BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 173, No. 1,1990

Pages 283-288

November 30,1990

CALCIUM

ION-REGULATED PHOSPHOLIPASE C ACTIVITY BOVINE ROD OUTER SEGMENTS I. Panfoli,

Istituto

A. Morelli #

IN

and I. Pepe*

Policattedra di chimica Biologica dell 'Universita' degli Studi di Genova, V.le Benedetto XV i, 16132 Genova, Italy

* Istituto di cibernetica e Biofisica del C.N.R., Via Dodecaneso 33, 16146 Genova, Italy Received October 5, 1990 SUMMARY: B o v i n e r e t i n a l rod o u t e r s e g m e n t m e m branes are enriched in a p h o s p h o i n o s i t i d e - s p e c i f i c phosphodiesterase (phospholipase C) activity strictly modulated by free calcium ion concentration. The e n z y m e ( s ) was h i g h l y a c t i v e on p h o s p h a t i d y l i n o s i t o l 4 , 5 - b i s D h o s p h a t e : m a x i m a l h y d r o l y s i s rate was a t t a i n e d at 10-SM Ca 2+ a n d a c c o u n t e d f o r 91 ± 4 n m o l e s hydrolyzed/ min/ mg of protein. The results support the notion that in vivo the enzyme(s) is regulated so as to conform to the phototransduction events. ©1990AcademicPress.Inc.

Phosphatidylinositol minor a

component

of cell membrane

phospholipase

molecules

(I):

for

mobilizing

(2)

and

4,5-bisphosphate

C

(PLC)

to

lipids, two

calcium

ions

from

1,2-diacylglycerol,

is degraded

active

inositol trisphosphate

(PIP2) , a

(IP3), responsible

intracellular

known

to

by

messenger

activate

stores protein

kinase C (3). Evidence has been accumulating that PIP 2 breakdown might mediate in Limulus musca

weaker:

To

eye

(4),

for

phototransduction

squid photoreceptor

for PIP 2

transduction

is m u c h

there have been in the past years three

reports

whom

fly eye

in v e r t e b r a t e

(6,7).

(5) and

The case

involvement

#

ventral

and Drosophila

to support the notion

visual

correspondence

should

be

addressed.

Abbreviations: BSA, b o v i n e s e r u m a l b u m i n ; DOC, Sodium deoxycholate; IP3, inositol 1,4,5trisphosphate; PC, phosphatidyl choline; PI, p h o s p h a t i d y l inositol; PIP2, p h o s p h a t i d y l i n o s i t o l 4,5bisphosphate; PLC, p h o s p h a t i d y l inositol-specific phosphodiesterase.

283

0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 173, No. 1, 1990

whose

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

results

PIP2-PLC

indirectly

in frog ROS

However, inositol

to

claimed

at

a light-activated

(8,9,10).

conclude

that

lipids breakdown

rhodopsin

mediates

for

in vertebrate photoreceptors,

a lot more evidence has to be seeked for. We

show

(ROS)

of

herein the

that

disrupted

bovine

retina

[3H]labeled phosphoinositides light insensitive

rod

outer

hydrolyze

segments exogenous

in a calcium-dependent

but

manner. METHODS

Phosphatidyl[2-3H]inositol 4,5-bisphosphate, L-3phos~phatidyl[2-3H]inositol and L-3-phosphatidyl[N-methyl-~H] choline, 1,2-dipalmitoyl were from Amersham International. Rod Outer Seqments preparations. B o v i n e R O S w e r e i s o l a t e d f o l l o w i n g the m e t h o d of Schnetkamp and Daemen (ii). The resulting ROS suspension was homogenized in a small tight glass/glass potter with 2 ml of 41 mM HEPES pH 7.0, i00 mM KCI, 20 mM NaCI, 2 mM MgCl2, 2 m M D T T , 20 ~ g / m l l e u p e p t i n , to o b t a i n a suspension of disk membranes. Phospholipase C activity assay PLC was a s s a y e d in the p r e s e n c e of e x o g e n o u s phosphatidyl [2- H ] i n o s i t o l 4 , 5 - b i s p h o s p h a t e or L-3phosphatidyl[2-3H]inositol or L-3-phosphatidyl [N-methyl-3H] choline, 1 , 2 - d i p a l m i t o y l on small m i c e l l e s derived from pure substrate by sonication, a c c o r d i n g to the m e t h o d of Banno et al. (12), with minor m o d i f i c a tions. Hydrolysis of PIP 2 (or PI,PC) was assayed at pH 7.0 in a reaction mixture containing: 45 mM HEPES pH 7.0, i00 m M K C I , 20 m M N a C I , 0.33 mM MgCI2, 2 mM EGTA, .isl 50 ~g/ml leupeptin, 0.08% DOC, 0.250 mM PIP 2 (or PI, PC) p l u s i 0 0 , 0 0 0 D P M of [ 3 H ] P I P 2 (or [ 3 H ] P I ; P C ) (6600 DPM/nmol) and ROS suspensions (7.0 ~g) to a final volume of 60 ~i. Free Ca 2+ c o n c e n t r a t i o n was c o n t r o l l e d by a C a 2 t - E G T A b u f f e r (13). The s a m p l e s w e r e i n c u b a t e d at 37°C, for the time indicated in the Figure Legends, in dim red light except for the samples that were illumin a t e d - f o r 1.5 m i n t h r o u g h o p t i c a l f i b e r s w i t h an actinic light source of 150 W. P r o t e i n was d e t e r m i n e d by a m e t h o d b a s e d on C o o m a s s i e blue s t a i n i n g (14) with BSA as a s t a n d a r d protein.

RESULTS

Fig 1

shows

[3H]inositol linear PIP 2

that

DISCUSSION

hydrolysis

of exogenously

labeled phosphoinositides

with

Breakdown

that

AND

the

time

of

the

of PI was n e g l i g i b l e was

relatively

high 284

added

at 10-5M Ca 2+ was

experiment with r e s p e c t

(16

min) .

to that

(91 ± 4 nmoles

of

of PIP 2

Vol. 173, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

600/

150

/

/

o

50

0

4

8 time, m i n

12

i6

Fiqure i. Time course of PIP 2 and PI breakdown. ROS suspensions (7.0 ~g) wer~ incubated in the assay m~xture (see Methods) with [ -H]PIP~ ( • ), or with [ H]PI ( O ) as substrates; aliquo£s of 60 ~i were q u e n c h e d at the time indicated. Free Ca 2+ concentration was 10-5M.

hydrolyzed/ recovered tissues was

min/mg in

the

of p r o t e i n ) crude

in s i m i l a r

not

similar

result

membranes

was

PC

reported

(18.1)

0.7

nmol

consistently conditions PLC

fractions

(data

for

of

that other

PLC a c t i v i t y

not

rat

to

shown).

liver

A

plasma

(15).

The ratio

±

compared

(15,16,17).

against

between

(91 ± 4 nmol hydrolyzed/ (5

membrane

conditions

detectable

when

PLC a c t i v i t y

min/mg protein)

hydrolized/

higher

than

min/ that

mg

against

protein)

observed

for the bovine brain three

PIP 2

and against

in

forms

the

PI was

same

of p u r i f i e d

(18). The assay was conducted

and

required

proteolysis late

(DOC)

the

on the ROS d i s k h o m o g e n a t e

presence

inhibitor

of

leupeptin

the

calcium-dependent

and of sodium d e o x y c h o -

for reproducibility.

The p r e s e n c e of DOC six-fold

activation

previous

reports

(0.08 %) at 10-5M Ca 2+ p r o d u c e d a

of PIP 2 h y d r o l y s i s

(16,19),

consistent

w h i l e the d e t e r g e n t

was

with less

e f f e c t i v e on PI, as shown in Fig 2. The activation of PI hydrolysis

by DOC

responsive

(Fig 2) and amounted only to two-fold.

(0.08 %) at 10-4M Ca 2+ was not d o s e -

The strong dependence of hydrolysis PI

in

disrupted

concentration

bovine

is shown

ROS

on

of both PIP 2 and

free

in Fig 3. M a x i m a l 285

calcium activation

ion of

Vol. 173, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

100

5

ce8o

4o

~6o 3~ o 40 r~

20

2~ / 1 0

%.00 o'.o2 o.'o, o.06 o.68 o.lo o.12 o.14 o.1~ DOC, Z

Fiqure 2. Effect of DOC concentration on PLC activity. ROS s u s p e n s i o n s (7.0 ~g) were i n c u b a t e d for 5 min in the a s s a y m i x t u r e (see M e t h o d s ) wit h [ 3 - ~ ] P I P ~ in 10-5M Ca z+ ( • ) or with [~H]PI in 10-~M Ca zT ( A ~ .

PIP 2 h y d r o l y s i s was observed

in 10-5M Ca2+.

A reduction

in Ca 2+ levels below 10-BM as well as an increase up to millimolar

concentrations

Hydrolysis

rate

of

PI

leaded to a loss of activity.

was

maximal

at

10-3M

Ca 2+

and

almost n e g l i g i b l e around the resting values of free Ca 2+ inside the p h o t o r e c e p t o r suggesting enzyme

that

might

(i.e.

in basal be

active

about

5,I0-7M,

physiological only

d e p e n d e n c e on free calcium ion

on

ref.

20),

conditions

P I P 2.

A

concentration

the

similar has

been

120 ~I00

6

80

40 ~

20 0

8

7

6

5 pCa

4

3

2

0

F i q u r e 3. E f f e c t of free Ca 2+ c o n c e n t r a t i o n on PLC activity. ROS s u s p e n s i o n s (7.0 #g protein) w e r e i n c u b a t e d , for 5 m i n in the a s s a y m i x t u r e (see M e t h o d s ) w i t h [ 3 - H ] P I P 2 ( • ) or w i t h [3H]PI ~ • ). F r e e Ca 2+ c o n c e n t r a t i o n was controlled by a Ca ~T_EGTA buffer. 286

Vol. 173, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o b s e r v e d also in other tissues

(6,12,18),

however

influence of free calcium ions on PIP 2 h y d r o l y s i s the case of Free

Ca 2+

shown

ROS,

of extreme physiological

concentration

(21)

to decrease

(to less than 10-8M) on

the

if levels 10-8M

or

is,

after

ROS

was

illumination

the effect of this rapid has

in

significance.

vertebrate

functioning

although

of

the

significantly

and

receptor

investigated

inside

the

been

not yet clarified.

fall

widely

For example,

internal Ca 2+ should fall from 5,10 -7 to

less,

PIP 2 h y d r o l y s i s

almost s u p p r e s s e d

would

result

(about 85% reduction,

in b e i n g

Fig 3).

Such r e s u l t w o u l d be in a c c o r d a n c e w i t h the p r e s e n t model

of p h o t o t r a n s d u c t i o n :

caused

indirectly

would

stop

caused

by

the its

intracellular In

our

presence activity

by

light

basal

inactivation

by the

undesirable

lowering

increase

in

IP3-dependent

of

PLC

of Ca 2+,

Ca 2+

level

extrusion

from

stores.

experimental of

the

DOC,

even

light

after

conditions, did

not

extensive

that

affect

is

in

bovine

rhodopsin

the

ROS

bleaching

PLC (data

not shown). We

examined

activity of the

enzyme

120,000

This

and

by g

in the p e l l e t centrifuging

distribution

for

1 hour

and

and

the

might

mean

that

localization.

characterize

the

specific

activity

supernatant

fraction

suspension

30 m i n

were

68.4

of protein, ROS

PLC

and

are

putative

activity

in progress various

at

22.1

respectively.

isozymes with different

Studies

PLC

disk

bovine

to two or more

of the

ROS

PIP 2 h y d r o l y z e d / m i n / m g

ascribed lular

subcellular

for PIP 2 and found that the

obtained

nmoles

the

forms

can

be

subcel-

to separate of

PLC

of

the bovine ROS.

ACKNOWLEDGMENTS

W e w i s h to t h a n k Dr. D r o e t t o from the Servizio V e t e r i n a r i o della XV U.S.L. di Genova, for g r a n t i n g the availability of f r e s h l y enucleated bovine eyes. I.Panfoli is g r a t e f u l to t h e F o n d a z i o n e A n n a V i l l a Rusconi for a Fellowship. REFERENCES

I- B e r r i d g e , 315-321.

M.J.

and

Irvine,

287

R.F.

(1984)

Nature

312,

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2- Majerus, P.W., Connolly, T.M., Deckmyn, H., Ross, T.S., B r o s s , T . E . , Ishii, H., Bansal, V.S. and Wilson, D.B., Science (1986) 234, 1519-1526. 3- Nishizuka, Y. (1986) Science 233, 305-312. 4- Brown, J.E., Rubin, L.J., Ghalayini, A.J., Tarver, A.P., Irvine, R.F., Berridge, M.J. and Anderson, R.E. (1984) Nature 311, 160-162. 5- Wood, S. F., Szuts, E.T. and Fain, A. (1989) J. Biol. Chem. 264, 12970-12976. 6- Inoue, H., Y o s h i o k a , T. and H o t t a , Y. (1988) J. Biochem. 103, 91-94. 7- Devary, Heichal, O., Blumenfeld, A., Cassel, D., Suss, E., Barash, S., Rubinstein, C.T., Minke, B. and S e l i n g e r , Z. (1987) Proc. Natl. Acad. Sci. U S A 84, 6939-6943. 8- Hayashi, F. and Amakawa, T. (1985) Biochem. Biophys. Res Commun. 128, 954-959. 9- G h a l a y i n i , A. and Anderson, R.E. (1984) B i o c h e m . Biophys. Res Commun. 124, 503-506. 10-Waloga, G. and Anderson, R.E. (1985) Biochem. Biophys. Res. Commun. 126, 59-62. l l - S c h n e t k a m p , P.P. and Daemen, F.J. (1982) M e t h o d s Enzymol. 81, 110-116. 12-Banno, Y., Nakashima, S., Tohmatsu, T., Nozawa, Y. and Lapetina, E.G. (1986) Biochem. Biophys. Res. Commun. 140, 728-734. 1 3 - F a b i a t o , A. and F a b i a t o , F. (1979) J. P h y s i o l . (Paris) 75, 463-505. 14-Esen, A. (1978) Anal. Biochem. 89, 264-273. 15-Uhing, R.J., Jiang, H., Prpic, V. and Exton, J.H. (1985) FEBS Lett. 188, 317-320. 16-Claro, E., Wallace, M.A., Lee, H.M. and Fain, J.N. (1989) J. Biol. Chem. 264, 18288-18295. 17-Cockcroft, S. (1986) Biochem. J. 240, 503-507. 18-Ryu, S.H., Suh, P.-G., Cho, K.S., L e e , K . - Y . , and Rhee, S.G. (1987) Proc. Natl. Acad. Sci. U S A 84, 6649-6653. 19-Chung, S.M., Proia, A.D., Klintworth, G.K., Watson, S.P. and Lapetina, E.G. (1985) Biochem. Biophys. Res. Commun. 240, 411-416. 20-McNaughton,P.A., Cervetto, L. and Nunn, B.J. (1986) Nature 322:261-263. 21-Cervetto, L., Lagnado, L., Perry, R.J., Robinson, D.W. and McNaughton, P. (1989) Nature 337, 740-743.

288

Calcium ion-regulated phospholipase C activity in bovine rod outer segments.

Bovine retinal rod outer segment membranes are enriched in a phosphoinositide-specific phosphodiesterase (phospholipase C) activity strictly modulated...
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