Red cell membrane ATPase: Radiation inactivation estimates of “size”

Vol. 23, No. 2, 1966

BIOCHEMICAL

RED CELL MEMBRANE ATPase:

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RADIATION

INACTIVATION

GORDON R. KEPNER** and ROBERT I, Department

of Physiology,

Received

March

been reported

system the

quantitative

enzymatic

"target

"equivalent

molecular

and ouabain

insensitive

Materials

(collected

paper

ghosts.

an inactivation weight" ATPase

system

1 x lo6

enzyme

suggests

that

mechanism

ATPase

on

of

conventional

preparation,

the

of -1.5

x 10 -1acm3

for

the ouabain

both

that

experiments

sensitive

to this

volume

of-

of this

describes

Assuming

can be applied

The

the transport

of the Na, K, ouabain

with

an

sensitive

systems.

membranes

were

prepared

in ACD) by a modification

1mM NaH2P04,

et.

al.

1960.

Cells

solutions

and finally were

20 min.

adjusted at 20,000

from

were

hemolysed

washed

washed twice

to pH 7.5 with

Fellow:

Group

in Biophysics

202

et,

al.

in (6m~ Na2HP04, in

(1QmM Tris,

HCl or NaOH.

x g in a refrigerated

human blood

of Dodge,

and stored

by NSF GB 1928

NIH Predoctoral

fresh,

of the methods

1mM EDTA, 1mM glycylglycine),

1mM glycylglycine),

**

This

ior

has

preparations.

transport

as a marker

1964).

1946)

ghost

1963 and Post,

*Supported

of the Na-K active

activity

and Methods

Fragmented

for

the kinetics

(Skou,

(Lea,

suggest

were

between

human erythrocyte

experiments

All

correlation

inactivation

theory"

ATPase

membrane

can serve

fragments

sensitive

of cellular

activity

the radiation

ouabain

variety

and the kinetics

freeze-dried

California

stimulated,

in a wide

in membrane

of California

23, 1966

Sodium-Potassium

striking

OF "SIZE"*

MACEY

University

Berkeley,

ESTIMATES

centrifuge

in

1mN EDTA,

(1omM Tris).

Centrifuge (2-4'C).

runs

Vol. 23, No. 2, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

--Results FIG. ATPase

1

INACTIVATION

TOTAL

I .2

.Ol 0

I .4

I .6

DOSE

Each data fitted

point

to a single

Weighting

had no significant in the plot, calculated

represents

effect

on the calculated

lo5

I

1.4

using

a least

squares

according slopes.

megarads

Data were

for

procedure.

to their

Although the total

variances

not

included

ATPase

was on the

line.

frozen

Once dried, dried

I 1.2

or inversely

at three

Two ml of suspension quickly

I 1.0

by computer

equally

point

I .s

- Megarads

points

the data

ACTIVITY

fram 4 to 18 experiments.

exponential

the data

ATPare

ghosts

was pipetted

in a dry-ice

preparations were

;Xoentgen/minute

bath, remained

irradiated (in

with

air , &6OC)

into

and dried stable

a 25ml Erlenmeyer in a Virtis

and active

7.5 Mev electrons with

203

an electron

flask

vacuum

for

weeks,

system. Freeze-

at a dose rate linear

of

accelerator.

Vol.

23, No.

2, 1966

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

TABLE 1 *

Calculations

from

the data

of figure

1

ATPase Activity

v 0 x lo5

TOTAL

.12 + .01*

1.50

1

OUABAIN INSENS.

.13 + .Ol

1.65

1.1

OUABAIN SENS.

.12 + .Ol

1.50

1

*

Equations

1)

Jr*

V x 1018cm3

"Equiv.

M. Wt."

x 10

-6

used:

activity

= e-'

e D

V = inactivation

volume

0 = conversion

factor

cm3 = p gm/cm3x 1.6

lo-l2

x

lOOergs/gm-rad ergs/ev

x 85 ev Xctivating

event

D = Dose rads 2, **

P= density

***

glass Ghosts

'Three

chips

preparation:

were

(Bausch assayed

ml of incubation

was added shaker.

for

assumed = 1,d

ATPase

medium

to each flask

insensitive

ATPase

(5mM Tris,

stopped

NaCl,

at 37'C for

after

one hour

204

of ouabain

irradiation.

20mM KCl,

20 minutes

pH 7.4) in a Dubnoff EDTA, MgC12)

by addition

of Dreisbach,

by duplicating

The addition

after

(&nM each of Na2-ATP,

the method

was determined

the dosimetry.

a few hours 115mM

suspension

following

of 10w5M ouabain.

used for

activity

and incubated

and the reaction

P1 was measured

presence

weight"

and Lomb) were

One ml of substrate

was added TCA.

of dried

molecular

= .Ol

+ 2u

Cobalt

= "equivalent

7 v Navog.

of 2ml of 18%

1965.

the above assay at this

Cuabain in

the

concentration

Vol. 23, No. 2, 1966

gave ATPase the

BIOCHEMICAL

values

incubation

between

similar

and reaction

the

ATPase.

very

total

control

ghosts

insensitive

fia-K

Ghost

was less

(calibrated

with

for

ATPase

ATPase

suspension

BSA) gave

when K was omitted we identify

insensitive

activated

.l% that

than

obtained

Accordingly,

media.

values

for

ATPase.

themeoglobin)

to those

and the ouabain

Typical

freeze-dried

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the difference

as the Na-K stimulated

activities

were:

25pg Pi/hr./ml

ATPase

and 8&g P,/hr./ml

for

hemoglobin

(determined

by cyanme-

of whole

"protein"

from

values

blood

ouabain

and the Biuret

&mg/ml

reagent

ghosts.

--Discussion Kadiation molecular

inactivation

weight

of dried

of macromolecules Haanen,

et.

a)

Inactivation

of indirect

of

to those

The energy

secondary

released

inactivating

1961) al.

and

1966;

to biological

(Augenstein,

1963;

Brustad,

in the material

to the separation inactivations

between

and the

initial

is minimtzed

if

ionizing the

specimen

irradiation. biological within

in the initfal

if

et. theory

distributed

occurring

is highly

function

per

during

ionizations

ionizations

the biological

of target

the highly-specific

released

(Alper,

the

include:

compared

state

and Pollard,

of assumptions

are randomly

small

in a dehydrated

due only c)

these

The effect

(Hutchinson preparations

on a number

is

been used to estimate

The application

events

volume

events.

b)

rests

Inactivating

target

is

1965).

In particular

1962),.

enzymes

in biological

al.

preparations

has frequently

occurs

event

is

the

ionizing

localized

it

function target

event

assumed

target

is

volume.

and the ensuing

and sufficient

in the

assayed

to inactivate

volume.

to be ~~70-100

The energy ev (Hutchinson,

1965) ,, The presence excitations the energy

leading release

of impurities,

the possibility

to thermal

inactivations,

in the target

volume

205

of radiation-induced and the errors

result

in

a significant

in estimating degree

Vol.

23,

No.

2, 1966

BIOCHEMICAL

of uncertainty

as to the results

has suggested factor

the method

that

of 2-3 of the

countered

(Hutchinson

the basis

for

"true

imply

that

data

could

of figure

to a single

also

from ATPase

systems

steps

would

correspond

target

arranged

inactivate

two interpretations

II)

en-

to improve

does not

molecular

species.

The

Inactivation

coupled of any one

Our calculated

of the entire

The appearance

necessarily

of a tightly

sequence.

of the molecular

single

This

consisting

volume

the

sequence

of parallel

slopes

configuration

of the

volumes.

The two ATPase

inactivation

a

and to investigate

with

in series.

to the inactivation

inactivation

sometimes

preparation

theory.

the entire

may have one or many steps.

ATPase

work

to within

in progress

1 correspond

corresponds

steps

suggests

on this

each ATPase result

are

Work is

from one-hit

of these

I)

1961).

weight

errors

predicted

of biochemical

which

larger

Other

in membranes.

curves

sequence

volumes

but

COMMUNICATIONS

technique.

the molecular

the results

mechanisms

RESEARCH

hy this

estimates

and Pollard,

The inactivation

BIOPHYSICAL

obtained

value";

interpreting

radio-biological

exponential

AND

sites

event

in

The two ATPases

occur

in a single

the volume have

inactivation

inactivates

separate

but

both

equal

volume.

An

enzyme functions.

inactivation

volumes.

Acknowledgements The expert acknowledged. in providing critical

technical

contribution

The authors

appreciate

accelerator comments

time

of Mr.

Doug Pounds

the kindness

and thank

Dr.

Ernest

is

gratefully

of Dr.

C.A.

Tobias

Dobson

for

valuable

on the manuscript.

References Alper,

T., Haig, D. and M. Clarke, Biochem. Biophys. Res. Commun., 2, (1966). Augenstein, L., Progr. Biophys. Biophys. Chem., l3, 3, (1963). Brustad, T., Advan. Med. Phys., g, 161, (1962). C. and D. Hanahan, Arch. Biochem, Biophys., 100, Dodge, J., Mitchell, (1963).

206

278,

119,

Vol. 23, Neo. 2, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Dreisbach, R., Anal. Biochem., l.0, 169, (1965). Haanen, C., Morselt, G., Schoenmakers, J., Maters, M. and R. Braams, Stand. J. Haematol., 2, 248, (1965). F. in 18th Symp. Fund. Can. Res. Anderson Hosp. & Tumor Inst., Hutchinson, Cellular Radiation Biology, Williams & Wilkins Co., Baltimore, (1965). Hutchinson, F. and E. Pollard, Mechanisms in Radiobiology, 1, 71, Academic Press, New York and London, (1961). Lea, D., Actions of Radiations on Living Cells, Macmillan, London, (1946). Post, R., Merrit, C., Kinsolving, C. and C. Albright, J. Biol. Chem., 235, 1796, (1960). Skou, J., Progr. Biophys. Biophys. Chem., l.4, 131, (1964).

207