Target size analysis of rhodopsin in retinal rod disk membranes

Vol.

122,

No.

July

18, 1984

1, 1984

BIOCHEMICAL

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

TARGET

SIZE ANALYSIS Simon

Department

Received

AND

OF RHODOPSIN

M. Hughes,

of Biochemistry,

1'!ay 23,

Glyn

IN RETINAL

Harper

and

ROD

Martin

DISK

56-61

MEMBRANES

D. Brand

University of Cambridge, Cambridge CB2 lQW, U.K.

Tennis

Court

Road,

1984

SUMMARY: Radiation inactivation of rhodopsin in situ using high-energy -__ electrons gave a value for M of 20,200 by spectral assay, but 47,100 by assay of rhodopsin regenerat& from opsin and ll-cis-retinal (sequence M =38,840). No light/dark differences were seen. We conclude: (a) radiation insctivation measures the size of the functional unit, and the single hit hypothesis does not hold in our experiments; (b) 500 nm absorbance requires only about half the rhodopsin molecule to be intact, but reconstitution of rhodopsin from opsin requires the whole molecule; (c) we find no evidence for functional interactions between rhodopsin monomers in darkness or light. Target to

size

the

estimate

underlying single

analysis size

assumption hit

by

a

completely

destroy

by internal

energy

This

hits. We

be

proposed

that

it

transport

across

depends does

not

interdependent

hold

in

rhodopsin

a

linear

segment

disk

membranes.

or

polymeric

multimeric

rhodopsin in

in

situ

understanding that

of assay. system. polymers

the This We

protein protein

and

to tight

target

will

electron

that

find

no

the

of

has been

which

catalyse

ion

(17-19).

The

it

not

of

issue

of

resolved,

and

of vision.

rhodopsin

single

evidence

darkness

size

been

mechanism size

shows

in either

a

has

therefore the

the

Khodopsin

(lo-16),

is

that

complex,

the

investigate

complexes

in response

The

(2-S).

outer

(6-9)

(1).

accelerator)

on the

challenged

be used

states

a multisubunit

of where

can

membranes

which

to

report

this

often

that

in

analysis

of

method

(from

been

membranes

we

situ

size

form

state

in

target

may

paper

and

techniques

hypothesis

electron

monomeric

disk

few

hit

regardless

of importance

on the

single

has recently

rod

to

this

the

very

complexes

energy

use

suggested

In

is

transfer

in intact

is potentially

protein

a polypeptide,

to

aggregation

of the

of

high

hypothesis

wished

rhodopsin

the

is one

hit for

or light.

in

situ

hypothesis functionally

Vol.

122,

No.

1, 1984

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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EXPERIMENTAL segments Rod outer were prepared from fresh bovine eyes by a suEose/Ficoll 400 flotation procedure as described previously (20,21). They were stored in darkness on ice. A was routinely 2.7-3.5 and yield 2-5 280/500 nmol rhodopsin/rctina. Tarqet size analysis. Irradiations were carried out as described by Huqhes & Brand (21). Samples were prc-incubated in 1.5 ml Sarstedt tubes for 5 min either in darkness or in daylight at 21°C. They were then rapidly frozen in liquid nitroqen and freeze-dried in darkness in medium supplemented with 10 mM KH2P04 and 5 mM dithiothreitol, then placed in liqhtand air-tight containers and evacuated to under 0.1 mm Hg. Irradiation with 16 MeV electrons from the Phillips MEL SL75-20 linear accelerator at New Addcnbrookc's Hospital, Cambridge was in an air-cooled apparatus in vacua in darkness. The dose administered to the sample was accurately calibrated (21). After irradiation samples were resuspended in Ii20 to the volume present before freeze-drying and assayed. Rhodopsin assay. ,,;502;+& wy;5 ;e=;;~fche~ b; ~;;;s=,~'mty 557 q~cctrophotometer. room was remeasured. The absorbance at 500 nm due to l?zop$," ~~~"~%?&ated according to: (A500-A650)d-(A where d is 500-A650)l' before and 1 is after full bleaching. Regeneration of Rhodopsin. After rhodopsin bleaching the all-trans-retinal formed detaches from opsin lcavinq the chromophore site empty. If ll-cis-retinal is added to the system it recombines with opsin reforming the rhodopsin peak at 498 nm (22). Subsequent addition of 50 m8'1 hydroxylamine converts free excess ll-cis-retinal to retinaloxime which has no siqnificant absorbance at 498 nm. Hence addition of ll-cis-retinal to a fully bleached sample will allow the assay of all the remaining functional opsin molecules. Freeze-dried bleached rod outer segments
. . . .

OKl”al

RhodoC)s,n

(nmol)

Fig. 1. Regeneration of Rhodopsin. Freezedried, bleached rod outer segments were resuspended as described in the text. Aliquots containing various amounts of bleached rhodopsin (calculated from ASP7.4. of They the original preparation) were added to 1 ml of 10 mM Hepes(K), p were then regenerated to rhodopsin by addition of ll-cis-retinal followed 7 by hydroxylamine and assayed for rhodopsin as described in the text. A500-A650 for 2 nmol original rhcdopsin was 0.081.

Vol.

122,

No.

1, 1984

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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In fact recovery was 1128, probably because of the pool of even in dark-adapted rod outer segments. of data. Individual points are averages of all estimates at that dose. Lines are least squares fits to the individual estimates. The values were calculated by interpolation. 95% confidence emits of Djg D37 were calculated taking into account errors in both slope and intercept o the interpolated line. full bleach. opsin present Treatment

Assay

A500. Irradiation

by

unexpected

absorbance

at

signal, so direct

spectral

samples.

shows

Fig.

2a

of

short

RESULTS rod outer

segments

wavelengths

which

assay was restricted that

unbleached

0

5

10 Radmtion

Dose

had

to

an

the

380

nm

a target

unbleached size

20

25

(Mradl

A: Rod outer segments (l-3 nmol size anafysis of rhcdopsin. were pre-mcubated in darkness, frozen, freeze-dried and 'Experimental', then irradiated with various doses as described under resuspended in 0.1 ml of 67 mM KH PO , pH 6.4, 2% (w/v) diqitonin. Rhodopsin remaining after irradiation w& &sayed spectrally as described under 'Experimental' by addition of 0.025 ml to 1 ml of 10 mM Hepes(K), pH 7.0. In both A and B points are averages of triplicates from 4 separate The line represents M =20,200. B: Rod outer segment experiments. as described suspension from A after rhodopsin ass&y was regenerated under 'Experimental'and regenerated rhadopsin was assayed (closed circles, solid line, M =51,800). Rod outer segments (l-3 nmol rhodopsin) were pre-incubated ';m daylight for 5 minutes to bleach all rhodopsin present opsin then frozen, freezedried, irradiated and resuspended as above. remaining after irradiation was measured after regeneration as described under 'Experimental' (open circles, dashed line, Mr=53,700). ;~G~p~jrqet

58

of

when assayed by A500

limit)

15

obscured

rise

to A5oof i.e.

rhcdopsin

20,200 + - 4,600 Mr (mean -+ 95% confidence

gave

Vol.

122,

No.

BIOCHEMICAL

1, 1984

remaining

after

known

that

irradiation.

rhodopsin

increased

by

remaining

A5OO does

molecule, loss

of this

Assay

of

used

(Fig.

target

axis

the

the

which

vrvo.

Clearly,

of

the

it

is

must

tie

assay

whole

by

rhodopsin

is destroyed

is there

seen

the

lines

+ 14,000

and

size

This

protein

may

Jarvis

that seen the

of

by

the

from

four

such

experiments

size

change

induced

et --

al

true

light

bleached

Mr

(Gpp(NH)p) rhodopsin (results by light

loss

in

not

shown).

as detected

lines

the was

-+ 12,400. the

Closer

intercept

to

also

presence

and

were point Mr

was

assessed

absence

guanine

a

a free

intercept

We have

technique.

of

sizes

dark

there

the

amount

the

no difference

by this

41,000.

target

removes

Thus

sizes

due

the

after

target

a small

species)

which

(29). we saw

about

the

+ 16,400,

assayed Both

the

When

value

as for

assay.

of rhodopsin

(28).

43,800

rhodopsin

exactly

spectral

either

a single

was

ll-cis-retinal

was 51,800

of

to

segment

size

that

100%

represented

outer of

circles).

indicate

or a small

rod

addition

value

shows

without method

pre-incubation,

2b, open

2b

A50O spectral

regeneration

measured

light

to be ,a

rhodopsin

The

simple

The

known

of the

the

target

expected

Fig.

is

fragment

then

by the

ll-cis-retinal.

existence

The

(Fig.

the

aggregate

as

opsin

by the

was

after

-+ 19,700

100%.

and

used

circles).

in

and

irradiation.

This

than

opsin

can

regenerated

rhcdopsin

assuming

target

after

data

rhcdopsin

effect

the

proteolysis

5'-guanylylimido-diphosphate bindinq

a value

domain

from

than

was found

53,700

below

recalculated

47,100

of

higher of

that

(24),

a critical

integrity

than

was

slightly

radical

sequence

ll-cis-retinal -

2b, closed

size

examination

Le.

from

2a were

higher

Mr

primary

intactness

of rhodopsin

500 nm peak.

assay.

vertical

when

COMMUNKATIONS

the

in

(27). We therefore

Fig.

the

regeneration

higher

only

remaining

in

substantially

were

that

careful

opsin

reform

The

on the

regeneration the

simple

depend

although

preventing

to

not

of opsin

(25,26),

samples

38,840

qlycosylation

rhodopsin

indicator

assess

of

to

by reconstitution

better

Mr

from

RESEARCH

signal.

regeneration

peak

an

owing

suggesting

BIOPHYSICAL

However

has

5%

AND

of

nucleotide

in

tarqet

is

no

size

significant

in

Vol.

122,

No.

1, 1984

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

DISCUSSION The

value

of

regeneration

appears

studies this

(32).

It

the

use

with

small greatly. protein

or

may not

destroy

the hit

inactivation,

at

functional

entity

of

there

is

affects

no A500

transfer exclude

in

from

undetected

of

is

alter the

preliminary

not

the

holding

low

chain

and

in

size

molecule

alter

is

Indeed,

even

removal

its

cytoplasmic

involved

media

spectrum.

(331 do not

in

target

rhodopsin of the

large

not

our

before

or pre-incubation

opsin

on the

data

(24), showing must

inadequate least

to

under for

agrees

our

between

monomers

the

of

spectrum

domain

of the

chromophore

was

for

our

binding

of the

conclusions

of

the

between

from

opsin.

any

putative

rhodopsin

assay

polymers

to

the

that

radiation

the

size

invertase

maximum

size

shows

that

This

rhodopsin

monomers

polymer. exist

is

there

which

great

However, in situ

the

activity

for

The

single

of

remaining

monomer.

Neither

M

that

reached (4,5).

the

regeneration

and

measures

iNa,K)-ATPase

that

close

successful

results,

conditions,

(3) and

that

for

similar

are

We conclude

subsequent

with

of

that

account

interaction

reconstitution

assay

be intact.

some

studies

functional

in our

not

proteases

reconstitution

molecule

possibility

period

of the

the

or

the

in

hypothesis

did

alpha-helices

dehydrogenase

rhodopsin

obtained

and qeneration

with

rhodopsin

500 nm absorbance.

This

(2), glutamate

not

region

incident

needed

protein.

small

cleavage

the

rhodopsin is

regimes

protein

sequence

hypothesis

was

unbleached

single-hit

binding

that

the

from

of the

of

values

whole

a

electrons

some

Mr

expected

only

of the

Hence

The

assay

(27,31,32)

portions

the

of

of freezedrying

chromophore

is known

Mr

and

that

various

Perhaps

involved

the

anomalous,

Variation of

observed.

for

suggests

experiment.

or

it

20,200

energy

we but

cannot

remained

experiments.

ACKNOWLEDGEMENTS We thank This work studentship

for Professor M. Akhtar was supported by the to SMH from the Science

suggesting the Wellcome trust and Engineering

reconstitution assay. and by a research Research Council.

r

Vol.

122,

No.

1, 1984

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

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