Chemistry of lens nuclear sclerosis

Vol.

35, No.

6, 1969

BIOCHEMICAL

CHfMISTRY S.

OF LENS

Zigman,

Departments University

AND

J.

BIOPHYSICAL

COMMUNICATIONS

SCLEROSIS*

NUCLEAR

Yulo

Schultz

end

T.

of Biochemistry

and

Surgery-UPhthalmology

of Rochester

School

Rochester,

Received

RESEARCH

of Medicine Nem

and Dentistry

York

May 19, 1969 forms of insoluble protsin their solubility in 78 uree. of ths lens, contains nearly

Two baaed upon the cortex and appsars

isolated from the rat lsns soluble form, found mslnly in levels of elpha and gamma crystsllina,

have been The urea

squel

to be associated by hydrogsn and hydrophobic bonds. The urea insoluble form, found mainly in ths nucleus of the lens contains mostly gama crystallin, and is held together by covalent linkages+ such as SS bonds. The data strongly indicates that nucleer sclsrosis during aging is dus to the conversion of the urea-soluble form to the Ursa-ineolubls form in the nucleus dependsnt upon these fectorst dehydration, SH unmasking, pressure , and gemme crystallin content. As

This

reduced. level

of

the

s consequsnce chenga

of in

insoluble

the

aging, lens

proteins

A @ 0 .

lenticular is

than

elasticity

accompanied of

by

soluble

the

transparency

and

a more rapid proteins

are

increase

(Figure

in

1).

RATIO OF SOLUBLE : INSOLUBLE TOTAL PROTEIN SOLUBLE PROTEIN INSOLUBLE PROTEIN

0

IO

20

30

40

50

WEEKS OF AGE Figure

1.

Changss

estimated

*

Thi5 lnvestigstion U.S.P.H.S. Grant

wa5 number

in

ret lens protein by the Lowry method

supported

by

the

NB-08360-01

931

levels (15).

Rochester

with

Eye

age.

Bsnk

Pratcins

Society

oers

and

the

Vul.

35,

No.

Fulhorst

6,1969

and

wrkara

have

related

to

reported

the

Recant

cow lens

, end not that

insoluble

insoluble The

$naoluble

studias

In we

quantitated

soluble

in rat

diecrapancias

tha

uaad

cow

l nalyaia.

far

of

protains

are

closaly

proteins,

between

tha compeaitian

others

have

gamme crystallin

insoluble

the

with

t3ota crystallin

most closaly

reeamblas

that

shawn

Prom Aany

protein

while

(2,3,4),

lens

darkirsd

materisl.

the soluble

lens.

In

the

total

the

ralabd

An

alpha

fraction

af

prsdominsting

also

in

seemed to be prasent

rtud$es,

work

bath

left

ths whole which

aster

ass

en ths

inaolubla

not

insoluble

fraction

a camplately

cwmblned

mare

(TIP)

the water

of which

producad

procadures

irtsoiubla protein

water

was

lens

in praparative

portion

rwsidua

of SS and SH bonds

@resent

of cow and rst

to diffarances

solubilitsd

inaalubla

JR the rat

by sulfonation

product.

portions

have

tha gin uraa

studias,

or characterized.

IMS diasolwd

(8)

soluble

wara at laast pmtly

proteins

trutfan

rat

COMMUNKATIONS

synthesized is

the

RESEARCH

proteins. apparent

pracadllres.

of

of thsse

newly

corn lans

tha

Practien

both

lens

crytitallin

of

immunochemicel

contains

from

alpha

fraction

BlO&t%CAt

insoluble

that

and gammaprsdominating

both

AND

shown

rapertad

insoluble

that

spscias

hsve

proteins

alao tha

(5,6,7).

in

(1)

Young

soluble

preexisting

both

BIOCHEMKX

basis

water

to obtain

tmo

in

UP solubflity

7 m uraa. homegeniratian

After

mmaininq

soluble

at lOoC, th

protein

TW wae

uraa

insoluble

(SOj,

S40&

7N urea,

insoluble umakly

cevalent in

the

asseciatsd

waa

pli 9.9)

aolubilirad

which

protein

bands,

such ameunts

as

SS bridges

d

these

reprasants

probably

and

held

Prwction by

at 2000 rpm

se&&la (US)

Thw mafdua , or 7A

with Bailey’s

932

by hydregen

is firmly

others

two portiolw

a nancevalsntly

together

not of

held

togathar

as yat characterized.

the TIP

of

ret

roagsnt

Addition

in rsformation

rsaultad

af all

rammml

011 SS and SH groups.

therefore

UI

and

and rwcentrifugation

by treatment

UI fraction

whereas the

in water

xg Per 3 hr).

sulfanatad

sggragata,

lanaas

3 hr and the 78 urea

far

(200,OUO

The US fraction

attrwctions,

relative

in 7Auraa

to the swlubllized

product.

hydrephobic

(UI)

rat

raauapenwian

by centr$fugatlan

partian

msrcapbethanol

by rapawted

mapar&+

was ramavad

partian

af Holttmemt

lames

of

of an

linked, bonds by

end etrang

Changes

are shown

in

Vol.

35, No.

BIOCHEMICAL

6, 1969

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

0 OUREA-SOLUBLE 0 UREA- INSOLUBLE

lb

2b

io

4b

;o

WEEKSOF AGE Figure

Changes

2.

in

Proteins

Figure tha

2. The

predominance

UI in

of

slowdown

rapid

mast of

the older of

gamma

level

tc belcw

cloud

at temperatures

emphaaieas

the

rat

were

change

US in

takes

lenses

of

the

Coincidental

crystallin

synthesis tote1

below

marked

decrease

between

in the

level

fairly

Loss

of

age.

age,

when

a predominance

by changeover

is

the

gamma crystallin

of

the ability at this

of soluble

20

abrupt

of

precisely

with

(15).

rsplaced

in a drop

0 SOLUBLE . INSOLUBLE ---CORTEX

0

is

results

UI)

and

10 weeks

5 and

this

protein.

occurs

(US method

animals

with which

also

proteins Lowry

the

younger

soluble

10%

by

place

animals.

50% of the

ineolublc

lens

estimated

age

ef the (S),

lens

a fact

to which

gamma crystallin.

2

40

60

WEEKS OF AGE Figurm

Changes in cortical were extimated by

5.

During the

ratio

of

total

aging

of

lens

cortex

tha

and tha

lens

protein

nuclear protein Lowry method (15).

a harY

central

to total

933

nucleus

nucleus

levels

forms.

protein

age.

with

As changes

Proteine

seen from

in

Figure 2.3

J

Vol.

in

35, No.

old

6 week

during

this

amount

of

of

TIP

the

from

6, 1969

BIOCHEMICAL

rats

to 0.8

in

the

amount

of

in

the

interval insoluble

in

protein

nucleus

the

10% to 65$ (Figure

52 week

to only 4).

AND

It

old

BIOPHYSICAL

rats.

soluble

The

figure

protein

nucleus.

in

During

25% of

TIP,

clear

seems

RESEARCH

the

cortex

time

this

the

illustrates

further

concomitant

that

COMMUNICATIONS

drops the

tuith in

below

the

US drops

from

incrsase

of

an

increase

that

size

and

85%

UI

hardness

OUREA-SOLUBLE l UREA-INSOLUBlE ---CORTEX NUCLEUS

40

20

60

WEEKS OF AGE figure

4.

Changes

Proteins

in

cortical and nuclear were estimated by the Table

Amino

Amino

Acid

Analyses*

Acid

Urea

Insoluble (U.I.1

of

US and Lowry

UI method

I Rat

Lens

Urea Soluble

Sulfa-proteins Gamma

Alpha

Crvstallin

Crvstsllin

of

Amino

Acid

per

1000

Residues 42 30

L sine HiMdine

Acid Acid

Proline Gl ycine Alanine Ualine

Kethionine Isoleucine Leucine Tyrosine

Phenylalanine *Hydrolysed Average

with

(15).

(U.S.~ Residues

Arginine Aspartic Threonine Serine Glutamic

distribution

of

119 107 27 80 137 49 89 25 43 30 56 72 81 44

102 102 31 108 130 56 89 38 50

at llO°C; 24 hrs.; two detsrminations.

6N HCL

111 106 30 76 127 48 87 29 50 30 36 70 83 57

15

36 75 50 49

934

l

zs

35 107 110 73 74 39 63 15 39 87 38 77

age.

of

the

BIOCHEMICAL

Vol. 35, No. 6, 1969

lens

nucleus

closely

urea-soluble

accompanies

fractions

by

100% gamma

amino

amount

higher

than

US

composition

analysis

of

US

is

of urea-insoluble

both

alpha

has and

preaent

proteins

(Table

approximately

contamination

alpha crystalline

absorption

of the

while

of beta those

identification acid

cryatallin

small

nuclear

accumulation

the

caused

I>

50%

gamma

gamaa crystallins.

anelyaia

(Figure

contains

60% gamma; cortical

5).

cortical

aimilar

US appaara

of

UI

contains

A

Saphadsx

G

100

ralationship

chranatography

crystallins

alpha

of

in

the

acid of

i’O$

and

nuclear

gamma; to as

ganana crystsllin

~11. similar

aubractions.

to be gamma

by ultraviolet alpha;

alpha

lnsolubls

A

only

indicated

havs

alpha.

values

ths

nearly

is

to contain

UI exists in 6 week old rat lens cortex and nuclaua

gamma and

50%

UI

glutamic

When

and

on

for

that

in 52 wssk old rat lenses ware determined

cryatallin.

UI

and

values

the

gamma

betwesn

expense

in the US and UI

indicates

00%

US and

at the

pratein. Preliminary

and

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

UL

is

in US

Ssparations

of

rslationships

The prsssnce

PERCENT ALPHA 100 204

80 L

60 t

40 I

20 I 8 ’

6 WEEK RAT NUCLEAR S-UREA INSOLUBLE

,

52 WEEK RAT NUCLEAR S-UREA INSOLUBLE

6

52 WEEK RAT CORTICAL S-UREA INSOLUBLE

’

6 WEEK RAT NUCLEAR S-UREA SOLUBLE

4

52 WEEK RAT NUCLEAR S-UREA SOLUBLE

3

6 WEEK RAT CORTICAL S-UREA lNSOLU8LE

2

6 WEEK RAT CORTICAL S-UREA SOLUBLE

I

52 WEEK RAT CORTICAL S-UREA SOLUBLE

0

PERCENT GAMMA Figure 5.

Ultraviolet analysis lens nucleus and cortex. calculating and

along

S-gamma

the

gamma crystallin lsvals in rat (closed circles) wsrs cbtsinsd by of purified S-alpha mvaluss et 280 ny~ of mixtures % lins. The US and UI sxtinction valuss are marksd

ths El cry&a curve with

of

alpha Points

x’s.

and

Vol. 35, No. 6, 1969

of

both

alpha

BlOCHEMlCAL

and

immunoslectrophoresis

(Figure

Sclsroais

whereby loose

tha gel

gsl.

The

and

seams

held

held

rolubla

in the

corticsl

loose to

be

msinly togethsr

Figure

6.

whsrs gel

together

nucleus,

of

lens

tha nuclaus

into

by

crystallin

gamma

made

of &p

the

region

of

lsns

of

the

loosely

the

cortex

the

first tha

is

and

hydrophobic

protsin

also

was

Immunselectrophoretic of

rat

crystallina ware cellulose-purified

lens.

into

mainly levsls

attractions. , consists

proteins, prepared by antigans

a two

shown

the

the

of

by

the

of

procass

associated

cortex into

is

compressed

a firm

tight

urea-soluble

alpha The

mainly of

of

stap

weakly

a

is convsrtsd

gsl

equal

be

aging,

With

compossd,

analysis lens

to

converted

formation

by SS bridges.

fractisns

UI fractions

appears

associated

approximately

Ursa-insoluble part19

US and

are

proteins

hydrogan

in the 6).

nucleus

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tight gamma

firmly-bound

and

fraction,

gamma

gel

of the

crystallin gsl

crystallins

and

structure

1% agarose of a) US and b) UT Antisera to alpha 8nd gamma subcutaneous injections 0f DEAEinte rabbits in Freund’s adjuvant. on

is

No.

BIOCHEMICAL

Vol.

35,

6,1969

seams

to

which

must combine

depend

or as other

upon

of

groups;

folding

gel

increasing

is

the

solubls

It

has

the

in the

nucleus

(i.a;

diacusaion

free

of

the

is

Ths

work

of the which

human lane,

and

alpha

cryetallin

which

gaana

groups

in the reactive

glutathiona

(9); due

to

capaula

All

a change

in

pressure

in

elasticity

nuclear

and

of

(13)

be

however,

nuclaua,

nuclear

and

insaluble to

the

of

even

protein

lost

from

a much

of the higher

in

in awn

(14)

human

lenses phase

alpha

crystallin

937

perfectly

also of

that contains the

lens

content

easy

is

gema

Recent

chicken mall the

the with has

with

that

lens

have

as a result

markedly

cryatrllin

cryatallin

gamete

old

indicates

soluble

the

present

Studies

repart

of

age.

the

applls~

group

this

has no gamma

old

enable

preducts

impertanca

which

is

(ll),

we&d

of

of

it

aggragstss.

in

phase

seama to posaaaa

structure

to insoluble

the

lmna,

its

shorn

fermatien.

(lo),

content

importance

sclerosis our

irradiation

soluble

of UI are present

amounts

it

to fcrm

ia also

Further

central

lenm proteins;

acid

itself

crystallin

gamma protein

cortex;

amino

amphaaizs

in the chicken

hard

scheme

in

aclerasis.

illustratad no

It

facts

tha

that

urea-insoluble

aggregataa

S’S (12,5).

of these

in

with

crybtallin

idea

praviaus

uitheut

and

crystsllin

gauwaa

tha

the esluble

apolar

of Piris

appears

all

of

with

predicate

high

contant)

trace

water

prapertiee

than

of

This

is consistent

levels

and

only

that

whose

of SH to

of

SH content,

above

higher

(10).

sclerosis

involved

as

nucleus

groups

passes,

time

surrounding

such

lens

that

as

be

water

lens

decreased

so

other may

molecules,

in the

from

at much

the level

process

resulting

even

in

depressed

of

more reactive

axposes

co&ant

the oxidation

aging

levels

radical

with

reported

which

SH content

low helical

association

exclusion

or exchanging

each

which

Factors the

at higher

nuclsua

with

first,

volume.

greeteat

S’

includes

gal

bound

interact

can

proximity.

into

moleculea

protein

a high

gel

SH masking

in the

lane

Tha

been

of

of protein

the protein

them

to be present

known

is

firm

the

US to UI could

exclusion

the

which

bring

the

of the weakly

astablfahment

to farm

forces

conversion

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

in nuclear

cryatallin, studies

law indicate

lens.

to the human urea

lens.

insoluble

pmbt&on

gamma crystallin aging. been

In the found

in

both

Vol. 35, No. 6, 1969

the

urea

soluble

the rat.

This

between

the

BIOCHEMICAL

and urea further

compoation

human

lens

fully

charactarizsd

These

other

the

tyrosine

oxidative

types

of

other

insoluble

of

bonding

bonding

residues

which

are

found

seem to cause

Fulhorst,

2.

Themann,

3.

Ruttsnberg,

4.

Rae,

5.

Zigman,

S.

6.

Lerman,

S.,

Zigman,

7.

Zigman,

S.,

Bfochim.

8.

Manslgi,

W.,

Presentad Nijmegen,

H.W. H.,

Lerman,

11.

Zigman,

12.

Dishe,

S., Z.,

The

Pirie,

14.

Zigman,

S.,

15.

Lowry,

O.H.,

A.,

W.F.,

W.F., Eye,

Unpublished

L.O.,

Zigman, Tutzing,

Such

298

lenses.

(1966).

18 (1965).

A,

Experimental Acta,

Exp.

Eye

Eye

Rss.,Q,

154,

423

(1968).

444

(196R).

Res.,

2,

36 (1965).

319 (1969).

&3l,

at Symposium on The Netherlands,

merola,

Biochem.

of

June,

1968

American S.,Kirman, August

J.

the

Ophth., J.,

Lens,

46,

Symposium

36

on Biochem,

1966.

E.,

end

Zalmanis,

C.,

A.M.A.

Arch.

Ophth.

47 (1956).

Annals

(19%).

data

Borenfraund,

Invest.

yet

of

human

I,

Biophys.

Acts,

groups

of

Ophth.

S.N.,

Forbas,

not

219 (1962).

Eiochim.

Biophys.

and

55, 13.

S.,

are

of SS bridges.

coloration

Eye Research

S.,

distribution.

crystallin.

gamma

165,

species,

which

phenolic

Invest.

Cooper,

Lerman,

in

to brown

R.W.,

P.D.,

Forbes, of

yellow

E xpsrimental

J.H.,

S.,

abundance

Ophthalmol.,

Mehta, and

as the formation

in

differences

protein

molecules

oxidized

than

different

of

soluble

protein

between

Young,

Arch. G.,

S.S.,

10.

and

lenses

the

protein

qualitative

than

and

between

in

the

1.

Kinashita,

of

fraction

are probably

insoluble

rather

protein

to be as important

of

9.

of the water

quantitative

the

type8

appear

the

insoluble

the

between

products

portions

emphasizes

and the relationship In the

insoluble

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Ophth.

z,

674 (1968).

of Ophthalmology

(in

press

, 1969).

Rosebraugh, N.J., Far-r, A.L., and Randall, Journal Biol. Chem., 195, 265 (1951).

938

R.J.,