Isolation and recombination of bovine rod outer segment cGMP phosphodiesterase and its regulators

Vol.

92, No.

Jonuory

2, 1980

29,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 505-510

1980

ISOLATION AND RECOMBINATION OF BOVINE ROD OUTER SEGMENT cGMP PHOSPHODIESTERASE AND ITS REGULATORS J. B. Hurleyl Department

of Physiology and Biophysics, University Urbana, Illinois 61801

October

Received

26,

of Illinois

1979

SUMMARY: cGMP phosphodiesterase extracted from rod outer segments can be activated by GTP in the presence of phospholipid vesicles containing I have separated the phosphodiesterase from a phosbleached rhodopsin. phodiesterase inhibitory protein and a GTPase also present in the crude extracts from rods. The GTPase can be activated by bleached rhodopsin. However, in the absence of the GTPase and inhibitor, the phosphodiesterase was not activated by GTP in the presence of bleached rhodopsin. Recombination with these proteins partially restored the activetion by GTP and bleached rhodopsin. Rod outer cular that

contain

several

a cGMP phosphodiesterase activation

light

cidated. which

the detailed

segment tried

and a GTPase.

step

here

to play

not

aware

the visual

the isolation

from

Recent

enzymes, evidence

transduction of this

process

and recombination role

by light

such a reconstitution

activated

by the bleaching

mechanism

an essential

phosphodeisterase

were

in

molecular

I report appear

light

of the phosphodiesterase

may be an important

However,

they

segments

in

(3). components

of the presence MATERIALS

isolated

suggests

of rhodopsin process

has not been

of bovine

et al. from

by

(1,Z).

of several

the regulation Shinozawa

in parti-

(4) frog

elu-

proteins rod

outer

have previously rods,

but

of the inhibitor. AND METHODS

cGMP, GIP, trypsin, trypsin inhibitor, and snake venom (Ophiophagus hannah) were obtained from Sigma Chemical Company; Cellex D(DEAEcellulose) from Bio-Rad Laboratories; Se hadex GZOO and Se harose 6B from Pharmacia Fine Chemicals. c3H) cGMP, ( 1E C) 5'GMP and (y- 33 P) GTP were purchased from Amersham Corporation; (r3*P) GTP was also purchased from New England Nuclear. Dodecyltrimethylammonium bromide was obtained from Eastman. Protein concentration was accomplished using a single hollow fiber concentrator obtained from Bio-Med Instruments, Chicago, Illinois. 1Present Building,

Address: Stanford

Department University

of Structural Biology; Sherman Fairchild School of Medicine, Stanford, California

94305.

0006-291X/80/020505-06801.00/0 SOS

Cop.vright 8 1980 b.v Academic Press, Inc. All rights of reproduction in any form reserved.

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cGMP phosphodiesterase was assayed as described elsewhere (5). was assayed by measuring 32P release from (y-3*P) GTP using activated charcoal to isolate the free phosphate from nucleotides (6).

GTPase

Extraction of the rod outer segment phosphodiesterase and DEAE-cellulose chromatography were carried out as described elsewhere (6). Bovine retinae obtained from Hormel Co., Austin, Minn. were isolated by flotation on 42% sucrose, followed by a 15% sucrose wash. Phosphodiesterase was extracted by homogenization of the rods under dim red light in 10 mM Tris pH 8.0 and 1 mM dithiothreitol. The extract was applied to a 1.5 x 15 cm DEAE-cellulose column at 40 mM Tris pH 8.0, 1 mM MgC12 and 1 mM dithiothreitol. Inhibitor was eluted by washing with 40 mM Tris pH 8.0, 1 mM MgCl , 1 mM dithiothreitol and 100 mM NaCl. Phosphodiesterase and GTPase were t i en eluted together with a gradient from 100 mM to 500 mM NaCl. Rhodopsin solubilized in from rod outer segment were prepared (8). Vesicles (9) to purified rhodopsin in the detergent.

ified

dodecyltrimethylammonium bromide was pur(7) by conconavalin A affinity chromatography by adding purified egg phosphotidyl choline the ratio 1OO:l and dialyzing 4 days to remove RESULTS

As reported hibitor

elsewhere

is present

be separated

a trypsin

(3),

in extracts

from

from bovine

the phosphodiesterase

by DEAE-cellulose

chromatography.

phosphodiesterase

and GTPase from

and efficiently these

separated

proteins

described

could

by gel

column

was concentrated

G200 column. is

not

recovered

below.

off

this

better

than

of purified

phosphodiesterase

independent

and ranges

GTPase.

extracts free

I isolated

recombination

is

experiments,

the DEAE-cellulose

shown in Fig.

rather

from

column. preparations

was used

GTPase yields

506

to a Sephadex GTPase activity

1.

a Sepharose

phosphodiesterase

several

6B column

from

of 1 to 2 ml and applied

20 to 100 nmoles

column.

can

can be quickly

chromatography.

the Sepharose from

from

A Sepharose

from the DEAE-cellulose

but

was used for

resolution

which

inhibitor

column

so that

in-

out.

profile

column

segments

show that

phosphodiesterase

elution

The G200 column

provided

filtration

to a volume

A typical

outer

and the GTPase in the

procedures

Pooled

phosphodiesterase

the DEAE-cellulose

be carried

Phosphodiesterase.

rod

Here I first

by the following

below,

sensitive

cGMP/min to further appear

6B column;

isolation

because

The specific is

see

activity

concentration

mg. purify

the GTPase

to be significantly

it

Vol.

92, No.

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AND

BIOPHYSICAL

40

40

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32-

16

24

32 FRACTION

Figure

1.

on agarose

I find

that

the

based

columns

the presence

than

on dextran

of 1 mM 5' GMP causes

enhancing

column,

Nevertheless,

the

separation

as can be seen in Fig.

phosphodiesterase

activity.

is

typically

The GTPase

fractions

Tris-Cl

at pH 8.0,

1 mM MgC12 and 1 mM dithiothreitol

On SDS polyacrylamide the phosphodiesterase

39,000

daltons

cellulose tively only

The major

3b).

column

(Fig.

appears

identified rhodopsin

bands 3~).

gels,

associated

with

as a monomer

major (3d). at 39,000

507

not

recovered

to elute

very

completely

late

with

free

about against

to remove

5'GMP.

protein

and 88,000

the GTPase appear inhibitor

50 mM

bands

of

daltons at 36,000

and

of the DEAE-

bands,

none of which

is

Purified

rhodopsin

vesicles

and as a dimer

of

20%

overnight

the major

at 84,000

The phosphodiesterase as several

the GTPase

and dialyzed

as a doublet

as the inhibitor both

pooled

electrophoresis

appear

In addition,

columns.

2, the GTPase is

GTPase were

based

of GTPase from phosphodiesterase.

yield.

(Fig.

64

Typical elution profile of the phosphodiesterase on the Sephadex G200 kolumn as described in the text. The column was 2.5 x 36 cm and was run at a flow rate of 5 ml/hr at 4°C with 50 mM Tris pH 8.0, 1 mM MgC12 1 mM dithiothreitol, and 100 PM phenylmethyl sulfonyl fluoride.' Approximately 3 ml fractions were collected. Blue dextran was used to make the void volume and DNP-glycine to mark the included volume.

higher

off

56

NUMBER

at 80,000

yet

posishow

daltons

(3a)

Vol.

92, No.

BIOCHEMICAL

2, 1980

AND

BIOPHYSICAL

RESEARCH

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t 20 FRACTION

Figure

2.

NUMBER

Typical elution profile of phosphodiesterase and GTPase off the Sepharose 6B column used to isolate GTPase for recombination The column was 2.5 x 38 cm experiments as described in the text. and was run at a flow rate of 7 ml/hr at 4°C with 40 mM Tris pH 8.0, 100 mM KU, 1 mM MgC12, 1 mM dithiothreitol, 100 uM phenylmethyl sulfonyl fluoride, and 1 ti 5' GMP. 3 ml fractions were collected. GTPase was assayed in the presence of bleached purified rhodopsin vesicles (5 ug rhodopsin).

~

88,000

84,000---

.

z

.-

80,00039,000

39,000-

36,OOOT

DYE FRONT-

0

Figure

3.

C

b

d

SDS polyacrylamide gel rlectrophoresis (10) of a) purified rhodopsin vesicles (-5 ug), b) gel filtration purified phosphodiesterase c) Sepharose 68 purified GTPasr (-30 ug), d) DEAE-cellulose (2 ug), 12.52 acrylamide: inhibitor (-50 ug). a), c), d) were run with b) with 10.5%.

508

Vol.

92, No.

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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Table 1. RECOMBINATION OF PURIFIED ROD OUTER SEGMENT cGMP PHOSPHODIESTERASE WITH ITS REGULATORS. -5 pg purified phosphodiesterase (PDE), -6 pg purified GTPase (27 pmoles GTP/min), and -30 ug DEAE-cellulose inhibitor were added where indicated. The numbers in parentheses are activities due to added PDE corrected for residual PDE activities in the isolated GTPase. __Phosphodiesterase activity (nmoles cGMP/min) Recombination Bleached Bleached Bleached Bleached Bleached Bleached

mixture rhodopsin rhadopsin rhodopsin rhodopsin rhodopsin rhodopsin

The agarose rhodopsin not

+ + + + + +

vesicles

However,

Sephadex

by

(Table

Addition

of inhibitor

rhodopsin

vesicles

absence

and presence

cluded

in

this

terase

activity

the presence

the

purified

carried

recombination the absence

and GTPase restores

However,

mixture,

the

rhodopsin

of bleached

does not

restore

the

the GTPase is

decreases

the combination

in-

phosphodies-

not when GTP is present.

vesicles

the GTP effect

experiments.

in both

when

is

sensitivity

activity

inhibitor

of GTP but

this

the presence

alone

GTP sensitivity.

of bleached

in

inhibitor

of bleached

purified

recombination

the phosphodiesterase

of GTP; i.e.,

31 19 3.8 74 (55) 59 (55)

bleached

to restore

out

~$1 GTP

phosphodiesterase

presenceof

to the phosphodiesterase

plus

in

in

100 I:!

in the presence

In an attempt

I have

decreases

rhodopsin

GTP

1).

to the phosphodiesterase,

bleached

activated

alone.

with

61 35 14 2.6 68 (54) 29 (26)

GTPase is

significantly

rhodopsin

GTP

PDE PDE + inhibitor GTPase GTPase + inhibitor GTPase + PDE GTPase + PDF + inhibitor

purified

vesicles

activated

w/o

Thus,

in

of inhibitor

on the phosphodiesterase. DISCUSSION

Hurley hibitor

et al.,

protein

from This

chromatography.

terase

and GTPase.

filtration

have

crude

extracts

procedure Here I report

chromatography.

the phosphodiesterase phodiesterase

(3,6)

regulation

recently

of bovine

allows that

Furthermore,

inhibitor

reported

isolation

segments

of inhibitor enzymes

I provide

and GTPase

by rhodopsin

rod

recovery these

the

play

and GTP.

of an in-

by DEAE-cellulose free

phosphodies-

can be separated

evidence an essential

suggesting role

by gel that

in phos-

Vol.

92, No.

2, 1980

BIOCHEMICAL

Isolated (3)

phosphodiesterase

on SDS polyacrylamide

at 36,000

and 39,000

phospholipid a molecular

weight

appears

gels.

This

containing

for

lation These the

activated during restored

phosphodiesterase results inhibitor

GTPase appears

GTPase fraction

here

by recombination

protein

maintains

basal

state.

Activation

tiich

in the presence

occurs

bands by assigned

rhodopsin with

on activation

rhodopsin

tht

phosphodiesterase

vesicles

phosphodiesterase

1) suggesting

The results

for

as two major

are based

of bleached

way (Table

GTP and bleached

a mechanism

doublet

I have not yet

Isolated

purification.

suggest

dalton

inhibitor.

the presence

in this

COMMUNICATIONS

can be activated

rhodopsin.

successfulrecombination.

proteins

I have partially

88,000

presented

by GTP in

significantly

regulatory

as an 84,000;

to the phosphodiesterase

of phosphodiesterase

is not

RESEARCH

bleached

The recombinationexperiments

as a criterion

BIOPHYSICAL

Isolated

daltons.

vesicles

AND

in Table activation

the loss 1 show that

to the iso-

GTPase and inhibitor. regulation

in which

the phosphodiesterase

at low activity

by bleached

activating

of GTP, counteracts

rhodopsin the effects

of

in the

the GTPase,

of the inhibitor

on the

phosphodiesterase. ACKNOWLEDGEMENT I thank advice. This fellowship.

Prof. Thomas Ebrev for his support, encouragement, and work was supported by NIH EY01323 and a NIH NRSA predoctoral

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Hubbell, W.L. and Bownds, M.D. (1979) Ann. Rev. Neurosci. 2, 17-34. Keirns, J.J., Miki, N., Bitensky, M.W. and Keirns, M. (1975) Biochemistry 14, 2760-66. Hurley, J.B. and Ebrey, T.G. (1978) Biophys. J. 2, 314a. I ., Wheeler, G., and Bitensky, M. (1979) J. Supramol. %,;Zo,zawa, . lo, T&%F? Thompson, W. and Appleman, N. (1971) Biochemistry lo, 311-316. for publication. Hurley, J., Barry, B., and Ebrey, T. (1979) submitted Papermaster, D. and Dreyer W. (1979) Biochemistry 13, 2438-2444. van Breugel, P., Daemen, F., and Bonting, S. (1977) Exp. Eye Res.4, 581-585. Singleton, W., Gray, M., Brown, M., and White, J. (1965) J. Oil Chem. 42, 53-56. Laemmli, V. (1970) Nature 227, 680-685.

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