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
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M.J.
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Irvine,
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R.F.
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312,
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