Rapid diglyceride phosphorylation in isolated bovine rod outer segments

Vol. 123, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 121-127

August 30, 1984

RAPID DIGLYCERIDE PHOSPHORYLATION IN ISOLATED BOVINE ROD OUTER SEGMENTS* Mark

A. Seyfred,

Department Received

July

Russell E. KohnkenA, Christine and David G. McConnell** of Biochemistry, East Lansing,

Michigan Michigan

State 48824

A. Collins*

University,

6, 1984

When isolated bovine rod outer segment fragments were incubated with [Y-~~P]ATP, 32P, as revealed by autoradiography, was rapidly incorporated into rhodopsin bands on sodium dodecyl sulfate polyacrylamide gels, and into a low M, lipid band. Incorporation of 32 P into rhodopsin was light-dependent, but labeling of the lipid band was not. A single phosphorylated product, phosphatidic acid, was identified by 2-dimensional thin layer chromatography and by high pressure liquid chromatography of the corresponding glycerophosphate ester. Incorporation of label into phosphatidic acid was detected as early as 15 set following start of incubation and the product was stable for at least 30 min. No other products were detected, indicating that under the experimental conditions phosphatidic acid was not metabolized to other phospholipids. up to 1 mol phosphatidic acid was formed per 18 to 40 mol rhodopsin present.

When light-dependent and rod outer dodecyl

segments

sulfate

(SDSI)

phosphorylation (ROSI)

are

analyzed

polyacrylamide

common to see a low Mr radiolabeled not

stain

suggest

with that

ROS incubated

Coomassie this with

radioactive

gel

rhodopsin

for

band just Frank

rapidly

were lipid.

tracking

it

earliest

We report

here

Foundation

is

dye which

among the

32P into

does

to that

isolated

phosphatidic

Grant

BNS-8210414.

Experimental

'The abbreviations used are: ROS-rod outer segment(s); BHT-butylated hydroxytoluene; HEPES-4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; HPLC-high pressure liquid chromatography; sodium dodecylsulfate; PAGE-polyacrylamide gel electrophoresis; PA-phosphatidic acid; PC-phosphatidylcholine; PE-phosphatidylethanolamine; PS-phosphatidylserine; PI-phosphatidylinositol; TLC-thin layer chromatography.

SDS-

0006-291X/84 121

(1 1

by sodium

(PAGEI)

above the

incorporated

by National Science should be addressed.

in vitro

32P incorporation

et a1.(2)

address: Worcester Foundation for Shrewsbury, Massachusetts 01545.

is studied

electrophoresis

band contained

[Y-~~P]ATP

* Research was supported ** To whom correspondence 'Present Biology,

Blue.

of

$1.50

Vol. 123, No. 1, 1984

acid

(PAI),

BIOCHEMICAL

apparently

through

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

action

of a diglyceride

kinase

on endogenous

diacylglycerol. METHODS ROS were isolated from sucrose gradients as previously described (3) and either frozen in liquid nitrogen or used directly. [v-32P)ATP was prepared from [32P]inorganic phosphate by the method of Glynn and Chapel1 (4) as modified by Walsh et al. (5). ROS containing 40-80 ug protein (6) were incubated at 300 for 15 set to 30 min in a final vol of 60-100 ~1 containin 50 mM Tris-Cl or HEPEST buffer, pH 7.5; 5 mM MgC12, and 0.01-1.0 mM [Y- ! 2P]ATP (200-300 cpm/pmol). The reaction was carried out in darkness or room light, and was terminated by addition of either 1.5 ml chloroform:methanol (1:2) containing 0.01% BHTl (if lipid extraction followed) or 0.5 ml of'lO% trichloroacetic acid/l0 mM sodium pyrophosphate (if SDS PAGE followed). A Gelman ACD-18 scanning densitometer was used to quantitate autoradiograms, made on Kodak XAR-5 films. Phospholipids were extracted from ROS with chloroform:methanol (1:2) or with acidic chloroform: Inethanol using the method of Schacht (7). The extracts were then spotted directly on TLC plates or deacylated by mild alkali treatment as described by Kates (8) if HPLC followed. Two-dimensional TLC employed Silica 60 plates (Merck) developed in the first dimension using chloroform:metha‘nol:28% NH40H (13:7:1) and in the second dimension using chloroform:acetone:methanol: acetic acid:H20 (10:4:2:2:1) as described by Sickotos and Rouser (9). The time course of 32P incorporation was measured by densitometric scanning of autoand by scintillation counting of areas scraped from radiograms of SDS PAGE gels, TLC plates after autoradiography of one-dimensional chromatograms developed in the acid solvent only. Lipids were visualized by exposure of the TLC plates to TLC phospholipid,standards were purchased from Boehringer-Mannheim iodine vapor. or Sigma. HPLC of deacylated glycerophosphate esters was performed with a Bio-Rad Aminex A-27 anion exchange column as previously described (10). A polyphasic gradient was used beginning with 20 II+! ammonium borate, 100 mM ammonium formate, 850 mM ammonium formate, pH 9.5. pH 9.5 and ending with 20 mM ammonium borate, SDS-PAGE was performed by the method of Fairbanks et al. cases bands were visualized by staining with Coomassie Brilliant others, dried gels were directly subjected to autoradiography.

(11). In some Blue.R, and in

RESULTS 32P incorporation incorporated shown

in

stained

32P into

mM Ca2+,

ROS.

multiple

the autoradiogram with

Coomassie

light-dependent effect

into

bands in Fig.

Blue.

whereas

on labeling

of the

that

was included

peripheral

proteins

from

of rhodopsin

1.

Labeling of the

with

in the

[Y-~~P]ATP

and into

of rhodopsin

monomer

low M, band was not

calmodulin, reaction

a low M, band

The low M, band was not

low M, band was observed

1 mM EGTA, 3.3 ug/ml

calmodulin

ROS incubated

or 33.3 mix

ROS by hypotonic

(data

washing 122

when ug/ml not

present

as in gels

and oligomers

(data

not

was

shown).

No

1 r+l GTP, 1 IIM cGMP, 1 antibody

shown).

in darkness

to Prior (3)

did

depletion not

of

prevent

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 123, No. 1, 1984

- rhodopsin monomer

dye front

FIGURE

1. 32P incorporation into ROS bands on SDS polyacrylamide gel. Autoradiogram of unstained SDS polyacrylamide gel following 1 min incubation 300 of ROS (75 ug protein) depleted of peripheral proteins (3) with 100 uM [y-32P]ATP, 5 mM MgC12, 50 ti Tris-Cl pH 7.5. In parallel gels stained with Coomassie Blue, all three of the upper bands (rhodopsin Imonomer and oligomers) stained but not the lipid. The radioactive spot corresponding to dye front contained ATP and phosphate.

labeling

of the

emulphogene the

low M, band,

or sodium

nor

cholate

incubation

not

with

[Y-~~PIATP,

chromatogram.

ROS were

mix

the

presence

inhibited

by TLC and HPLC.

extracted

in acidic

The single

of added&carrier

The material

reaction

product

in 2 dimensions.

by co-migration

However,

at

the

of labeling

of

shown).

of low Mr labeled

and chromatographed PA

of rhodopsin.

at 0.5% in the

low M, band by 90% (data Identification

as

- lipid

-

(Fig.

remaining

labeled

After

chloroform: product

2) and of PA run

at the origin

was most

methanol was identified

on a parallel likely

free

phosphate. After

products same point

alkaline

were

separated

were

extracted

3 times

as

described

by HPLC (Fig.

as glycerol

fractions

remained

deacylation

phosphate

treated

with

with

1:l

in the aqueous

in

3).

Methods,

A single

and inorganic

labeled

phosphate

0.4% ammonium molybdate, :benzene.

isobutanol

phase

indicating

stoichiometry

of

their

the

water-soluble

product

eluted

standards.

0.5

Peak

N H2S04 and then

Approximately

association

80%

with

of

the

organic

(12). Time course illustrates

and

the time

course

32P

incorporation.

of 32P incorporation

123

Figure

into

at the

4A

PA in one experiment.

counts

phosphate

Vol.

123,

No.

BIOCHEMICAL

1, 1984

AND

1s I

BIOPHYSICALRESEARCH

COMMUNICATIONS

dim.

< FIGURE

2 nd

1\

Dimension

origin

2. Autoradiogram of Z-dimensional thin-layer chromatogram of Lipids were extracted from ROS after 1 min incubation with Phosphatidic acid (PA) standard was described in Methods. Dashed lines indicate lipid spots before chromatography. iodine vapor. Had PI been detectable it would have appeared Standards were run in a parallel chromatogram.

cy

GrE

GPI

.

.

GrG

.

GgPi

GlfS

labeled ROS lipids. [Y-S*P]ATP as added to the extract after development with close to PS.

GPIP 12

GqlP

600

200

loo:&

01

IO

20 Fraction

30 No. (l.Oml)

40

I 5:

GURE 3. Separation of glycerophosphate esters by anion exchange HPLC. Incubation of ROS with [v-3zP]ATP as before was followed by acidic chloroform/methanol extraction and mild alkaline deacylation as described in Methods. Along the top of the figure are indicated the points at which unlabeled standard samples of the indicated glycerophosphate esters eluted. GPC-glycerophosphorylcholine; GPE-glycerophosphorylethanolamine; GPI-glycerophosphorylinositol; GPG-glycerophosphorylglycerol; GP-glycerol phosphate; GPS-glycerophosphorylserine; GPIP-glycerophosphorylinositol 4-phosphate; GPIPZ-glycerophosphorylinositol 4,5-bisphosphate.

124

BIOCHEMICAL

Vol. 123, No. 1, 1984

012

5

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IO

3(

20 Time (mid

FIGURE 4A. Time

course of 32P incorporation into PA. After different incubation times with 1 m+l [Y-~~P]ATP as before, ROS lipid extracts were spotted on thin layer chromatograms and run in one dimension with chloroform:acetone:methanol: acetic acid:H20 (10:4:2:2:1).+ The chromatogram was exposed to X-ray film for 12 h. 32P-positive areas were scraped off the plate, hydrated in 0.5 ml H20 before addition of 7.5 ml scintillation cocktail and counted in a Beckman LS 7000 scintillation counter.

48. -

The

Similar incubation

inset

(40)

experiment times.

shows

incorporation

a time

appeared

experiment

of

1 lnol

was

which

incorporation

course

linear

about

incorporation

in

during

for

the

to

per

be

5 min, 40 mol

1 mol

assayed at shorter

32P was

another,

first

PA labeled

calculated

of

shorter

experiment.

reaching

a final

rhodopsin

phosphate

in

In value In

protein. PA per

18 mol

4A in

46

this

final

rhodopsin.

DISCUSSION In into

isolated

phosphatidic

synthesized This

label

the

absence

per

18

could of

However,

these

activity

are Although

we observed

acid.

incorporation

3*P

(13,14),

ROS,

not

In

rhodopsin

was

not

results

indicate

present

in

rapid

turnover

or

approximately upon any

of

3*P up

incorporation to

1 mol

1 nmol

bleaching

other

synthetic that

isolated

of

incubation,

in

and/or

rate

min

dependent

detected

precursors

identification

a 30

mol

be

a rapid

of

lipids. enzymes

of

the Whether

and

mg ROS

ROS

by

be

protein.

visible

this to

ATP

PA was

PA per

remains

diacylglycerol

from

light.

reflects determined.

a diacylglycerol

kinase

ROS. of

enzymes

ROS

lipids

has

involved

has

125

been been

surmised elusive.

for

a decade Fliesler

or and

more

BIOCHEMICAL

Vol. 123, No. 1, 1984

Anderson

(15)

individual

have marshalled

discs

Phospholipids, another

over

and between

decarboxylation

evidence

and above disc

especially

PI1,

discs

and the

incorporation

the

layer

retinas, bulk

and not

phospholipid

PI. 3*PO4

in the

(19)

level also

photoreceptor were

presence

PI pool

(including (15).

PI in

in our

PA) is

The high

components

restricted

rate

Recently,

kinase

activity

in photoreceptor

finding

which

underlies

Acknowledgements

the

cells possible

- We thank

for

that

de novo

incorporation

of hydrolyzed,

of

pre-existing

of [14Clglycerol,

(19).

in 30 min)

A low

was

of a phospholipase

isolated

bovine

ROS.

possibility in turnover

have

W. W. Wells

reported

The size

location

PC and PE as well the

phosphorylation

and renewal absence

activity

for

of ROS disc

of diglyceride

mutants

and L. N. Roer

of the

on a molar

strategic

that

drosophila of this

may account

phospholipids

ROS phospholipids

importance

126

plexiform

precursors

mg retina

of blind

research.

PI in

outer

ROS fraction

action

the

et al.

as

(17,18).

in a crude

process

Yoshioka

slow

retinas

Following

the major

a significant

phospholipids.

in the

of 3*P and the

PS and PI raise

reflects

PI in toad

to 3% of the total

for

to

(19).

of incorporation

pathways

rod.

a

retinas.

localized

of diacylglycerol

as the

here

rat

mol 3*P per

was not

reported

and 3*P into

recovery

PI was detected

this

as well

incorporation

isolated

of the

In addition,

suggesting

a light-stimulated

PI by the

synthetic

observed

the

in

one disc

(16)

Moreover,

endogenous

of PA in the minor

only

(50 x 10-18

from

the

basis

from

was localized

incorporated,

reflected

although

Production

et al.

be renewed

base

(15).

into

cells.

not

some labeled

reported

the

freely

[3H]inositol

incorporation

into

of PA synthesis

Anderson

of both

reported

or C3Hlinositol

fractionation,

for

of this

and phosphate Schmidt

from

to diffuse

and of [3H]inositol

synthesis

inositol

replacement

must

of PSI to PEl in ROS has been observed

light-stimulated

However,

ROS lipids

ROS plasmalemma

of PE to PC1 (16).

laevis

that

appear

transmethylation

Xenopus

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(20),

a

in vision.

assistance

in the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 123, No. 1, 1984

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