Spinach aldolase interactions with rabbit, chicken, and fish muscle phosphofructokinase-1

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: prussell@ucsd.edu

Received 15 July 2013; revised 15 August 2013; accepted 19 August 2013

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

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

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

0 102030405060

minutes

Relative activity

cm PF

-1 eu/mL

(a)

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)

0.00

0.50

1.00

1.50

2.00

2.50

0.0 0.5 1.0 1.5 2.0

mM ascorbate

Relative activity

fm PFK

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

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

CONTROL+AA+rm

aldolase '+AA

aldolase

+sp

aldolase '+AA

+sp

aldolase

30 nM rm PFK-1 eu/mL

(a)

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)

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)

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)

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)

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)

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-

A. Williams et al. / Advances in Enzyme Research 1 (2013) 121- 131

130

stitutes of Health.

REFERENCES

[1] Liu, J., Zhang, X., Yang, F., Li, T., Wei, D. and Ren, Y.

(2006) Antimetastatic effect of a lipophilic ascorbic acid

derivative with antioxidation through inhibition of tumor

invasion. Cancer Chemotherapy and Pharmacology, 57,

584-590. http://dx.doi.org/10.1007/s00280-005-0073-9

[2] Russell, P., Williams, A., Marquez, K., Tahir, Z., Hos-

seinian, B. and Lam K. (2008) Some characteristics of

Rabbit muscle phosphofructokinase-1 inhibition by as-

corbate. Journal of Enzyme Inhibition and Medicinal

Chemistry, 23, 411-417.

http://dx.doi.org/10.1080/14756360701611621

[3] Russell, P.J., Williams, A., Amador, X. and Vargas, R.

(2004) Aldolase and actin protect rabbit muscle lactate

dehydrogenase from ascorbate inhibition. Journal of En-

zyme Inhibition and Medicinal Chemistry, 19, 91-98.

http://dx.doi.org/10.1080/14756360310001623309

[4] Struneck, A., Patočka, J. and Sarek, M. (2005) How does

lithium mediate its therapeutic effects? Journal of Applied

Biomedicine, 3, 25-35.

[5] Kajda, P.K. and Birch, N.J. (1981) Lithium inhibition of

phosphofructokinase. Journal of Inorganic Biochemistry,

14, 275-278.

[6] Nordenberg, J., Kaplansky, M., Beery, E., Klein, S. and

Beitner, R. (1982) Effects of lithium on the activities of

phosphofructokinase and phosphoglucomutase and on

glucose-1,6-diphosphate levels in rate muscles and liver.

Biochemical Pharmacology, 31, 1025-1031.

http://dx.doi.org/10.1016/0006-2952(82)90338-0

[7] Rodriguez-Gil, J.E., Fernandez, J.M., Barbera, A. and

Guiovart, J.J. (2000) Lithium’s effects on rat liver glucose

metabolism in vivo. Archives of Biochemistry and Bio-

physics, 375, 377-384.

http://dx.doi.org/10.1006/abbi.1999.1679

[8] Klein, P.S. and Melton, D.A. (1996) A molecular mecha-

nism for the effect of lithium on development. Proceed-

ings of the National Academy of Sciences of the United

States of America, 93, 8455-8459.

http://dx.doi.org/10.1073/pnas.93.16.8455

[9] Mann, L., Heldman, E., Shabltiel, G., Belmaker, R.H. and

Agam, G. (2008) Lithium preferentially inhibits adenyl

cyclase V and VII isoforms. International Journal of

Neuropsychopharmacology, 11, 533-539.

[10] Kemp, R.G. (1975) Phosphofructokinase from rabbit

skeletal muscle. In: Woods, W., Ed., Methods in Enzy-

mology, Vol. 42C, Academic Press, New York, 71-77.

[11] Russell, P.J., Williams, A., Abbott, A., DeRosales, B. and

Vargas, R. (2006) Characteristics of rabbit muscle ade-

nylate kinase inhibition by ascorbate. Journal of Enzyme

Inhibition and Medicinal Chemistry, 21, 61-67.

http://dx.doi.org/10.1080/14756360500043372

[12] Morrissey, J.H. (1981) Silver stain for proteins in poly-

acrylamide gels: A modified procedure with enhanced

uniform sensitivity. Analytical Biochemistry, 117 , 307-

310. http://dx.doi.org/10.1016/0003-2697(81)90783-1

[13] Anderson, R.L., Hanson, T.E. and Sapico, V.L. (1969)

D-Fructose-1-phosphate kinase and D-fructose 6-phosphate

from Aerobacter aerogenes. The Journal of Biological

Chemistry, 244, 6280-6288.

[14] Vassault, A. (1983) Methods of enzymatic analysis, en-

zymes I: Oxidoreductases, transferases Vol. III. Verlag

Chemie, Basel, 118-126.

[15] Lebherz, H.G., Leadbetter, M.M. and Bradshaw, R.A.

(1984) Isolation and characterization of the cytosolic and

chloroplast forms of spinach leaf fructose diphosphate

aldolase. The Journal of Biological Chemistry, 259, 1011-

1017.

[16] Wilson, K. and Walker, J. (2001) Principles and tech-

niques of practical biochemistry. Cambridge University

Press, Cambridge, 319-204.

[17] Bradford, M.M. (1976) Determination of protein concen-

tration in the manufacture and characterization of bio-

pharmaceuticals. Analytical Biochemistry, 72, 248-254.

http://dx.doi.org/10.1016/0003-2697(76)90527-3

[18] Russell, P., Williams, A., Abbott, A., Chadwick, J., Ehya

F., Flores, R. and Hardamon, C. (2010) Effect of lithium

salts on lactate dehydrogenase, adenylate kinase, and

1-phosphofructokinase activities. Journal of Enzyme In-

hibition and Medicinal Chemistry, 25, 551-556.

http://dx.doi.org/10.3109/14756360903357627

[19] Pelicano, H., Martin, D.S., Xu, R.-H. and Huang, P. (2006)

Glycolysis inhibition for anticancer treatment. Oncogene,

25, 4633-4646. http://dx.doi.org/10.1038/sj.onc.1209597

[20] Herling, A., König, M., Bulik, S. and Holzhütter, H.-G.

(2011) Enzymatic features of the glucose metabolism in

tumor cells. FEBS Journal, 278, 2436-2459.

http://dx.doi .org/10.1111/j.1742-4658.2011.08174.x

[21] Flurit, R., Ramasarma, T. and Horecker, B.L. (1967) Pu-

rification and properties of fructose diphosphate aldolase

from spinach leaves. European Journal of Biochemistry,

1, 117-124.

[22] Kruger, I. and Schnarrenberger, C. (1983) Purification,

subunit structure and immunological comparison of fruc-

tose-bisphosphate aldolases from spinach and corn leaves.

European Journal of Biochemistry, 136, 101-106.

http://dx.doi .org/10.1111/j.1432-1033.1983.tb07711.x

[23] Lebherz, H.G., Leadbetter, M.M. and Bradshaw, R.A.

(1984) Isolation and characterization of the cytosolic and

chloroplast forms of spinach leaf fructose diphosphate

aldolase. The Journal of Biological Chemistry, 259, 1011-

1017.

[24] Rutter, W.J. (1964) Evolution of aldolases. Federation

Proceedings, 23, 1248-1257.

[25] Nikoulina, S.E., Ciaraldi, T.P., Abrams-Carter, L., Muda-

liar, S., Park, K.S. and Henry, R.R. (1997) Regulation of

glycogen synthase activity in cultured skeletal muscle

cells from subjects with type II diabetes: Role of chronic

hyperinsulinemia and hyperglycemia. Diabetes, 46, 1017-

1024. http://dx.doi.org/10.2337/diab.46.6.1017

[26] Nikoulina, S.E., Ciaraldi, T.P., Carter, L., Mudaliar, S.,

Park, K.S. and Robert, R.H. (2001) Impaired muscle

glycogen synthase in type 2 diabetes is associated with

diminished phosphatidylinositol 3-kinase activation. The

A. Williams et al. / Advances in Enzyme Research 1 (2013) 121- 131

131

Journal of Clinical Endocrinology & Metabolism, 86,

4307-4314. http://dx.doi.org/10.1210/jc.86.9.4307

[27] Shulman, G.I., Rothman, D.L., Jue, T., Stein, P., De-

Fronzo, R.A. and Shulman, R.G. (1990) Quantitation of

muscle glycogen synthesis in normal subjects and sub-

jects with non-insulin-dependent diabetes by 13C nuclear

magnetic resonance spectroscopy. The New England

Journal of Medicine, 322, 223-228.

http://dx.doi.org/10.1056/NEJM199001253220403

[28] Henry, R.R., Ciaraldi, T.P., Abrams-Carter, L., Mudaliar,

S., Park, K.S. and Nikoulina, S.E. (1996) Glycogen syn-

thase activity is reduced in cultured skeletal muscle cells

of non-insulin-dependent diabetes mellitus subjects. Bio-

chemical and molecular mechanisms. Journal of Clinical

Investigation, 98, 1231-1236.

http://dx.doi.org/10.1172/JCI118906

[29] Halse, R., Bonavaud, S.M., Armstrong, J.L., McCormack,

J.G. and Yeaman, S.J. (2001) Control of glycogen synthe-

sis by glucose, glycogen, and insulin in cultured human

muscle cells. Diabetes, 50, 720-726.

http://dx.doi.org/10.2337/diabetes.50.4.720

[30] Chen, H., Karne, R.J., Hall, G., Campia, U., Panza, J.A.,

Cannon III, R.O., Wang, Y., Katz, A., Levine, M. and

Quon, M.J. (2006) High-dose oral vitamin C partially re-

plenishes vitamin C levels in patients with type 2 diabetes

and low vitamin C levels but does not improve endothe-

lial dysfunction or insulin resistance. American Journal

of Physiology—Heart and Circulatory Physiology, 290,

H137-H145.

http://dx.doi.org/10.1152/ajpheart.00768.2005

[31] Cunningham, J.J. (1998) The glucose/insulin system and

vitamin C: Implication in insulin-dependent diabetes mel-

litus. Journal of the American College of Nutrition, 17,

105-108.

http://dx.doi.org/10.1080/07315724.1998.10718734

[32] Will, J.C. and Byers, T. (1996) Does diabetes mellitus

increase the requirement for vitamin C? Nutrition Re-

views, 54, 193-202.

http://dx.doi .org/10.1111/j.1753-4887.1996.tb03932.x

上一篇:Dexamethasone treatment alters 下一篇:Optimized production and prope

我要分享到：

职称论文发表交流平台！

Spinach aldolase interactions with rabbit, chicken, and fish muscle phosphofructokinase-1

最新文章NEWS

推荐期刊Tui Jian

热点文章HOT