Increased purine nucleotide cycle activity associated with dietary zinc deficiency

Vol. 78, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

INCREASED PURINE NUCLEOTIDE CYCLE ACTIVITY ASSOCIATED WITH DIETARY ZINC DEFICIENCY M. S. Brody,

J.

Department

Received

R. Steinberg,

B. A. Svingen

of Biochemistry, East Lansing,

June 27,

Michigan Michigan

and R. W. Luecke

State

University

48824

1977

SUMMARY: Rat muscle 5'-AMP aminohydrolase (EC 3.5.4.6), adenylosuccinate synthetase (EC 6.3.4.4), and adenylosuccinate lyase (EC 4.3. 2.2) activities were elevated 50-6096 in zinc-deficient weanling rats when compared with restricted-fed zinc supplemental control rats. In addition, the activities of these enzymes were increased by 50-100% when zinc-deficient rats were compared with ad libitum-fed controls. There was no significant difference in totalyumotein or total muscle zinc among the three groups of animals. This increased activity of the purine nucleotide cycle may be responsible for the recently observed increase in blood ammonia in zinc-deficient rats when compared to controls. INTRODUCTION Zinc tissues

deficiency (l-4).

catabolism the

produces

There

is

catabolism

that (7)

and the fact

effects

of zinc

In addition,

recent

(10)

as well

as elevated

rats

as compared

activity

the activities pair-fed

supplemented

Copyright All rights

0 1977

reports

with

that

(8,

ammonia

zinc-supplemented

of

amino

several

sources

levels

(11) were

acid has

in muscle

activity.

acid

levels

in zinc-deficient suggestive

nucleotide

and adenylosuccinase.

zinc-supplemented,

In view

in muscle

aspartic

rats

enzymes

in protein

AMP deaminase

plasma

of the purine

of these

in various

9) we have investigated

muscle

of reduced plasma

6).

enzyme from

on rat

enzymes

(5,

may be involved

that.the

responses

an increase

deficiency

metalloenzyme

synthetase

zinc-deficient,

zinc

deficiency

of the other

adenylosuccinate measured

with

of enzyme

evidence

AMP deaminase

been shown to be a zinc the

recent

is associated

proposal

a variety

cycle

of increased (12,

13),

We have therefore homogenates

and --ad libitum-fed

from zinc-

rats.

by Academic Press, Inc. in any form reserved.

of reproduction

144 ISSN

0006-291x

Vol. 78, No. 1, 1977

BIOCHEMICAL

MATERIALS

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

AND METHODS

Male weanling rats of the Sprague-Dawley strain were obtained from a local breeding farm at 21 days of age and immediately placed on experiment. Rats were kept in individual stainless steel cages The zinc-deficient with free access to deionized distilled water. diet has been previously described (3). This diet contained, by analysis, 0.6 ppm zinc. Positive control diets contained added zinc carbonate to provide an additional 50 ppm of zinc. In the first experiment eighteen rats were divided into three equal groups. In the second experiment thirty rats were divided into three equal groups. For each experiment group 1 received the low zinc diet; group 2 the zinc-supplemented diet but with the intake restricted to the average amount consumed daily by group 1; group 3 received the zinc-supplemented diet in unrestricted amounts. After the three week experimental feeding period rats were killed by decapitation and upper hind leg muscle was dissected out and frozen at -20°C. Frozen muscle samples were thawed and individually homogenized in exactly 3.3 volumes of ice cold extraction buffer containing 0.18 M KCl, 0.054 M KH P04, 0.035 M K HP0 , pH 6.5. Extracts were stirred occasionally for Itine hour at roo ii; te $ perature and were then centrifuged at 14,000 x g for 20 minutes. The supernatant was filtered through Pyrex wool and all assays were performed on this fraction. AMP, IMP, GTP, aspartic acid, HEPES and MES were all purchased from Sigma Chemical Co. Adenylosuccinic acid was from Calbiochem. Enzyme assays were performed as follows: The conversion of AMP to IMP was followed spectrophotometrically at 265 nm. Adenylosuccinate synthetase and adenylosuccinase were assayed at 280 nm. AMP deaminase was assayed at 30°C with either 0.05 MM AMP (experiment 1) or 0.10 M-AMP (experiment 2), 0.1 M KCl, 0.05-M MES-Tris, pH 6.5. Adenylosuccinase was also assayed Zt 30°C with-O.05 mM adenylosuccinate, lOn+J potassium phosphate, pH 7.0. Adenylosucci-nate synthetase was assayed at 37°C in an assay mixture containing 5 ti-aspartic acid, 0.15 mM IMP, 0.06 mM GTP, 1 mM MgCl 0.05 M HEPES, pH 7.5. Basal rates &f reaction prior to the addifion of an aliquot of extract were subtracted from post-addition rates to yield synthetase activity. Protein determinations were by the method of Lowry et al (14) using bovine serum albumin as a standard. Atomic absorption analysis was performed using a Perkin-Elmer 303 Atomic Absorption Spectrophotometer and Harleco standardized zinc solutions. The muscle samples for analysis were ashed in a mixture of nitric acid and perchloric acid. Data were examined by analysis of variance, with statistical significance of treatment differences being determined by the multiple range test (15). RESULTS Table and diet depressed. that

I shows the consumption. Growth

effect

The growth

deficiency

controls

on total

of the zinc-deficient

of the restricted-fed

of the --ad libitum-fed

the zinc-deficient

of zinc

control but

group.

145

significantly

group

body weight rats

is

less

greater

is

severly than

than

Vol. 78, No. 1, 1977

TABLE I:

BIOCHEMICAL

Effect

of Zinc

Deficiency

Group

Experiment

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on Growth

and Diet

Initial Weight (9)

Consumption

Final

of Rats.

Average Daily Diet Consumed (9)

l*

1.

Zinc-deficient

2.

Restricted-fed

control

54.7

+ 1.8

68.5

f 3.7

4.9 + 0.3

54.5

-+ 1.9

92.8

2 5.2"

4.9 +- 0.1

3.

Ad libitum-fed control 54.7 t 1.9 190.8 ~12.1~ 12.6 + 0.6b -____ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*......f

Experiment

2**

1.

Zinc-deficient

2.

Restricted-fed

3.

Ad libitum-fed -~

* Each group shown.

value

56.2

+ 1.0

72.2

+ 1.6

5.3 -+ 0.1

control

55.8

+ 1.0

91.1

f 0.8a

5.3 -+ 0.1

control

56.2

+ 0.9

200.5

+ 2.5b

is

**Each group value deviation shown.

the average

is the

average

for

six

rats

with

the

standard

for

ten

rats

with

the

standard

a Significantly

greater

than

least

value

b Significantly

greater

than

least

two values

Table significant protein

II sumnarizes difference

concentration

ase activity cient

the

rats

is

elevated

muscle

by 50% in upper with

increase

cannot

be attributed

but must

reflect

an increase zinc-deficient

30% when zinc-deficient

animals

zinc

levels

to changes in the

total

Units

hind

muscle

146

leg control

in muscle units of activity with

is

no

or homogenate extract

are compared

controls.

There

However,

restricted-fed

rat.

(P < 0.01).

1.

AMP deaminof zinc-defi-

animals. protein

This content,

of activity are

of this increased

ad libitum-fed -~

+ 0.2b

deviation

(P < 0.01).

of experiment

among the groups.

when compared

enzyme in the

results

in whole

13.2

by

Vol.78,No.

1, 1977

TABLE II:

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Experiment 1; Effect of Zinc Deficiency on Whole Muscle Content and 5'-AMP Aminohydrolase Activity.

Muscle Zinc Content

Group

Extract Protein Content

(ppd

1.

Zinc-deficient

2.

Restricted-fed

3.

Ad libitum-fed -~

aExpressed

values

plus

are

for

there

8.60

+- 2.1

.033 f

control

8.14

& 0.77

8.70

+ 0.9

.021 + .003

control

9.05

_+ 1.13

11.40

+ 1.3

.019 + .003

AMP deaminated/min/mg

standard

(not

muscle

enzymes

cantly

elevated

least

of experiment

five

the

summarized

that

purine

when zinc-deficient

III.

in Table

nucleotide are

of all cycle

compared

zinc-supplemented

concen-

are with

three signifi-

either

control

animals.

DISCUSSION The purine results

in the

trated

in Figure

catalyzing with

the

the reported

aspartate The reasons

nucleotide net production 1. cycle

as described

of ammonia

The observation are

increase

in zinc-deficient for

cycle,

elevated

elevated

that

rats purine

by Lowenstein

from

aspartic

the

activities

in zinc-deficient

in plasma

ammonia

as compared nucleotide

147

1;

Again,

protein

the activities

rats

or --ad libitum-fed

in group

(P < 0.01).

among homogenate

It is clear

constituting

individuals

2 and 3.

two values

difference

shown).

restricted-fed

for

2 are

.008'

protein.

in groups

than

was no significant

trations

deviation

individuals

greater

The results

hg/ml)

2 0.93b

six

'Significantly

5'-AMP deaminase Specific Activitya

8.38

as vmoles

b Mean values

Zinc

acid

cycle

as illus-

of enzymes rats

and decrease with

(12),

controls activity

is consistent in plasma (10, are

11). not

Vol. 78, No. 1, 1977

TABLE

III:

BIOCHEMICAL

Experiment 2; Effect of Purine Nucleotide Cycle 5'-AMP Specific

Group 1.

Zinc-deficient

2.

Restricted-fed

3.

Ad libitum-fed -___

+ .265'

control

.118

+ .015

.304

+ .050

.645

+ .149

control

.107

+ .105

.237

+ .054

.618

+ .lOl

greater

deficiency

two

zinc

are

without

values

reason

for

catabolism cycle

are

of increased

16)

in liver

dehydrogenase,

responses protein

of resulting

liberated

nucleotide

cycle

does not seem to be the

activity.

is unchanged

apparently

metabolic

of the purine (7,

catabolism

liver,

muscle

not a result

function

of glutamate

enzyme in

protein

reutilization

increased

protein

the activity

controls

probably

acid

muscle

Therefore,

subsequent

of amino

(P < 0.01)

and total

The proposed

increased

Despite

the evidence

of zinc-deficient the major

amino

when such rats

are

rats

(6)

acid

deamin-

compared

to

(4).

Recently

evidence

the purine

nucleotide

(17,

That

18).

ATP production in the

least

muscle

acids.

ating

protein

by zinc-deficiency.

primary for

protein

than

Total

in terms

Adenylosuccinase Specific Activityb

Activityb

1.083

'Significantly

amino

Adenylosuccinate Synthetase Specific

of

+ .045'

as umoles/hr/mg

catabolism

of Enzymes

.483

bExpressed

to zinc

deaminase Activitya

on Activities

+ .023'

as pmoles/min/mg

unaffected

Deficiency

.159

aExpressed

clear.

Zinc

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

energy

of the purine fructokinase to be effective

is,

has been accumulating cycle

as a regulator

the AMP deaminase

by the myokinase charge nucleotide activity

cycle need

activators

reaction

may serve

the

(19)

ratio

to stimulate drops

of components

specifically,

Ammonium ions

of

charge

to sudden

effects

and,

of phosphofructokinase

148

functioning

energy

in response

on glycolysis

be considered.

the

of the

reaction

Additionally,

ratio.

for

are

phosphoknown

and oscillations

BIOCHEMICAL

Vol. 78, No. 1, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ASPARTIC ACID -,

NH3 Ir

GDP +

Y-----a

FUWATE

FIGURE1, THE FIJRINENUCLEOTIDE CYCLEINDICATINGTHE RELATIONSHIP BETWEEN ASPARTIC ACID UTILIZATION ANDAF/MONIAPRODUCTION

in the

purine

nucleotide

of AMP may have potent A high

correlation

activity that

cycle effects

between

among different AMP deaminase The manner

in the discussed ACKNOWLEDGEMENTS. Agricultural

on phosphofructokinase

muscle

fiber

Experiment

types

zinc

deficiency

work Station

remains

was supported as Journal

adds

(20).

and phosphofructokinase support

of glycolytic is

of levels activity

activity

is a regulator

enzyme activities This

in fluctuations

AMP deaminase

activity in which

resulting

responsible

to the flux for

hypothesis (21).

alterations

unsolved. in part

by the Michigan

Article

No. 7788.

REFERENCES 1. 2. 3. 4. 5. 6. 2 1;: 11. E:

J. G. and Simonian, S. J. (1968) J. Kfourv, G. A., Reinhold, Nutrition 95, 102-110. J . P. (1967) Prasad, A. S., Oberleas, D., Wolf, P., and Horw itz, J. Clin. Invest. 46, 549-557. Luecke, R. W., Olman, M. E., and Baltzer, B. V. (1968 ) J. Nutrition 94, 344-350. 103, 1175-1181. Huber, A. M. and Gershoff, S. N. (1973) J. Nutr ition Macapinlac, M. P., Pearson, W. N., Barney, G. H ., and Darby, W. J. (1968) J. Nutrition 95, 569-577. . HSU, J. M. and Anthony, W. L. (1975) J. Nutrition 105, 26-31. Lowenstein, J. M. (1972) Physiological Reviews 52, 382-414. Raggi, A. Ranieri, M., Taponeco, G., Ronca-Testoni, S., Ronca, G. and Rossi, C. A. (1970) FEBS Letters 10, 101-104. Zielke, C. L. and Suelter, C. H. (1971) J. Biol. Chem. 246, 2179-2186. HSU, J. M. and Anthony, W. L. 1977) Fed. Proc. 36, #4590. Rabbani, P. and Prasad, A. S. I 1977) Fed. Proc. 36, #4591. Lowenstein, J. M. and Tornheim, K. 1971) Science 171, 397-400. Tornheim, K. and Lowenstein, 3. M. t 1972) J. Biol. Chem. 247, 162-169.

149

Vol.78,

14. 15. 16. 17.

20. 21.

No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Farr, A. L., and Randall, R. J. Lowr 0. H., Rosebrough, N. J (l&l) J. Biol. Chem. 193, 2&275 Steel, R. G. D. and Torrie, J. H. (1960) Principles and Procedures of Statistics pp. 107-109, McGraw-Hill Co., New York. Turner. L. V. and Fern. E. B. (1974) Brit. J. Nutrition 32. 539-548 Chapman, A. G. and Atkinson, 0: E. (1973) J. Biol. Chem. 248, 8309-8312. Coffee, C. J. and Solano, C. (1977) J. Biol. Chem. 252, 1606-1612. Abraham, S. L. and Younathan, S. (1971) J. Biol. Chem. 246, 2464-2467. Tornheim, K. and Lowenstein, J. M. (1976) Fed. Proc. 35, #588. Winder, W. W., Terjung, R. L., Baldwin, K. M., and Holleszy, J. 0. (1974) Amer. J. Physiol. 227, 1411-1414.

150