BIOLOGY

OF

REPRODUCTION

16,

(1977)

377-384

Characterization

of Sea Urchin DAVID

Departments

of

Vanderbilt

Sperm

Adenylate

Cyclas&

L. GARBERS2

Pharmacology

University

and School

Nashville,

Physiology,

of

Tennessee

Medicine,

37232

ABSTRACT

(Lytechinus pictus) sperm adenylate cyclase was inhibited by the methylxanthines, 1-methyl-3-isobutylxanthine (MIX), caffeine and theophylline. MIX was the most potent inhibitor (I, = 1.4mM) while caffeine and theophyiline were about equal in potency (I, = 15 mM). A more detailed kinetic analysis with theophylline indicated that it was a linear competitive inhibitor Sea urchin

with

respect

to

MnATP. but not

30#{176}, and Mn2+, plots were linear these

data

which

also

both that

The Mg2

sperm or

as a function were

adenylate cyclase accelerated

of either

linear.

Thus,

free

the

Mn2+

was inactivated by temperatures this rate of denaturation. Double

or MnATP,

and

slope

and

sperm

adenylate cyclase conforms a Mn2+ regulatory site exist.

intercept an

enzyme

as

replots

of

model in these data

substrate site and Furthermore, is not a potent inhibitor of the enzyme. When the concentration of total was less than the concentration of total AT?, Ca2+ and Mg2+ were capable of activating the enzyme, but when the concentration of total Mn2+ exceeded that of ATP, both Ca2+ and Mg2+ acted as inhibitors. Various phosphorylated nudeosides (GDP, AD?, GTP, dGTP) inhibited the suggest

a MnATP free AT?

to

as low reciprocal

enzyme, pymvate,

but adenosine and guanosine were without effect. A number of carbohydrates (acetate, glucose), fatty acids, and fluoride also had little, if any, effect. Two polyamines, cadaverine (2mM) and putrescine (2 mM) caused a slight stimulation (‘\30 percent) of enzyme activity. Under a variety of conditions, solutions containing factors released from sea urchin eggs either had no effect or inhibited the sperm adenylate cyclase.

Hoskins, well-known many types

INTRODUCTION

Although adenylate 1971;

spermatozoa cyclase (Casillas

Gray,

1971;

1975;

Garbers

1976;

Towns

and

et

Tash,

1976; in the

slightly

regulation

increase

AMP

in

1970),

but if any,

little, hormones

monkey

on

al.,

the

1975a;

1976)

studies effects

sperm

adenylate

of

al.,

in spermatozoa plished by the phosphodiesterase

phosphodiWells and mechanisms

1971).

AMP

sea

accumulation

various direct

et

metab-

and

Hoskins,

1

obtained

mm

after

1976); this any substance,

However,

despite

sperm

cyclic

addition

had been on sperm AMP

accom-

or other et al., from

to increase sperm much as 100-fold

1975b, that

methylxanthines, marked effects

(Casillas

Accepted November 4, 1976. Received September 28. 1976. ‘This study was supported by NIH grants HD and HD 10254. 2lnvestigator for the Howard Hughes Medical tute.

substance(s)

eggs were shown AMP levels by as

Hardman, first time

since have shown a wide variety of cyclases

Recently,

within

cyclic

generally has been of methylxanthines inhibitors (Garbers

use

urchin

cyclic

Thyroxshown of

a in also

has been shown not to stimulate sperm adenylate cyclases (Casillas and Hoskins, 1971; Gray, 1971). Thus, the elevation of cyclic AMP levels

1973;

adenosine

remain unclear. were initially (Casillas

1970,

Gray

and

1976) the of cyclic

sperm

contain

Albagli,

(cyclic AMP) (Gray et al., 1976;

olism in the sperm cell ine and triiodothyronine to

and

Luck,

3’,S’-monophosphate esterase activity Garbers, involved

are known to and Hoskins,

Morton

Braun,

1971; Gray, 1971). Fluoride, activator of adenylate cyclase of broken cell preparations,

and

(Garbers

and

represented including

the the

shown cyclic

these

marked

levels,

solutions

to have such AMP levels. effects

on

containing

the egg factor(s) either had no effect or inhibited the adenylate cyclase of broken sperm cell preparations (Garbers and Hardman, 1976). These apparently inconsistent results could be

05797

due, about

Insti-

sea

377

at

least the

urchin

in part, properties or

other

to our of sperm.

lack

adenylate

of information cyclase

from

GARBERS

378 MATERIALS

AND

METHODS

creatine

L. Pictus sea urchins were purchased from Pacific Bio-Marine, Venice, CA. The unlabeled nucleotides and fatty acids were obtained from Sigma or Boehringer and the tritiated [311-2]-ATP (25 Ci/nmole) from Schwartz/Mann. The metal chlorides were from Fisher, 1-methyl-3-isobutylxanthine from Aldrich, and theophylline and caffeine from Merck. Preparation

ence tions

pellet was resuspended in the TEA-DTT buffer and centrifuged again at 36,000 X g for 1 h. The resulting pellet was diluted to 0.01 percent based on the original wet weight with the TEA-OTT buffer, and then frozen in 5-mI aliquoss at -70#{176}.The enzyme was stable at -70#{176} for at least 3 months, but was not stable to repeated freezing and thawin& For this reason, enzyme not used after one thawing of a 5-mI aliquot was

of

of 1.0 mM ATP, adenylate cyclase were linear for at least 10 mm. Thus,

reacboth

could

effects on adenylate NaN3 (20 mM) nor

direct

Neither

mM)-creatine initial

(data

not

enzyme centrations the

shown).

general adenylate cyclase assay mixture con40 mM TEA at pH 7.9, 1.0 mM cyclic AMP, 20 mM sodium azide (NaN3) 0.5 mM ATP, 6.0 mM MnCI3, 333 ig sperm particulate protein, and 1.01.5 X 10 dpm of l HI AT? in a final volume of 0.25 ml. In some instances, 10 mM creatine phosphate and 300 Mg/rnl of creatine kinase replaced the NaN3. Enzyme reactions were linear for at least 20 mm at 23#{176}under these conditions, and were linear as a function of added protein between 67 and 660 g. Reactions were stopped by the addition of 2 ml of 0.5 N perchloric acid, and the cyclic AMP was then purified on acid alumina and neutral Dowex-50 (l1-form) columns (Jakobs et al., 1976). Of the 6 ml collected from the Dowex-50 column, 1 ml was used The

tained

for

the

determination of the remaining cyclic AMP (absorbance at 259 nm) while counted for cyclic E3H] AMP in a liquid counter. Cyclic nucleotide recoveries after were generally about 65 percent. Prior to the selection of the above tions, a number of potential constituents adenylate cydase assay mixture were

determine directly presented

whether affected in the

or not enzyme Results.

any activity.

of

kinase

or

Two

of ATP systems

percent

tion under

remained standard

at

NaN3

and

creatine

to

represent

preservation

when

of

With

the

initial

the assay

the

end of conditions. useful

ATP assay

the

et

al.,

1971),

The

1976);

the

some

instances

inhibit adenylWeinryb and of

the

primarily sperm

in

at low

cyclase

sea

hydrolysis the

homogenate

thus

adenylate

in

for

exists

of

are inhibi-

phosphodiesterase

responsible

Garbers,

the

sperm

methylxanthines

also have been reported to cyclase (Sheppard, 1970;

fraction

urchin

phosphodiesterase

(Robison

AMP

for

Degradation

they ate

cyclic

kinase

in sea

AMP

various

1971).

incubation Thus both

mixtures.

to be effective

sperm

system,

concentra-

systems

levels

of Cyclic

urchin

either

phosphate-creatine

cyclase

Michel,

either

ATP

tors

of soluble

(Wells

protein

activity,

and

concentrawhich

is par-

>

06

TI-4EOPHYLLINE

CArFINE

assay

40 20

2

FIG.

cyclase

Levels to preserve

conditions

when using phosphate-creatine

below).

of

appear

adenylate

obtained creatine

(see

80

scintillation purification

condiof the studied to the added agents These data are

NaN3, a nucleoside and 2) creatine

altered

these

4

6

8

0

12

[IN4I8lr0R1

designed

were studied: 1) phatase inhibitor

pg/mI)

under

Furthermore,

were the

system

over

tions,

conditions

cyclase activity. creatine phosphate

unlabeled the rest was

RESULTS

Maintenance

these

(300

rate

in

kinetics were studied at MnATP conranging from 20 pM to 2.0 mM, the

results NaN3

known

under

kinase

reaction

Although Assay

tested

and

In

pres-

be

system

for

same

system.

the

Prevention

discarded.

ATP-regeneration

either

systems

the

Sea urchin sperm (Lytechinus pictus) were collected and washed as described previously (Garbers et al., 1975a). The washed cells were diluted to 0.01 percent (wet weight/volume) in a solution containing 25 mM triethanolamine (TEA) and 2 mM dithiothreitol (DTT) at pH 7.6, and homogenized with an Ultraturrax (10 sec intervals, 10 times) at 0-4#{176}. The suspension was centrifuged at 36,000 X g for 1 h, and the resulting supernatant fluid was discarded. The

an

absence

(10 Enzyme

kinase,

the

ATP

levels

triphosphosphate-

1.

by

Inhibition

of

methylxanthines. mixtures contained

sea

6

4

IS

20

Ir,,M)

urchin

sperm

Adenylate 0.4 mM AT?

adenylate

cyclase reaction and 4.0 mM total 4j2 and were otherwise as described in Methods, except that various concentrations of methylxanthines were added as indicated. The control enzyme activity in these experiments was 9.1 pmol cyclic AMP formed/mix/mg protein.

SPERM

ADENYLATE

CYCLASE

Under

379

the

same

or theophylline Theophylline was

to

respect

10.1

mM.

tus

2. Linear competitive inhibition adenylate cyclase with respect

of sea urchin to MnATP by

sperm theophylline. Adenylate cydase reaction mixtures contained 4.0 mM free Mn and were otherwise as described in the text except that theophylline and AT? were varied as shown. Reactions were for 10 mm. at 23#{176}.The inset is a replot of the slopes of the primary double reciprocal plot.

ticulate, both

can

AMP

“cold

cyclic

AMP

reaction

estimated

was 1.4

mM

all 1);

or

absence most an

the

absence

did

and

these not

the

shown).

and

1-methyl-3-isobutylxan-

of

ATP

1.0

mM

concentration

inhibitor

2)

with

a

Ki

of

(1976)

labile,

rates

were

However,

We

370

sea not

that sperm

that

linear

possible

at

23#{176}the

product

was

that

S-purpura-

reaction

found

urchin

and

at

linear

of time

reported

cyclase

rates the

was

for

reaction

either

30#{176}or

was

quite

accumulation

possible

extremely

enzyme

enzyme

as

at least

were

adenylate

37#{176}.

stable,

a function

20 mm.

of

initial

However,

inhibitor,

(Fig.

mM. and

a cyclic

not

potent

caffeine

15 detail

conditions,

alter

inhibited adenylate this occurred in

the at

Under

mM)

(data

(MIX) (Fig.

presence MIX

trap”.

caffeine

thine activity

in inhibitors

(1.0

rate

phylline,

of

be

phosphodiesterase

al.

from

heat

and

for

competitive

MnATP

adenylate at

cyclase

FIG.

be a linear to

et

sperm

non-linear

I/MnATP

150

Stability

Gray 0

the

was approximately was studied in more

shown

with

Thermal

conditions,

theo-

cyclase either the

cyclic

AMP.

with of

an 0.4

mM.

3

S

60 PREINCUBATION

g

FIG.

the

FIG. 3. The effects of Ca2 and Mg2 on sea urchin sperm adenylate cydase at various Mn2+ concentrations. Reaction mixtures contained 0.5 mM AT? and were as described in Methods except that or jg2 (6.25 mM) was added to some of the reactions as shown.

and the total All reactions

Mn2+ concentration were for 15 mm.

was

varied

at 23#{176}.

rate

adenylate protein) no added mM (2+

4.

The

TIME

(mm)

AT

30#{176}

effects

of Mn2, Mg2 and Ca2’ on denaturation of sea urchin sperm cyclase. Sea urchin sperm particles (5.3 mg were preincubated at 30#{176} in the presence of metal, 1.8 mM Mn2, 1.8 mM Mg2 or 1.8 in a final volume of 0.55 ml. At the times

of thermal

indicated in the Fig., 50 td aliquots were removed from the preincubation mixture and assayed for adenylate cyclase activity as described in Methods. The basal adenylate 0-2#{176}) was 20.2

protein.

cyclase

pmol

activity

cyclic

AMP

(preincubation

formed/mm/mg

at

380

GARBERS

Metal

Requirement

The

sea

highly detectable

Thermal

urchin

sperm

dependent (rates

on less

Mn2+.stimulated observed mM not

Mg2, shown).

putrescine absence or

activity ceeded (putrescine tions when

the

(2

mM)

Mn2.

Mn26 ATP

activity

0.5

mM

Co2’, activity

presence

of

or

cadaverine

The

addition

increased

when total the total

of

Ca2, Ba2, Enzyme

of

Mg2+

enzyme

presence

in

cyclase

apparent

ATP

The

No the

preincubation

6

polyamines, (2 mM)

of

either

enzyme

concentrations concentration,

increase

enzyme

< [ATP1.

addition

of

This

the

rapidly

Mn2,

enzyme form

and that

of

of

assuming

that

changes

the

is less

not

during

assay

(Fig.

Mg2

or

denaturation

effect

by

to

but

rate

destabilizing

explained

inactivated

30#{176} prior

stable

(Fig.

Mn2 Mn2

binds

higher

4).

could

be to

conformation

to

4).

Ca2,

the

to

a

temperatures.

Ca26 activity

were less than but depressed

apparent

was

at

in the

when total Mn2+ concentrations extotal ATP (Fig. 3). The polyamines and cadaverine) at 2 mM concentra-

did not [Mn2Jt

The

enzyme

accelerated

was and

or Ni2 (data also was not the

Denaturation

was

Mn26 for activity. than 0.5 percent of

rate)

in the

detectable

adenylate

activity

Kinetics

With kinase

either the creatine ATP-regeneration

shown)

or

of

NaN3,

MnATP

intersecting sperm MnATP

and

(Fig. adenylate catalytic

phosphate-creatine system (data

reciprocal free

5a,

plots

Mn2+

b).

site

were

This

cyclase

as

indicates contains

and

a

separate

not

a function linear

and

that

the

both

a

Mn26-

C

C V

LI.

a. 4 ‘I 5-

U I’

0

a. >.

6 U

0 -J

Lu >

20 I/MnATP

2.0

30 (rnMi1

I /Mn(mM)1

FIG. 5. Double reciprocal plots showing the effects of M.n2 and MnATP on sea urchin sperm adenylate cydase activity. Reactions were as described in the text except that [ATPltotai and [Mn2i free were varied as shown in the Fig. Analyses of the kinetic data were as described by Gathers et al. (1974) and Gathers and Johnson (1975). Replots of the slopes and intercepts of the primary double reciprocal plot are shown as insets. Although these data were obtained using NaN3 as an AT?ase inhibitor, the sante type of linear and intersecting double reciprocal plots were obtained when using the creatine phosphate-creatine kinase ATP-regeneration system.

SPERM

regulatory

site

Secondary

plots

(Garbers

of

and

slopes

were linear; thus free act as a potent enzyme Johnson,

1975).

fined

according

to

sperm

adenylate

cyclase

Johnson,

and

kinetic

not appear (Garbers parameters,

(1963),

Cleland were

CYCLASE

1975).

intercepts

ATP does inhibitor

The

ADENYLATE

for

381

theophylline

also

nucleosides

to and

known

facieus

de-

1965,

1967),

the

drates

did

(Table

1).

as follows:

(Table

1),

but

inhibited

the

enzyme.

activator

of

adenylate

not

=

MnATP

=

O.9SmM;KM

sea

3.6mM

=

mM;

and

KMn

8.9

=

elevate

sperm

the

sperm

of

Other

Agents

Adenosine ranging

or

from

appreciable in

affect

either

the

TABLE

1. The

presence

guanosine

0.02 the

to

sea

concentrations 2

urchin

or

presence

effect

at

mM

of various

mM

did

sperm

not

enzyme

absence

of

compounds

4

of

Ca2,

Mg2

only

observed

tion

sperm

to

The were

NAD,

and of

solutions

tested

adenylate

acids

cyclase

cyclic

(arachidonic,

AMP

form

ed/mm/mg

Theophylline

phylline

...

74±3

72

±

4

68

± ±

1 2

71

Cyclic

liMP

2.0

80

IMP TM? XMP dGMP

2.0 2.0 2.0 2.0

76±6 74±1 69±4 71 ± 5

S-Ado-Met GD? AD? GTP dGTP Cadaverine

2.0 1.0 1.0 1.0 1.0 0.2 2.0

96±3

Putrescine Acetate Pyruvate Glucose Triton X-100

2.0

92

2.0 2.0 2.0 2%

76 83 86 66

Fluoride

8.0

86

KH2PO4 FRE

2.0

79

...

...

aReactions

as

described

listed and of

82 79 68 80 83 64

... ... ... ...

...

68 76 59 64

43

...

54±3 52±2 55±2

56

57 51 28 78 96

... ...

2.0 20.0 were

protein No Theo-

GM?

various compounds with theophylline tus eggs.

arachi-

activitya.

4mM

+

Cyclic

Theophylline

the inhibi-

was

tration mM

0.02 0.20 2.0 0.02 0.2 2.0

Guanosine

the GTP,

shown).

of fatty

Pmol

None Adenosine

in

instances

factors

Concen-

Compound

and inhibit

of

in all

the

known

(Garbers

concentrations

effect

A number

on sea urchin

from

are

shown

cyclase.

various

not

levels

also

enzyme

released

were

factors

and

(data

mM

AMP

1976),

the

carbohythe

or all of which

adenylate

containing Effects

affect

cyclic

1975b,

Hayaishi,

other

factor(s)

some

a lique-

and

various

appreciably

eggs,

Hardman,

mM.

(Hirata as

containing

urchin

to 2.3

well

Pyruvate,

Brevibacterium

cyclase as

Solutions KMnATP

the

phosphorylated

±

4

94

±

±

2 5 2

81 73 84

±

5

72

±

5

88

±

2

...

±

35 78 41

... ...

in

Methods

above were 2 observations

added

([ATPI

total

to the reaction

without

theophylline.

=

0.4

mixtures. FRE

mM;

1Mn2I

Data represents

total

are means factors

4.0

mM)

except

of 4 observations released from

that

the

(± SEM) S. purpura-

382

GARB

dic, n-caproic, palmitoleic, myristic, and above

fatty

were

tested

activity; 10%

effects

effects

inhibition. to

on

(0.25

ml

sulfoxide, cyclase

which activity.

final

the

vol.)

acids

5

il

inhibit

to were

of

dimethyl

adenylate

obtained

cles

(see

bath

for

during

Methods)

10

were

in the

In

also

in

the

the

parti-

a boiling

tested

of

some

supernatant of

heated

and

effects

tightly or that

sperm.

first

preparation was

mm

No

fluid

were bound not eluted,

do not exist experiments,

fractions.

mixture

basal

or that activators column and were

activators additional fluid

effect

reaction

in

not

no

fatty

cyclase

did

cyclase

from

of

adenylate

also

adenylate

ranged All

the

the

at 24 j.zM concentrations

for the

added

filtration to the

palmitic, 11, 14-eicosadienoic, oleic, 1 1,14,17-eicosatrienoic, all of the methyl esters of acids)

ERS

on

water

the

heated

column

supernatant

observed.

DISCUSSION

Detergent-Dispersion The and

enzyme

applied

The

and was

to

peak

nation

of

(#7)

near other

resulted

6).

Thus,

separated

it from

Filtration

solubiized

BioGel

with

A0.5

detergent-dispersed

single

Gel

m

void

fractions

essentially appears the

that

WX

(Fig.

migrated

volume. with

Lubrol

columns

activity the

The

The

peak

the

in additive activators

adenylate

6).

as a recombi-

fraction

by

with the consistent a MnATP site exist

substrate (Garbers of

enzyme

gel

by

model.

a

ATP.

This

with

earlier

and

tion

by

the

model

brain 8

suggested

by

and

in

Hammes

The

FRACTION

(Lin

et

al.,

inhibi-

activator

site

agree

with

detergent-dispersed

with

and

the

Johnson,

model

(1976)

proposed

with

respect

to

site.

mechanisms

involved

in

cyclase factors

the

regulation

remain associated

unclear. with

eggs, although causing marked elevations in the sperm cyclic AMP levels (Garbers and Hardman, 1975b, 1976) appear only to inhibit adenylate

(9.1-mi)

FIG. 6. Gel filtration of the Lubrol WX-dispersed sea urchin sperm adenylate cyclase on a BioGel A0.5m column (2.6 cm X 34 cm). Sea urchin sperm particles were prepared as described in Methods, then resus-

in a solution containing 25 mM amine, 1mM dithiothreitol, and 1% Lubrol 7.6. The suspension was allowed to set on mm. and then centrifuged at 36,000 g for

cardiac

potent

(Garbers

Rodbell

free agree

adrenal

liver

metal

the

of the sperm adenylate Solutions containing

2

pended

for

general

and

the

essentially

cyclase

a metal-activator 4

or

no

does

not

1974),

where

and

but

by

for

1975)

cyclases,

adenylate

1975),

6

models

ATP

evidence

does

(deHaen,

the above

no

model

Rodbell,

free

the

(K1