Spin label studies on synaptosomal membranes of rat brain cortex during aging

Vol. 117, No. 3, 1983 December 28, 1983

SPIN

LABEL

BIOCHEMICAL

1

STUDIES

ON SYNAPTOSOMAL CORTEX DURING

Nagy,

International Hungarian

Pal

October

Laboratory Section, Debrecen,

29,

MEMBRANES AGING

Simon1

Central Research Institute Pusztaszeri of Sciences,

Received

RESEARCH COMMUNICATIONS Pages

I
AND BIOPHYSICAL

and for

OF

Imre

BRAIN

Zs.-Nagy

Experimental Medical

University

RAT

688-694

Gerontology, School, H-4012

Hungary for Chemistry, 59-67. H-1025

ut

Hungarian Budapest,

Academy Hungary

1983

Summary: Membrane lipids of brain synaptosomes of 2, 12 and 24 months old rats were labeled by stearic acid spin probes with a doxyl group in the C-5 and C-16 position,respectively. We demonstrated that the hydrophobic region of synaptosomal membranes b&came more rigid during the life span studied, and the region located nearer the surface of membranes displays a significantly decreased fluidity only in the oldest group. Membrane proteins labeled with a nitroxide derivative of maleimide suggest that conformational changes take place during aging.

Functionally logical

one

membranes

gions.

This

chemical

region

rat

anisotropy

the

the

a

catalyzed

brain

in cortex

naptosomal

studies membranes

our

results

of

synaptosomal

obtained

important of

has

demonstrated

on

been

the

These

aging. spin

by

series

of

lipids

of

bioage-

synaptosomes fluorescence a

state present

re-

An

suggested

The labels

a

of

results

bio-

lipid

enzymes.

physico-chemical

during with

in

membrane-bound microviscosity

of

hydrophobic

role

membrane

(1).

parameters

the

regulatory by

measurements

further

most

fluidity

plays

increase

from

for

is

events

dependent

of

of paper and

need

the

sy-

describes proteins

membranes.

MATERIALS

AND

METHODS

N-oxyl-4’,4 ‘-dimethyloxazolidine derivatives of stearic acid with the doxyl groups in C-5 and C-16 positions (5NS and 16NS,respectively) were purchased from Syva Co.U.S.A.The protein label, N-(l-oxyl-2,2,6,6,-tetramethyl-4-piperidinyl)-maleimide (MSL) was prepared by Prof.Dr.K.Hideg,PBcs, Hungary.

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688

Vol. 117, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Synaptosomes were isolated from the brain cortex CFY rats of 2, 12 and 24 months of age,designated adult and old groups,respectively.The brain young, 2 rats was pooled together in each experiment. The synaptosome preparation was that of Gray and ‘.dhittaker applied by vlhittaker and Barker (3). Protein content synaptosomal fractions was determined by a modified action (4). Ultrastructural studies were performed ing the purity of the synaptosomal fractions (1).

of

male

feas

cortex method

of of

(2) of the folin-refor check-

as

Incorporation of the labels was carried out as follows: the necessary amount of label was dissolved in ethanol, and the solvent was evaporated from the test tube in vacuum.Known amounts of synaptosomes were added in aqueous suspemsions to the tubes, stirred for 2 min rigorously,then kept at 4’C overnight. The necessary label concentration was selected during preliminary experiments carried out in 5 steps between 1 and 16 pg label per mg of synaptosomal protein.The best iabel to protein ratio was 8 i-g/mg. Similar concentrations of label were used also by others (5, 6) for the lipid probes. For the MSL label others have used somewhat lower concentrations (6), in order to remove the excessive label, we nevertheless, washed the synaptosomes with phosphate-buffered saline (PBS, Since the 3rd washing medium contained insignificant pH 7.4). the measurement was carried out amounts of the label (Fig.3), routinely in the 4th dilution of PBS. ESR

spectra whereas by means

+o .5Oc, 5.5’C

of those of a

30

~1

of JEOL

lipid probes the MSL-labeled spectrometer

were

recorded fractions type JES FE

at at 3X.

37.0 22.0+ -

The polarity-corrected order parameter S was calculated from the spectra according to Hubbell and McConnell (7) for the 5NS-labeled samples. For the 16~~ labeled ones,we used the motion parameter toI corresponding to the rotational coras calculated and explained by Eletr and relation time, Tc, Inesi (8).For evaluation of the results obtained with MSLlabel, we used the ratio of the spectral amplitudes of weakly (w) and strongly (s) immobilized label molecules bound to described by Butterfield et al. the SH-groups of proteins as (9). It should be noted that although this method of evaluation was criticized (10). claiming that the w fraction is the free label in solution displaying a line broadening due only the fact that the third washto the viscosity of the medium, ing medium did not contain significant amount of free label clearly indicates the validity of this Ikind of spectral evaluation for membrane preparations. The parameter w/s has been used by other authors in recent years (11-13) for evaluation of ESR spectra of maleimide-derivatives incorporated into biological membranes.

RESULTS ESR

spectra

in

Figs.l-3

The

results

shown

summarized periments

in were

of

spin for

AND labeled

carried

synaptosomal

various

age

calculated Table

DISCUSSION

1.

from It out

is on

are

spectra

are

groups. the

respective

remarkable

that

samples

prepared

689

membranes

although each

the time

exin-

Vol. 117, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Figure

1. Typical examples of the ESR spectra of 5NS probe in and old samples of synaptosomes. Instrumental pa8 adult rameters were: 9.127 GHz microwave frequency, 0.2 mT modulation amplitude at 100 kHz modulation frequency, response time 0.3 set, sweep 10 mT/8 min. microwave power 5 mW, amplitude 1x1000. The arrow in the upper right corner indicates the scale and direction of low to high magnetic field. Two-ended arrows at the bottom show the localization of the measured values in the spec t ra .

young

Young/

Adult’

Old’

Figure 2. ters were calculated

expanded more

precise

ESR spectra the same from the

magnetic reading,

of the as indicated central

field

sweep as

shown

16NS

probe. Fig.1 of spectra

at line

(+2.5 in

690

mT), the

right

Instrumental except that recorded

in

order side

W

to of

paramewas %t an

assure the

figure.

Vol. 117, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-

Figure 3. ters were corresponds

ESR spectra the same to the

dividually,

the

rather

narrow

noticed

a

ran

slight

values

as

sons,

a

we

reliable The

ficant

difference was

With

samples

the

with and

relative

all

three

old

technical

rea-

incubation

the

we we

adult

For

a

label

time

the

same

MSL

in

Therefore,

each

one.

fell

the

time.

young

paramecurve

labels

expressed

keep

lipid

came

more

rigid

that Only

between increasing

the

of

the

demonstrate

an

that

face

old and

always

dependence.

data

the

and

lipid

group.

therefore,

results age

in

incubation

Instrumental The lowest

time values

in are

statistically.

played

there

with

of

experiments,

the age

times,

not

label. for Fig.1. medium.

each

adult

percentage

could

different more

change

incubation

MSL

regarding for

young,

identical

of the indicated washing

results range

parallel

as 3rd

1mT

young

more

hydrophobic

the

membrane.

5NS

label

showed

and

adult

rats,

tendency. layer

during

the

of aging, region

parameters

One the

can

than

conclude

691

insigni-

from membranes

this nearer

an although

synaptosomal

however,

dis-

is to

expressed the

hydrophilic

these bemore

Vol. 117, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

Table Order rat

parameter (S), brain synaptosomal

Age

group

n

S

6 +o

(young-adult)

<

p

<

0 .Ol

7

0.5636 +o .0038

OLD

Note:

(young-old)

of is

the

bound

both

an a

of

(16, of

the

21

9

animals. and

physicochemical

s

9 2

conformational

0 .Ol

p indicates Student’s w/s ratio

SH-groups

in a

79.12 4.82

0.01

yielding

and

88.51 -+ 3.29 0 .Ol

1.303 0.017

the

decrease

w

w/s

ratio w,

of

the

a

the “t” was

and

an

sensitive

15).Since

the

of

changes

is

14,

.oo 3.70 0 .Ol

1.261 0.019

(9,

decrease

of

are 17).

loose

consistent

This

and

the s

MSL

fractions

of

resulting can

be

from

interpreted

proteins

water

water basis

a

the

in

and

the

result

this

is of

the

consequence, the for

an

the cells

in the

free an basis

nerve

radical increased for

cell

a

space.

increased

condensation

de-

membrane

accumulate

intracellular

692

hypothesis

that

may

permeability

to

membrane

predicts

components

components,

As

with

hypothesis

membrane of

ion

old

the

100 2

0 .Ol

protein

SH-groups

findings

cross-linking

sium

16

conformation

to

w/s (%I

membranes.

aging

creased

protein

increase

The

in

9

0 .Ol

membrane

label,

sign

damage

of

bound

synaptosomal

of

1.210 +0.025

in of

0.01

0.5563 - +0.0025

<

st,ate

reflection

as

n

n indicates the number of measurements; significance level as calculated using test. The absolute value of the young average of 17.

The

label

10

To (ns)

N .S.

7

(adult-old)

and w/s ratio and 24 months

n

0.5537 .0031

ADULT

p

1.

motion parameter (TV) membranes of 2, 12 age (mean 2 S.D.)

YOUNG

p

RESEARCH COMMUNICATIONS

potasThis

is of

the the

Vol. 117, No. 3, 1983

chromatin

and

a

experimental Both

the

red be

BIOCHEMICAL

reduced

rate

approaches fluidity of

RNA

of

resulting

lipid

and

in

the

induced

serious

Various (1,

layer

proteins

free-readical in

(18).

assumption

the

membrane

RESEARCH COMMUNICATIONS

synthesis

this of

the

manifestation

membrane,

of

support

decreased

conformation a

AND BIOPHYSICAL

of

20-22).

the old

damage

impairment

19,

alterats

to

may

the

membrane

func-

tions.

ACKNOWLEDGEMENTS: Authors are very much indebted to Prof .Dr. Robert A.Floyd (Oklahoma Medical Research Foundation,Biomembrane Research Laboratory, Oklahoma City,USA) for the discussion of this material and critical reading of the text. This work was supported in part by a grant of the Hungarian Academy of Sciences, No .360/82/3 .l.

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