Vol. 91, No. 1, 1979 November

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

14, 1979

Pages 338-344

Ca2+-DEPENDENT CYCLICNUCLEOTIDE PHOSPHODIESTERASE FROMRABBIT LUNG RAJENDRA K. SHARMAand ERWINWIRCH Department of Biochemistry, University of Manitoba Winnipeg, Manitoba, Canada, R3E OW3 Received

October

9, 1979 SUMMARY

Studies are presented which demonstrate that rabbit lung contains both Ca*+-activated cyclic nucleotide phosphodiesterase and calmodulin activity. The Ca2+-activatable cyclic nucleotide phosphodiesterase is different from the commontype in that it contains tightly bound calmodulin. The bound calmodulin is not dissociated from the enzyme even in very low concentrations of Ca2+ after DE&?-cellulose and Sephadex G-200 column chromatography. INTRODUCTION A Ca2+-dependent cyclic rat brain extracts

nucleotide

phosphodiesterase was discovered in

by Kakiuchi and Yamazaki [1],

also in other mammaliantissues [2].

The activation

the presence of a Ca2+-binding protein by Cheung [5] as an activator

and later

protein

[3,4]

shown to be present

of the enzyme depends on

which was originally

discovered

of phosphodiesterase and recently

this

protein was renamed calmodulin by Cheung. The mechanismof the enzyme activation has been proposed as according to SchemeI: CM+ Ca2+ r--m-

Ca2++

CM+- Ca2+

(1)

(11)

Cl@ - Ca2+ SchemeI

where E and Cl4 stand for the enzyme and the calmodulin, respectively, and Cl8 denote the active

forms of the respective

proteins.

and E*

The main feature

of the schemeis that the calmodulin exists as an independent protein molecule which associates with the enzyme only upon binding of Ca2+ . Recently, Hidaka et al. [6] have shown the separation of two Ca2+-dependent phosphodiesterases Abbreviations used are EGTA, ethylene glycol bis (p-amino-ethyl ether) N,N'tetraacetic acid; CAMP, cyclic adenosine 3':5'-monophosphate; cGMP, cyclic guanosine 3'5'-monophosphate; CM, calmodulin. 0006-291X/79/210338-07$01.00/0 Copyright All rights

@ 1979

by Academic Press, Inc. in any form reserved.

of reproduction

338

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Vol. 91, No. 1, 1979

from humanaorta,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and both forms are activated

by the calmodulin.

Also Dedman

2+ [7] have shown the presence of a calmodulin independent, but Ca -

et al.

activatable

cyclic

nucleotide

These observations' indicate

phosphodiesterase among testis sertolis proteins. 2+ that not all Ca -dependent cyclic nucleotide

phosphodiesterases are regulated by calmodulin. In the present study, we have found the existence of a Ca2-k-dependent phosphodiesterase in rabbit calmodulin. even after

lung which is not activated

The enzyme preparations,

of

however, contain the calmodulin activity

several column chromatographies.

enzyme contains tightly

by the addition

The result

suggests that the

bound calmodulin which mediates the Ca2-k-activation

of the enzyme. Thus, schemeI should not be considered as a general mechanism for the calmodulin mediated enzyme activation. MATERIALSANDMETHODS Bovine heart calmodulin deficient phosphodiesterase and bovine brain calmodulin were prepared according to the previously described methods [a-lo]. The assays of phosphodiesterase and calmodulin were also as previously desfrozen cribed [lo]. To prepare the (NH ) SO fraction of rabbit lung extracts, rabbit lung (Pel Freeze) was thawe 4 5 akd homogenized in 2.5 volumes of a buffer containing 20 m&lTris-HCl and 1 mMEDTA, pH 7.5. The homogenatewas centrifuged at 3,500 rpm for 30 min, the supernatant was collected and brought to 55%saturation of (NH4)2SO4. The solution was then centrifuged at 8,000 rpm for 20 min and the pellet was suspended in minimal volumes of 20 mMTris, 1 mMimidazole buffer, pH 7.5,containing 1 mMMg2' 0.1 r&l EGTAand 10 mM2mercaptoethanol (buffer A). The solution was dialyzed against the samebuffer overnight. The dialyzed sample was centrifuged at 40,000 rpm for 30 min and the clear supernatant was collected. RESULTS AND DISCUSSION Table I shows that rabbit leotide activities

lung contains both Ca2+-activated

phosphodiesterase and calmodulin activities. in humanand guinea pig lungs [ll-131

ever the mechanismof Ca2+ activation tionship

cyclic

nuc-

The presence of these has been shownbefore.

How-

of the phosphodiesterase and the rela-

between the enzyme and the calmodulin have not been investigated.

A commontype of Ca2+-activatable

phosphodiesterase that is found in

many mammaliantissues has been shown to depend on the presence of the calmodulin for its activation.

The enzyme and calmodulin exist as separate pro-

339

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TABLE 1 CYCLIC NIJCLEOTIDE PHGSPHODIESTERASEAND CAIMODULJN IN CRUDE EXTRACT OF RABBIT LUNG Activity Units/mg Calmodulin activity Phosphodiesterase activity +ca2+ +Ca2' + CM -Ca2+ (EGTA)

protein

Units/KG

(Wet tissue

45.oa

weight)

2,75o,oOo

o.oub o.onb o . oo4b

221.0 221.0 78.0

20 gns rabbit lung was homogenized in 20 mM Tris-HCl containing 1 mM The homogenate was centrifuged as described in "Materials EDTA, pH 7.5. and Methods." The supernatant was analyzed for phosphodiesterase activity in the presence O.lmM Ca2+ or O.lmM Ca2+ with 60 units of exogenous bovine brain calmodulin, when in the absence of Ca2+ O.lmM EGTA was used. For the assay of calmodulin, an aliquot from the supernatant was boiled for two minutes, centrifuged to remove the precipitate and then dialyzed prior to use. "One unit of the calmodulin is defined as the amount which give rise to 5% of the maximal enzyme activation of the standard phosphodiesterase from bovine brain. bOne unit of phosphodiesterase is defined as the amount of enzyme which, when fully activated, hydrolyzes 1 p mole CAMP per minute at 30°. teins

in buffers

containing

low

concentration

in the medium,

metal

thus

(see

complex

associates

containing Consequently,

the is

enzyme and the

in the

(NH4)2S04

pellet

column

in a buffer

terase

activity

be activated

common type fraction containing could

or by gel

calmodulin

enzyme

are

is no longer

in Ca2+

protein-

in enzyme activation of this

filtration separated

enzyme in buffers

from

activatable

is

each other.

2+ unless by Ca

assay. from

of Ca2+ -activatable of lung 0.1

be resolved

by Ca2+ without

and the

preparation

CAMP phosphodiesterase

The Ca2+ -activatable from the

column

Upon increase

calmodulin

enzyme resulting

When a crude

fractionated

added

of Ca2+.

to the

the

on a DEAE-cellulose

EGTA, the

calmodulin

binds

with

scheme I in Introduction).

chromatographed

ferent

Ca

concentrations 2+

extract

the addition

340

lung

phosphodiesterase.

was chromatographed

mM EGTA, (Fig.

rabbit

three

IA).

All

peaks three

of calmodulin.

appears

dif-

When an on DEN%cellulose

of CAMP phosphodiespeaks

of activity

When the

calmodulin

could

BIOCHEMICAL

Vol. 91, No. 1, 1979

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0.6

0.4 -2 Q 5 0.2 z 5 5 4: 2 zi 5 0.4

0.2

20

40

60

80

FRACTION

Figure

was monitored

fractions Since

was pooled taining

still

in the

containing peak

eluents,

140

160

NUMBER

I phosphodiesterase

Fig.

activatable

by Ca2+,

was found

about

to be distributed

most of the enzyme activity,

on a DE&E-cellulose the enzyme was eluted 0.15

addition

in all

activity.

contained

1B shows that

concentrations,

it

phosphodiesterase

and rechromatographed

EGTA.

same salt

120

Elution profile of phosphodiesterase from DEAE-cellulose column. A) Supernatant was applied on a DEAE-cellulose column (2.8 x 40 cm) previously equilibrated with buffer A. The column was washed with two column volumes of buffer A containing 0.05 M NaCl followed by a linear salt gradient (x-x) from 0.05 to 0.35 M in the same Fractions were analyzed for phosphodiesterase activity in buffer. the presence of 0.1 mM Ca*+ with 60 units of bovine brain calmodulin (o-o) or without calmodulin (O-O) and with 0.1 mM EGTA (w). For the assay of the calmodulin, an aliquot of the fraction was diluted 100 fold and incubated in a boiling water bath for 2 min, then 30 ~1 of the boiled supernatant was used to activate the calmodulin-deficient phosphodiesterase (0.015 units) from bovine brain B) Peak I, from the first DEAE-cellulose, was dialyzed C-1. against buffer A overnight and rechromatographed on a DEAE-cellulose column under the same conditions.

1.

activity the

100

M NaCl. of the

341

As the calmodulin

column

in a buffer

from the chromatographed to the assay

it con-

column

at the

enzyme was mixture

did

Vol. 91, No. 1, 1979

BIOCHEMICAL

20

40

AND BIOPHYSICAL

60

80

FRACTION

Figure

2.

in changes

enzyme activity

modulin

activity.

nucleotide it

activity

depend

it

is

pooled

and assayed,

These

results

show that

the Ca2'

cellulose

in the

possible

To further

also

were

column

that

from

When the they rabbit

for

enzyme preparation enzyme

found

lung

Ca 2+ -activatable

activity. even

contains

containing

were

the common type

calmodulin

the

fractions

to contain

of the Since

cal-

cyclic

enzyme the

the

in that

calmodulin

in the rechromatographed

tightly

bound

calmodulin

which

effect. test

this

suggestion,

was concentrated

2 shows that, failed

differs

on the added

was found

mediates

Figure

NUMBER

Ca2+ -activation.

in the

phosphodiesterase

does not

sample,

120

Gel filtration of phosphodiesterase on Sephadex G-200. Fractions from the second DEAE-cellulose column containing the enzyme and calmodulin were collected (75 ml pooled fraction was concentrated to 4.0 ml by diaflo ultrafiltration using an Amicon PM 10 membrane). The concentrated sample was applied to a Sephadex G-200 column (2.5 x 80 cm) which bad been equilibrated and eluted with buffer A containing O.lM NaCl and lO$ sucrose. Phosphodiesterase activity in the presence of 0.1 mM Ca2+ with 60 units of calmodulin from bovine brain (o-o) or without calmodulin (O-O), phosphodiesterase activity in the presence of 0.1 x&4 EGTA (o-o) and calmodulin activity I-), other conditions were the same as in Fig. 1.

not result the

100

RESEARCH COMMUNICATIONS

to render

like

the

the cyclic

the

pooled

and applied previous

column

nucleotide

342

fraction

from

to a Sephadex chromatograph.es, phosphodiesterase

the

G-200

second

DEAE-

column. gel

filtration

Ca2+ insensitive.

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

The enzyme was eluted in a single peak and the enzyme activity Ca2+ to the sameextent either with or without bovine brain calmodulin.

The result

of the purified

was found to elute in two

closely with the enzyme activity

other was eluted at a position modulin.

the additions

The calmodulin activity

peaks, one of them correlated

was enhanced by

identical

whereas the

to that of the free bovine brain cal-

supports the notion that the Ca2+-activatable

further

phosphodiesterase from rabbit

lung binds the calmodulin tightly

irrespective

of the presence of Ca2+. The reason for the presence of free calmodulin in the enzyme preparation

(Fig. 2) is not known. However, since only about 45%

of the enzyme activity

of the second DEAE-cellulose column pooled fraction

recovered from the gel filtration enzyme was inactivated. dissociated

column, it suggests that about 55%of the

Consequently, the free calmodulin might have been

from the inactivated

enzyme.

2+ In conclusion, the present study suggests that the Ca -activatable nucleotide

phosphodiesterase in rabbit

in that it contains tightly dissociated

was

lung is different

bound calmodulin.

cyclic

from the commontype

This bound calmodulin is not

from the enzyme even in very low concentrations

2+ of Ca .

Thus, it

appears that the mechanismof action of the calmodulin described in schemeI should not be considered as generally

applicable to all

the enzyme systems.

A second mechanismis proposed as follows (scheme II):

Ca2+ + E -CM- -E-CM-Ca

2+ep

- *QJ _ (&2+

SchemeII where E and CM stand for the enzyme and the calmodulin respectively asterisk

indicates

the activated

and the

state of the protein species.

ACKNOWLEDGEMF3TS The investigation was supported by grants from Medical Research Council of Canada~1-2381 (to Dr. J.H. Wang, Department of Biochemistry) and 1x152 (to Dr. W.M. Thurlbeck, Department of Pathology). The authors are grateful to Dr. J.H. Wang for his suggestions and helpful discussion regarding this paper.

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REFERENCES 1. 2.

Kakiuchi, S. and Yamazaki, R. (1970) Proc. Jap. Acad. 46, 387-392. Wells, J.N., Baird, C.E., Wu, Y.J. and Hardman, J.G. (1975) Biochim. Biophys. Acta 384, 430-442. Teo, T.S. and Wang, J.H. (1973) J. Biol. Chem. 248, 5950-5955. ?: Kakiuchi, S., Yamazaki, R., Teshima, Y. and Uenishi, K. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3526-3530. Cheung, W.Y. (1970) Biochem. Biophys. Res. Commun.38, 533-538. 2: Hidaka, H., Yamaki, T. and Yamabe, H. (1978) Arch. Biochem. Biophys. 187, 315-321. Dedman,J.R., MacDougall, E. and Means, A.R. (1978) Fed. Proc. 37, 1302. 87: Ho, H.C., Teo, T.S., Desai, R. and Wang, J.H. (1976) Biochim. Biophys. Acta 429, 461-473. 9. Ho, H.C., Wirch, E., Stevens, F.C. and Wang, J.H. (1977) J. Biol. Chem. 10. 11. l.2.

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252, 43-59.

Teo, T.S., Wang, T.H. and Wang, J.H. (1973) J. Biol. Chem.248, 588-595. Bergstrand, H. and Lundquist, B. (1976) Biochemistry 15, 1727-1735. Davis, C.W. and Kuo, J.F. (1977) J. Biol. Chem.252, 4078-4084. Bergstrand, H., Lundquist, B. and Schurmann, A. (1978) J. Biol. Chem. 253, 1881-1891.

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