Effects of NaF on amylase in mung bean seedlings

Journal of Fluorine Chemistry, 41 (1988) 95-100

EFFECTS

OF NaF ON AMYLASE

M. YU, M. SHUMWAY Huxley

College

University,

95

IN MUNG

BEAN

SEEDLINGS

and A. BROCKBANK

of Environmental

Bellingham,

Studies,

WA 98225

Western

Washington

(U.S.A.)

SUMMARY

Extracts lings

obtained

treated

72 hours

have

centrations

been

studied

effect

for amylase found

of fluoride

of added

inhibition

CaC12.

seed-

activity.

NaF at con-

to inhibit

the enzyme.

was markedly

These

of amylase

due to the interaction

(Vigna radiata)

1.0, and 5.0 mM for 48 and

as low as 1.0 mM was

the presence

enzyme

bean

NaF at 0, 0.1,

The inhibitory

induced

from mung

with

data

in mung

of F- with

suggest

bean

Ca2+,

diminished that

seedlings

which

in

the F-may be

is required

for

activity.

INTRODUCTION

It is widely sources ergy

such as

as well

known

as basic

growth

and development

ample,

depend

pounds. with

degradation

in mung NaF

acids.

bean

previously

qualitative

In addition,

from these

tissues

Subsequently,

lipase

was

found

we observed

0022-1139/88/$3.50

leaves,

for ex-

exists

germination,

en-

The

energy-releasing

of literature

energy

to provide

purposes.

and primary

com-

dealing

reports

on the

are limited.

that NaF

(Vigna radiata)

showed

stored

down

for anabolic

of these

during

of F- in this process

germination

are broken

of radicles

a large volume

We reported

with

components

on the breakdown

While

starch

effects

that during

fats and starch

inhibited

fat metabolism

[ll.

Tissues

treated

and quantitative

changes

in fatty

seedlings

activity

in cotyledon

to be significantly

that. F--treated

extracts inhibited.

seedlings

contained

0 Elsevier Sequoia/Printed in The Netherlands

higher

amounts

water

in the F--treated

degradation

or decrease in an attempt

MATERIALS

AND METHODS

Mung

bean

seeds

previously

of 48 hours cotyledons crude

enzyme

buffer,

extract.

and the

x g for 10 minutes at 22,000 used

x g for 30 minutes

as crude Enzyme

enzyme

assay

solution,

and

carried buffer,

0.05 M of the buffer. except

mixture

was

unless

period,

containing

cooling,

pH 7.0,

otherwise

mixture

was heated

Protein

was defined

was

The amount

content

as pg glucose

containing

all comThe assay

at 30° C for

At the end of the incu-

blue

water

was

into a test solution

tube

[3-51.

for 10 minutes.

Upon

followed

was centrifuged,

read

of sugar

by and

in a spectrophotom-

produced

during

the

by use of a calibration

of the enzyme et al.

of

1:l with

as a blank.

The mixture

was determined

of Lowry

consisting

[3,4] was added,

[5].

by the method

supernatant

diluted

was pipetted

in boiling

of 12 ml water.

period

at 8,000

was recentrifuged

maintained

stated.

a 1.0 ml aliquot

at 520 nm

four lay-

3.0 ml of 0.2% starch

in a water-bath

eter

incubation

through

centrifuged

extract

was used

of the supernatant

minutes.

was

out in a mixture

the absorbance

curve.

strained

The of

in 50 mM Tris

and the resulting

1 ml of arsenomolybdate

the addition

at the end

started.

homogenized

1 ml of the Nelson-Somogyi

The mixture

was

The supernatant

A reaction

the substrate

incubated

30 minutes,

was

1.0 ml of the enzyme

ponents

bation

were

of

extract,

was

3.0 ml of 0.1 M Tris

was

the same as de-

harvested

for the preparation

filtrate

at 4' C.

were

were

F- treatment

pH 7.0, and the slurry

ers of cheesecloth,

This work

Germination

locally.

and used

Tissues

accumu-

in starch

this question.

obtained

after

with

sugar

utilization.

Seedlings

separated

treated

the observed

of the seedlings

[l].

or 72 hours were

tissues

is due to increase

to answer

were

and F- treatment

scribed

whether

tissue

in glucose

initiated

seeds

than control

It is not known

[2].

lation

of sugars

[6].

produced

extract

Specific

was determined enzyme

per mg protein

activity

per 30

97

Experimental differences ered

data were

between

significant

analyzed

two independent

by Student means.

r-test

Data were

for the consid-

at the 95% level of confidence.

RESULTS

The amylase ly affected

activity

by E treatment,

concentration-dependent. 0.1 mM NaF

decreases

lings

with

treated

At 72 hours,

and the effect

showed

were

appeared

in tissues

an 11% increase

of 13% and 31% were

inhibition

to 0.1 mM NaF.

TABLE

I

Inhibition

seedlings

marked-

to be

exposed

over

was

of amylase

in mung

concentrations

of NaF

shown

bean

even

cotyledons

to

the con-

observed

in seed-

1.0 mM and 5.0 mM F, respectively

posed

varying

bean

The activity

for 48 hours

trol, whereas

I).

in mung

(Table

in tissues

treated

ex-

with

for 48 and 72 hours

Specific (pg glucose/mg

activity

protein/30

minutes)

NaF

(mfa

Percent

Percent 48 hours

of

72 hours

control

0

of control

71 f 16.3a

100

150 + 22.4

100

0.1

79 f 10.1

111

132 ?: 18.8

88

1.0

61 f 14.3

87

131 ?

9.9*

87

5.0

45 f 12.3*

63

120 +

6.4"

80

aValues

are means

*p co.05.

+ SD.

98

It was activity

speculated

of fluoride

with

zyme activity thus

except

where

the effect

tion,

were

contained

used

mixture

as substrate

seedlings

for enzyme

treated

on enNaF was

with

to run

preparation. as described

previin those

To maintain

the

starch In addi-

accordingly. for 90 minutes,

a 1.0 ml aliquot

for sugar

5.0

The as-

the same as previously,

was allowed

mixture

with

of 5 mM or 10 mM CaC12

was reduced

of 30 minutes,

in amylase

CaC12

treated

of Ca2+ was to be tested.

the reaction

the reaction

of added

the same components

for the addition

of the assay

an interval

used

decrease

be due to interaction

from tissues

For this purpose,

ously

solution

might

The effect

calcium.

mM F- for 24 hours say mixture

the observed

seedlings

in extracts

studied.

volume

that

in F-treated

and at

was removed

determination

from

as described

previously. As shown alleviated

in Fig.

1, the presence

the inhibitory

5.0 mM CaC12

enhanced

an incubation

period

Interestingly,

effect

enzyme

of added

caused

activity

CaC12

Addition

by NaF.

of

by 73, 30, and 146%

of 30, 60, and 90 minutes,

addition

markedly

of 10 mM CaC12

for

respectively.

resulted

in much

small-

er increases.

DISCUSSION

Several during

glucosidase and starch breakdown

enzymes

These

(maltase),

the

final

the sugar

lings.

required

Inhibition an important

has on energy impaired

While

germination

from reactions

formed

limit

is used

of amylase

of starch

influence

for synthesis

observed

of

of cellular

in this

seeds.

in seedlings

source

of the seed-

this micromineral

in germinating

by other

in the germinat-

and development

that

and limit

as a primary

by F- observed

a-

dextrinase,

catalyzed

previously,

material

for growth

metabolism

enzymes,

the end products

As mentioned thus

of starch

cr-amylase, B-amylase,

are glucose-l-phosphate

product

and as a starting

components

in the breakdown

include

debranching

phosphorylase.

is glucose.

ing seed,

gests

are involved

by phosphorylase

dextrin,

energy

enzyme

germination.

study

sug-

element

The markedly

exposed

to even

low

99

concentrations bition

of NaF

[2] may be partly

that

ated

the F-- induced

Ca2+

ions may be required

earlier

reports et al.

biosynthesis ble that, Ca2+

of CaC12

of amylase

greatly

allevi-

(Fig. 1) implies

for its activity.

This

that cr-amylase is Ca-dependent

Recently,

the possible

role of calcium

of a-amylase

in rice.

absorbed

causing

into the tissue,

enzyme

of 5 mM CaC12

resulted

The reason

that high Cl-

is in progress

in much

for this

higher

with

in Fig. enzyme

is not known.

ion concentrations

to test

in the

It is possi-

F- may react

As shown

inhibition.

that

supports

171.

[El reported

10 mM CaC12.

possible Work

addition

inhibition

and secretion

once

ions,

presence than

to F- inhi-

of amylase.

The observation

Mitsui

attributed

1, the

activity It is

may be inhibitory.

this possibility.

70 r .i 60 E t

0

no added

CaC12

5 mM CaCl2 10 mM CaC12

/

4

01

0

I

I

I

30

60

90

Reaction

time, min.

Fig. 1. Effect of CaC12 on amylase activity in mung bean seedEnzyme extracts were prepared from seedlings exposed to NaF. Assay mixture conlings exposed to 5.0 mM NaF for 24 hours. tained 3 ml of Tris-buffer (pH 7.0), 2.3 ml of 0.2% starch solution, 1 ml of the enzyme, extract, and 0.7 mloof water or The reaction mixture was incubated at 30 C for 30, 60, CaC12. or 90 minutes.

As mentioned more

sugars

seedlings

previously,

than control

tissues.

ited by F-, it may be concluded lation

is not due to enhanced

may be due to impaired

exposed

Because

that starch

utilization

to F- contained

amylase

the observed breakdown.

of glucose

was

inhib-

sugar

accumu-

Rather, produced

it

during

germination.

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Chap-

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