The action of irehdiamine A on Escherichiacoli : Inhibition of the membrane response at low temperatures

Vol. 40, No. 2, 1970

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE ACTION OF IBEHMAMINE A ON ESCSEBICHIACOLI: INBIBITION OF TBE MEMBMNERESPONSE AT LCM TEMPERATURES Louis W. Wendt Department of Biology Washington University St. Louis, Missouri 63130

Received June 22, 1970 Lowering the incubation temperature reduces the rate of loss of potassium from cells of Escherichia coli produced by a given concentration of the steroidal diamine, Irehdiarm'i-nA. This effect can be explained as partly a reduction in the binding of the steroid, and partly a reduction in cooperative effects in the cell membrane which are involved in potassium loss. The steroidal

diamine,

which act on cell

Irehdiskne

E. coli

The initial

stage process (2).

death begins,

has been characterized of the steroid

(at 30”-35°C)

rate of loss of potassium,

time cell

cells

interaction

occurs in a 3-4 minute period

this

during which there

but no loss of cell

viability.

the rate of potassium loss falls

IDA concentration,

between IDA molecules shows cooperativity

in producing

report

tures

on the initial

tures

reduce the initial

and the extent

indicating

later

interaction

between IDA and the cells.

between steroid

varies

but this

of the ap-

tempera-

Lowered tempera-

the amount of steroid

molecules.

in-

loss of potassium

.of lowered incubation

rate of potassium loss,

of cooperation

During the

Binding

range,

than the initial

concerns the effect

and the

a cooperative

potassium loss.

over the same concentration

pears to depend on events occurring The present

change.

off,

period of rapid potassium loss the rate of potassium exit

steroid

for the steroid

After

initial

teraction

of the cells

as a two

is an acce-

characteristics

with increasing

Its

with the cells

binding

sigmoidally

transport

(1,2) and other energy dependent processes.

on membranes of intact

lerated

is one of a number of smines

membranes to produce leakage of ions and inhibit

of ions and small molecules action

A (IDA),

bound,

(2)

Vol. 40, No. 2, 1970

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MATERIALSANDMEZCHODS -The bacterial

strain

used was WI895

42 K using procedures

measured with the radioisotope in detail (1,2)

elsewhere

Briefly,

(1,3).

a few minutes termined

after

cells were grown in dilute

samples taken before

of IDA through

filters

millipore

in a gas-flow

filters,

counter.

IDA by centrifigation,

and by millipore

in the

potassium was ae-

and at intervals

after

the addi-

and measuring the 42K retained

Binding

broth

A was added within

Intracellular

shift.

tryptone

and resuspended

Irehdiamine

temperature.

the temperature

by filtering

Potassium loss was

which have been described

in the presence of 42 K at 37°C and centrifuged

same medium at the appropriate

tion

(HfrC, met-).

on the

of IDA was measured using 3H-dihydro filtration

(2).

RESULTS Lowering the temperature sively tion

the initial (Fig.

1).

of incubation

rate of potassium The Qlo for this

I

I

loss produced by a given IDA concentra-

effect

I

I

is approximately

u

I

I

I

I

2 (Table 1) suggest-

I

A 37"C,E~lll-~MIDA 0 23X,8 xW5 MIDA V WC,8 x~O-~MIDA 0 1°C.8 x~O-~.MIDA

t

0

from 37°C to 1°C reduces progres-

2

4

6

8

10

minutes

Figure 1.

Temperature

dependence of IDA-induced 490

potassium

loss.

Vol. 40, No. 2, 1970

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table 1 Comparative Temperature Dependence of m-Induced Potassium Loss -m-- sxd IDA B&!&g

Temperature

Rate of Loss(")

mM bound/1012 cells(b) Estimated by: Centrimation Filtration

("a

t-k)

37

-99

1.3 x 10'2

6.3

23

.38

7.9 x 10-3

3.1 x 10-3

10

.23

6.5

10-3

< 2 x 10-3

1

.l2

5 x 10-3

< 2 x 10-3

x

x

10-3

(a) measured at 8 x lo-5 M (b) measured at 5 x lo-5 M Initial

rate of potassium loss calculated from the data of Fig. 1: 2 Binding was measured 15 min after adding 5 x lo-5 M (42K(t) /4 K(o) = eekt).

5 x 10m6 M %I-dihydro-IDA

IDA containing Fig.

1.

At 37°C the amount of IDA bound atier

compared to that bound initially

ing that

in solution

with the cells.

same temperature potassium loss,

Although

described The relative

centrations

energy

in their

in-

of IDA is reduced over this

is not reduced as greatly

as the rate of

is not as effective

This conclusion

in inducing

is supported

by the re-

below. initial

rates

of the curves appears sigmdd

characteristics

by a decrease in the kinetic

that bound steroid

of IDA at 37", 23',

cates cooperation

in

15 min may be reduced slightly

the binding

potassium loss at lower temperature. sults

as described

(4) and a consequent reduction

range (Table 1) it indicating

premred

(2).

it might be accounted for solely

of the IDA molecules teraction

to cells

of potassium loss produced by various 10" and 1°C are shown in Fig. as has been reported

between IDA molecules

elsewhere

in producing

of these curves which are altered

con-

2.

The shape

(2),

and indi-

potassium loss.

by decreasing

Several

temperatures

Vol. 40, No. 2, 1970

BIOCHEMICAL

1.101.00-

0 0 v v

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

37°C 23°C 10°C 4°C

.90.80-

xlV

.20.10-

/-

0: 10-4

0

2x10-4

molarity IDA

Figure

2.

Concentration dependence of IDA-induced potassium loss. Kinetics of loss was measured as described in the legend to Figure 1. Initial rate of loss of potassium was calculated as for Table 1.

should be noted: celerated

potassium

3) the increase

The raising rate

1) The lowest concentration loss increases,2)

in rate per increment

of the 'threshold'

are consistant

that

produces significantly

ac-

the maximum rate of loss decreases and, in steroid

IDA concentration,

with a reduced binding,

492

concentration

is reduced.

and the decrease in maximum

ma also

may reflect

a reduced

Vol. 40, No. 2,197O

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

V

-1.6 -5.0

-4.8

-4.6

-4.4

-4.2

-4.0

-3.8

-3.6

log UDAI

Hill

Figure 3. effectiveness teristic

of bound steroid

is indicative

plot of some of the data in Fig. in producing

of a reduction

and is shown more clearly

in Fig.

3.

slopes of the curves obtained

at 37”,

potassium loss.*

in cooperation

are presented

!l?his indicates

a decrease in the cooperative

2

plot.

23',

The

than one; how-

less than that

interaction

charac-

molecules

for 37”,

in a Hill

23" and 10°C are greater

ever the slopes at 23' and 10°C become progressively

The third

between steroid

Here the data of Fig.

and lO"C, and from 2 x 10m5 to 10m4 molar,

2.

at 37°C.

between steroid

mole-

mis interpretation is complicated by the fact that we have experienced difficulty reproducing a given effect at a given concentration. hn extreme example of this problem is illustrated by the rates seen at 8 x 10-5 M in the Possible explanations for this have been experiments shown in Figs. 1 and 2. result of discussed elsewhere (2). However, it is clear that the qualitative a progressive reduction in the rate of loss as the temperature is lowered is reproducible. Interpretations based on comparative data obtained within an experiment such as shown in Fig. 2 are not subject to this complication.

Vol. 40, No. 2, 1970

cules,

the

ture

BIOCHEMKAL

degree

of

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

being progressively

cooperativity

reduced as the tempera-

is luwered.

DISCUSSION We have observed that tration

the rate of potassium loss at a giv6n IDA concen-

is reduced as the temperature

1°C.

This effect

lowering

at a given concentration

of the kinetic

of them interact

binding

and that

events following

affected

at low temperatures.

the temperature

is

lowered.

as intact

(2),

the physical lawdlii

that

this

reduction

nature

molecules

comes

observed as

reflects

as well

a membrane

is the consequence of an alteracould result

from a change in

such as has been reported

of the action

ing potassium loss can be explained proposed by Changeux -et al.

between lip-protein son at present

operativity.

subunits

to favor this

in only the lipid

for mcoplasm

which envisions the membrane.

cooperative However, there

from these data is that at the higher

of attack

the likelihoodS,or

effectiveness,of

494

inhibit

this

is no rea-

the observed cointeraction

must involve

molecules further

interactions

example, a change

the initial

temperatures

by the steroid

lowered temperatures

in induc-

on the basis of a model such as

over some other mechanism (for

with the cells

and that

molecules

phase of the membrane) which might underly

cess by which the effect is to increase

of the steroid

formally

(6),

within

What seems clear

of the steroid

molecules,

interpretation

(5).

The cooperative

that

for this

Such an alteration

of membrane lipids

the

loss may also be

Since IDA can act on membrane vesicles

membrane.

state

However,

suggest that

to potassium

between steroid

fewer

in the rate of loss,

and since potassium loss probably

is likely

in the cell

leading

Strong support

in cooperativity

tion

to the reduction

IDA binding

such that

potassium loss.

at the lower temperatures

from the reduction

alteration,it

in solution

so as to initiate

is not reduced in proportion

cells

might be accounted for by the

energy of the molecules

with the cells

measurements of steroid binding

is lowered over the range from 37°C to

a pro-

on the membrane attack

process.

by additional

Vol.40,

No. 2, 1970

BIOCHEMICAL

A delay in the initiation has been observed over this

in an intermediate

RESEARCH COMMUNICATIONS

of potassium loss following same temperature

the nature of the process underlying play a role

AND BIOPHYSICAL

K treatmnt

suggesting

that,

whatever

the observed cooperativity,

it

also may

step in colicin

range,

colicin

action

(3).

ACKNOWLEDGEMENTS Thanks are due to M. Baylor, repeated gifts of Irehdismine A.

H. Mshler and R. Goutarel

for generous and

This work was supported by NSF Grant GB 5922, and NM Grants AI 80862 and 59 07054. For part of the period of this work L. Wendt was a postdoctoral trainee of the Molecular Biology Program at Washington University, supported by NIH Grant 2Tl GM 714-11.

REJ?ERENcEs

1. 2. 3.

4. 5. 6.

1968. J. bcteriol. 96: 338-345. Silver, S. and E. Levine. Silver, S., L. We&t, P. Bhattacharyya and R. BeKchamp. 1970. Annals N.Y. Acaa. Sci., in press. Wendt, L. 1970. J. Bacterial., in press. Cell pnysiology, Chapter 10, W. B. Saunders Co. Giese, A. 1962. Steim, J. M., M. Exurtellotte, J. C. Reinert, R. N. McElhaney and Proc. Nat'l. Acaa. Sci. U.S. 63: 105-109. R. L. Rader. 1969. Changeux, J. P., J. Thiery, Y. Tung and C. Kittel. 1965-: Proc. Nat'l. Acaa. Sci. U.S. 57: 335-340.