
time.
Figure 4(a) shows that losses of activity were cm
PFK-1 concentration dependant. Loss of activities due to
dilutions to 30 nM cm PFK-1 (□) or to 100 nM cm
PFK-1 (Δ) showed an 80 percent activity loss, while ac-
tivity lose due to dilutions to 300 nM cm PFK-1 (O)
showed a 30 percent activity loss. Activities can be shown
to remain stable for more than 2 h. Prevention against
some of these activity losses due to dilution was pro-
vided by the presence of 5 µM rm aldolase (■, ▲, ●).
In Figure 4(b), fm PFK-1 shows a different pattern
from cm PFK-1. All dilutions (□, Δ, O) show about 70
percent activity losses. The presence of 5 µM rm aldo-
lase (■, ▲, ●) also affected fm PFK-1 differently from
cm PFK-1. Initially, 300 nM fm PFK-1 (●) always
showed stimulations of activity immediately after dilu-
tion in the presence of rm aldolase. Activity losses due to
dilution to 100 nM cm (Δ) and 300 nM cm PFK-1 (O)
were completely prevented by rm aldolase. Prevention of
30 nM fm PFK-1 (□) activity losses due to dilution were
small by comparisons with 100 nM PFK-1 and 300 nM
fm PFK-1. Neither cm PFK-1 nor fm PFK-1 were so
well protected from activity losses due to dilution by rm
aldolase as was rm PFK-1 [2] where it was shown that
dilutions below 200 nM rm PFK-1 resulted in tetramer
dissociations to less active dimers or monomers.
Inhibitions of cm PFK-1 and fm PFK-1 by AA were
examined. Figures 5(a) and (b) show the activity losses
of cm PFK-1 and fm PFK-1 due to dilutions and due to
inhibition by AA and effects of rm aldolase against these
losses. The relative activities in Figure 4 differ from the
relative activities in Figure 5. All activities of PFK-1
concentrations in Figure 4 are relative to 0-times active-
ties in the absence of AA and the absence (open symbols)
or presence of aldolase (closed symbols); each set has a
0-time value. Both cm PFK-1 and fm PFK-1 were more
sensitive to AA inhibition than rm PFK-1, which was not
inhibited by AA above 200 nM rm PFK-1 [2]. The rela-
tive activities in Figures 5(a) and (b) shows the preven-
tion of activity losses due to dilution and due to AA inhi-
bition of cm PFK-1 and fm PFK-1 by rm aldolase were
similar; the 0-time values are based upon the on the
0-time values in the absence aldolase only for each set.
It was previously shown that 0.1 molar monovalent
carbonates salts or sulfates inhibited rm PFK-1 [2,18].
Tables 1(a) and (b) show percent inhibitions of 30 nM
cm PFK-1 and 30 nM fm PFK-1 by monovalent salts; the
negative values show the percent stimulation. The con-
spicuous differences between cm PFK-1 and fm PFK-1
inhibitions were with Li2CO3 that almost completely
inhibited cm PFK-1, while fm PFK-1 was less than 50
percent inhibited. Lithium salts were particular of inter-
est because of their use in manic-depressive disorder
therapy and the many speculations for their therapeutic
value [4-9]. The Li2CO3 inhibition of PFK-1 and the de-
pendency of brain on glycolysis as an energy source add
the of inhibition glycolysis [5] as a possible mechanism
of its action. Glycolysis is also considered the major en-
ergy source in cancer cells [19,20] and the report [1] that
an AA fatty acid derivative prevented metastasis in mice
and cancer growth in cell culture prompted testing other
AA fatty acid derivatives on their abilities to inhibit 30
nM rm PFK-1.
Figures 6(a)-(d) compare the ability of rm aldolase
and sp aldolase to prevent activity loses of rm PFK-1 due
to dilution, due to inhibitions by AA or AA fatty acid
derivatives. Figures 6(a)-(d), show both rm aldolase and
sp aldolase prevented losses of activities due to dilution
in all instances. About half to two-thirds of the predicted
activity of 3 µM rm PFK-1 stock solutions diluted to 30

A. Williams et al. / Advances in Enzyme Research 1 (2013) 121- 131 125
A
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
0 102030405060
minutes
Relative activity cm PF
K
-1 eu/mL
(a)
B
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
0 102030405060
minutes
(b)
Figure 4. Loss of PK-1 activities due to dilution and the effect of aldolase. Figure 4 and
Figure 4(b) show activity loses with time of cm PFK-1 (a) and fm PFK-1 (b) at various
PFK-1 concentrations and the effects of 5 µM rm aldolase. Symbols for PFK-1 concentra-
tions are 30 nmolar (□, ■), 100 nmolar (Δ, ▲) and 300 nmolar (O, ●) for the absence of 5
µM rm aldolase (open symbols) and presence of 5 µM rm aldolase (closed symbols). Each
0-time value for a PFK-1concentration set was estimated activities from their 3 µM PFK-1
stock solutions independently from the others; In Figure 4(a), The 0-time estimates of the
cm PFK-1 dilutions were as follows: 30 nM cm PFK-1 = 0.0091 eu/mL; 100 nM cm
PFK-1 = 0.062 eu/mL; and 300 nM cm PFK-1 = 0.265 eu/mL. In Figure 4(b) the estimate
of the fm PFK-1 dilutions were 30 nM fm PFK-1 = 0.0192; eu/mL; 100 nM fm PFK-1 =
0.0755 eu/mL; and 300 nM fm PFK-1 = 0.168 eu/mL. The relative activities shown are
independently based on 0-time values of each set of dilutions. Other conditions are given
in Methods.
nM PFK-1 is lost [2] in the absence of aldolase. In Fig-
ure 6(a), differences between the two aldolases were in
their abilities to prevent inhibitions of 30 nM rm PFK-1
by AA. Rm aldolase partially prevented inhibition by AA;
sp aldolase not only prevented inhibition by AA but also
prevented activity loss due to dilution in the presence of
AA. In Figure 6(b), rm aldolase prevented inhibition by
AAP while sp aldolase not only prevented inhibition by
AAP but also prevented activity lost due to dilution. In
Figure 6(c), neither rm aldolase nor sp aldolase pre-
vented losses due to AAS. In Figure 6(d), rm aldolase
prevented inhibition by AADP but had no effect on pre-
venting losses due to dilution in the presence of AADP;
sp aldolase, on the other hand, prevented losses of activi-
ity due to dilution and prevented inhibition by AADP
simultaneously.
In summary, while both rm aldolase and sp aldolase
protect 30 nM rm PFK-1 from activity losses due to dilu-
tion, sp aldolase was superior in protecting against inhi-
itions by AA, AAP and AADP (Figures 6(a), (b) and b

A. Williams et al. / Advances in Enzyme Research 1 (2013) 121- 131
126
0.00
0.50
1.00
1.50
2.00
0 0.511.5 2
mM ascorbate
A
(a)
0.00
0.50
1.00
1.50
2.00
2.50
0.0 0.5 1.0 1.5 2.0
mM ascorbate
B
Relative activity
fm PFK
-
1
eu/m L
(b)
Figure 5. The effect of rm 5 µM aldolase on AA inhibitions. Symbols are the
same as in Figures 4. In Figures 5 the 0-time values of each PFK-1 concentra-
tion set is based on the 0-time value with no aldolase (open symbols) and no AA.
The 0-time values were as follows: in Figure 5(a) 30 nM cm PFK-1 = 0.0086
eu/mL; 100 nM cm PFK-1 = 0.056 eu/mL; and 300 nM cm PFK-1 = 0.258
eu/mL. In Figure 5(b), 30 nM fm PFK-1 = 0.0188; eu/mL; 100 nM fm PFK-1 =
0.0743 eu/mL; 300 nM fm PFK-1 = 0.165 eu/mL. Other conditions are given in
Figure 4 and in Methods.
(d)). Both rm aldolase and sp aldolase were ineffective
against inhibition by AAS (Figure 6(c)).
Figures 7(a)-(c) compare the abilities rm aldolase and
sp aldolase to prevent 30 nM rm PFK-1 inhibitions by
carbonate salts. Figure 7(a) shows that inhibitions by
K2CO3 were prevented by both rm aldolase and sp aldo-
lase and also prevented activity losses due to dilution in
the presence of K2CO3; In Figure 7(b), neither rm aldo-
lase nor sp aldolase prevented inhibition by Li2CO3. In
Figure 7(c), inhibition by Na2CO3 was prevented by rm
aldolase and sp aldolase but sp aldolase also prevented
losses of activity due to dilution in the presence of
Na2CO3.
5. DISCUSSION
The results demonstrated characteristics common
among PFK-1s purified from three species—mammals,
fish, and bird, supporting the role of AA as a facilitator of
glycogen synthesis [2,3] in animals. Both cm PFK-1 and
fm PFK-1 inhibitions were similar to rm PFK-1 activity
with respect to losses due to dilution (Figure 4), AA
(Figure 5), AA fatty acid derivatives (Figure 6), carbon-
ate salts, and sulfate salts (Table 1, Figure 7). The same
figures also showed sp aldolase was similar to rm aldo-
lase in preventing these rm PFK-1 activity losses with at
east the same effectiveness. These outcomes appear to l

A. Williams et al. / Advances in Enzyme Research 1 (2013) 121- 131 127
A
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
CONTROL+AA+rm
aldolase '+AA +m
aldolase
+sp
aldolase '+AA +sp
aldolase
30 nM rm PFK-1 eu/mL
(a)
B
0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
0.0300
CONTROL+AAP+rm
aldolase +AAP +rm
aldolase
+sp
aldolase +AAP '+sp
aldolase
30 nM rm PFK-1 eu/mL
(b)
C
0.000
0.005
0.010
0.015
0.020
0.025
0.030
CONTROL+AAS '+rm
aldolase '+AAS +rm
aldolase
+sp
adolase '+AAS +sp
adolase
30 nM rm PFK-1 eu/mL
(c)
D
0
0.005
0.01
0.015
0.02
0.025
0.03
CONTROL+AADP+rm
aldolase '+AADP +rm
aldolasse
+sp
aldolase +AADP +sp
aldolase
30 nM rm PFK-1 eu/mL
(d)
Figures 6. Comparisons of rm aldolase and sp aldolase effects on inhibitions of 30 nM rm PFK-1 by AA, AAP, AAS, and AADP.
Final concentrations of inhibitors were as follows: 2 mM AA; 20 µM AAP; 20 µM AAS; and 10 µM AADP. The activities of initial
stock solutions for dilutions to 30 nM PFK-1 were as follows: (a) 2.81 eu/mL; (b) 2.68 eu/mL; (c) 2.78 eu/mL; and (d) 2.64 eu/mL.
Other conditions are given in Methods.
reflect a conservation of PFK-1 characteristics among
other species and a conservation of a relationship be-
tween PFK-1s and aldolases.
Activity losses due to dilutions of cm PFK-1 were
more similar to rm PFK-1 than were dilutions of fm
PFK-1. At dilutions to about 300 nM cm PFK-1, activity
losses due to dilution (Figure 4) started to occur,
whereas activity losses due to dilution occurred below
200 nM rm PFK-1 [2], suggesting that rm PFK-1
etramer is less likely to dissociate than cm PFK-1. Fm t

A. Williams et al. / Advances in Enzyme Research 1 (2013) 121- 131
128
0.00
0.50
1.00
1.50
2.00
2.50
CONTROL+K2CO3 +rm
aldolase +K2CO3 +rm
aldolase
+sp
aldolase +K2CO3 +sp
aldolase
Relative activity
30 nM rm PFK-1 eu/mL
A
(a)
0.00
0.50
1.00
1.50
2.00
2.50
3.00
CONTROL+Li2CO3+rm
alldolase +Li2CO3 +rm
alldolase
+sp
aldolase +Li2CO3 +sp
aldolase
Relative activity
30 nM rm PFK-1 eu/mL
B
(b)
0.00
0.50
1.00
1.50
2.00
2.50
CONTROL+Na2CO3+rm
alldolase +Na2CO3 +rm
alldolase
+sp
aldolase +Na2CO3 +sp
alldolase
Relative activity
30 nM rm PFK-1 eu/mL
C
(c)
Figures 7. (a)-(c) Comparisons of rm aldolase and sp aldolase effects on inhibit-
tions of 30 nM rm PFK-1 by 0.1 M monovalent salts. Activities of initial stock
solutions for dilutions to 30 nM rm PFK-1 were as follows: (a) 2.91 eu/mL; (b)
2.77 eu/mL; and (c) 2.93 eu/mL. Other conditions were the same as in Figure 6.
PFK-1 appeared to lose about the same activity at all
dilutions tested, suggesting that it is most likely to disso-
ciate with dilution. It was previously determined that rm
PFK-1 activity loses were due to dissociations from
tetramers to less active dimers or monomers [2].
Figures 5(a) and (b) show that preventions of AA in-
hibitions by rm aldolase were similar for cm, fm PFK-1
and rm PFK-1 [2,3]. Figure 5(b) shows that aldolase
(closed symbols) protects against activity losses due to
dilution and activity losses due to AA inhibition.

A. Williams et al. / Advances in Enzyme Research 1 (2013) 121- 131 129
Table 1. Inhibition of 30 nM cm PFK-1 and 30 nM fm PFK-1
by monovalent salts. A minus sign (–) indicates the percentage
of stimulation. (a) Percent inhibition of 30 nM cm PFK-1 by
0.1 M monovalent salt; (b) Percent inhibition of 30 nM fm
PFK-1 by 0.1 M monovalent salts.
(a)
CATIONS ANIONS
Acetate Carbonate Chloride Sulfate
Lithium 5 95 4 72
Potassium −5 60 −13 48
Sodium 9 22 12 31
(b)
Cations Anions
Acetate Carbonate Chloride Sulfate
Lithium 4 43 6 63
Potassium 4 39 9 53
Sodium 18 46 4 58
Tables 1(a) and (b) show that cm PFK-1 and fm
PFK-1 carbonate and sulfate salt inhibitions follow inhi-
bition patterns similar to rm PFK-1 [2,18]. Generally,
acetates and chlorides were poor or not inhibitors and
carbonates and sulfates were good inhibitors of rm
PFK-1. Lithium salts inhibitions were of interest particu-
lar because of their therapeutic use in manic-depressive
syndrome [4-9] and because of brain dependence on
glycolysis.
Figures 4(a)-(d) show inhibitions of rm PFK-1 by AA
fatty acid derivatives and AA and compares the abilities
of rm aldolase and sp aldolase to prevent these inhibi-
tions. The AA fatty acid derivatives and AA showed dif-
ferent inhibition patterns. Sp aldolase appeared to be
superior to rm aldolase in preventing AA inhibitions. The
AA fatty acid derivatives were several-fold more inhibi-
tory to rm PFK-1 than AA. It was proposed by the au-
thors [1] that the therapeutic value of AA2P6L in mouse
cancer was due to its antioxidant properties. Since cancer
cells are so dependent upon glycolysis as an energy
source [19,20] and in view of the inhibitory character of
AA fatty acid derivatives here, an alternate view is that
the therapeutic value of AA2P6L [1] was due to its abil-
ity inhibit PFK-1 and glycolysis. In this regard, it may be
significant that rm aldolase does not prevent Li2CO3 in-
hibition of rm PFK-1.
Figures 5(a)-(c) show similarities between the ability
of rm aldolase and sp aldolase to prevent inhibitions of
rm PFK-1 by carbonate salts; as in previous Figures,
both aldolases prevented activity loses due to dilution. In
Figure 7(a), K2CO3 inhibitions were prevented by both
rm and sp aldolases and prevented losses due to dilutions
even in the presence of K2CO3. In Figure 7(b), neither
rm aldolase nor sp aldolase prevented inhibitions of rm
PFK-1 by Li2CO3. In Figure 7(c), sp aldolase was both
effective in preventing inhibitions by Na2CO3 but sp al-
dolase also prevented activity losses due to dilution even
in the presence of the Na2CO3, as with K2CO 3 in Figure
7(a).
In summary, it was shown that rm aldolase and sp al-
dolase interacted with PFK-1s from rabbit, chicken, and
fish in a similar manner. Both rm aldolase and sp aldo-
lase prevented activity losses due to dilution and due to
inhibitions by ascorbate, by AA fatty acid derivatives,
and by monovalent carbonate salts. In some instances, sp
aldolase was better than rm aldolase in preventing activ-
ity losses and in others sp aldolase was better.
It was shown by others [21-24] that aldolase from
spinach was closely related to the Class I (non-metallic)
aldolases found in eukaryotes. The close relationships
among Class 1 aldolases were based upon immunologi-
cal cross-reactions, chemical similarities, and structural
similarities.
The cm and fm PFK-1 inhibitions by AA and preven-
tions of inhibition by rm aldolase support the proposed
role of AA as a facilitator of glycogen synthesis [2] in
other species. Reports of faulty glycogen synthesis in
uncompensated diabetics may be explained by the lack
of AA in muscle [25-32]; AA requires the same mecha-
nism for entry into muscle as glucose. The muscle of the
uncompensated diabetic has been described as having
tissue scurvy [31].
This study supports reports of similarities of aldolase
isolated from spinach leaves and aldolases isolated from
mammalian muscle tissues. These similarities include
catalytic properties [21]; amino acid sequence [23]; and
immunological similarities [22]. This study adds to simi-
larities of rm aldolase and sp aldolase since both interact
with rm PFK-1, suggesting the existence a conservation
interrelationship between PFK-1s and the aldolases
among several species.
6. ACKNOWLEDGEMENTS
The projects described were supported by Grant Number P60
MD00220 from the San Diego EXPORT, NCMHD, NIH; by Grant
Number 1 R25 GM73590 of the UCSD CURE Program, National
Cancer Institute, NIH; by Grant Number R25 GM083275 UCSD IMSD
Program National Institute of General Medical Sciences, NIH; by Grant
Number D34HP18954 the Hispanic Center of Excellence, Health Re-
sources and Services Administration, and by Grant Number D18HP10623
Health Career Opportunities Program, Health Resources and Services
Administration. Its contents are solely the responsibility of the authors
and do not necessarily represent the official views of the National In-

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130
stitutes of Health.
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