Direct evidence for co-localization of adenylate cyclase, dopamine-β-hydroxylase and cytochrome b562 to bovine chromaffin granule membranes

Vol. 79, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS

DIRECT EVIDENCE FOR CO.-LOCALIZATION OF ADENYLATE CYCLASE, WPAMINE-S-HYDROXYLASE AND CYTOCHROME bSS2 TO BOVINE CHROMAFFIN GRANULE MEMBRANES Oren Zinder,l

Raymond Menard,' Walter and Harvey B. Pollard4

'Reproductive Research Branch, National Development; present address, Rambam 2Laboratory of Chemical Pharmacology, Bethesda, Maryland 20014 3Section on Biochemical Pharmacology, Bethesda, Maryland 20014 4Clinical Hematology Branch, National Digestive Diseases, Bethesda, Maryland

Received

October

Lovenberg,3

Institute of Child Health and Human Hospital, Haifa, Israel National Heart, Lung, and Blood Institute, National

Heart,

Institute 20014

Lung,

and Blood

of Arthritis,

Institute,

Metabolism,

and

3,1977

ABSTRACT : Adenylate cyclase, dopamine-B-hydroxylase and cytochrome b562 have been found to co-equilibrate on equilibrium sucrose gradients of lysed chromaffin granule membranes from bovine adrenal medulla. Peak activities for these enzymes, as well as maximum membrane protein concentration, were found to coincide at d = 1.11 gm cm-3, and the ratios of adenylate cyclase to both dopamine-E-hydroxylase and cytochrome b562 were constant across the entire peak. Adenylate cyclase activity has been reported previously to co-purify with chromaffin granule membranes, and we conclude, on the basis of these new data, that adenylate cyclase is also an intrinsic granule membrane enzyme. INTRODUCTION: isolated

Adenylate

bovine

chromaffin

Catecholamines granules

suppress

with

Wilson adenylate

results

in association this

Copyright All rights

issue

under

can be prepared

NH4Cl in order

cyclase This

and purified

adenylate

only

and Krishner

membranes. recent

granule

has been reported

(4)

activity

conditions

of others

in association

with

pituitary

unambiguously,

0 1977 by Academic of reproduction in any

also

Press,

able

Inc. reserved.

lysed

induce

activity

with

release,

NH4C1 (1,2). but

also

must

(3). inability

to detect

granules

or granule

own findings

to detect

in intact

catecholamine

activity their

of

(l-3).

of granules,

chromaffin

to our

neurosecretory we subjected

form

reported with

contrast

who were

lysis cyclase

have recently

membranes

or treatment

by hypotonic full

in preparations

and cyclase that

(l),

to detect

was in apparent

granule

cyclase,

to Mg2+ -ATP and chloride

membranes

be washed

activity

granules

can be activated

such as exposure Granule

cyclase

adenylate

(l-3)

and to

cyclase

activity

vesicle

membranes

(5).

granule

membranes

to equilibrium

To resolve

707 ISSN

0006-291

X

BIOCHEMICAL

Vol. 79, No. 3, 1977

centrifugation granule

on sucrose

membrane

cytochrome adenylate across

b562

marker

membrane

and analyzed

as adenylate

cyclase.

co-equilibrates

gradient,

and that

gm cm-3 .

fractions

for

conventional

such as dopamine-8-hydroxylase

activity

the entire

at d = 1.11

enzymes

as well

cyclase

gradients

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

We conclude

In this with

all

three

that

both

paper

we report

that

DBH and cytochrome

enzymes peak

adenylate

enzyme as are DBH and cytochrome

(DBH) and

cyclase

b562

in the same fraction

is

indeed

a granule

b562.

MATERIALS AND METHODS: Chromaffin Granules. Bovine adrenal glands were obtained at the slaughter house within 20 minutes of killing and transported to the laboratory on ice within two hours. The medullary tissue was dissected free of cortical contamination and highly purified, homogeneous chromaffin granules were prepared in osmotically intact form by differential centrifugation in 0.3 M sucrose as previously described (6,7). Chromaffin Granule Membranes. Granules were lysed by exposure to 20 volumes of 0.1 M NH4Cl containing 5 mM tris-HCl buffer, pH 7.4 and centrifuged at 100,000 xg for 30 minutes (2). The membrane pellet was resuspended in 0.3 M sucrose (30 mg protein in 5 ml) and layered on a continuous sucrose gradient (range: d = 1.04 - 1.17 gm cm-3) having a volume of 30 ml. The gradient was then placed in an SW 27 rotor and centrifuged at 100,000 xg for two hours. One ml fractions were then collected by needle puncture from below and subjected to enzyme and protein analysis. Chemical and Enzymatic Analysis. Protein was determined by the Bradford Coomassie Blue assay (8) using crystalline bovine serum albumin as the standard. Protein determined by this method is identical to that obtained using the Lowry method. Dopamine-8-hydroxylase (DBH; EC1.14.17.1) was assayed by the spectrophotometric method of Nagatsu and Udenfriend (9). Adenylate cyclase (EC 4.6.1) was assayed by the method of Salomon and Rodbell (10). Cytochrome b562 was determined from a reduced-minus-oxidized difference spectrum taken with the Aminco DW2 dual beam spectrophotometer using the absorbance difference between 416 and 429 run. A reduced-minus-oxidized scan revealed that cytochrome b562 was the only detectable cytochrome in the granule membrane preparation, and a reduced, carbon monoxide minus reduced spectrum verified the absence of either hemoglobin or cytochrome P450. The density of various fractions was determined from the index of refraction using an Abbe refractometer. RESULTS : equilibrium and with cytochrome adenylate fashion found

Lysed

chromaffin

sucrose coincident b562

gradient maxima

(Figure

cyclase,

1).

marker

to be activated

measured

(2,3)

with for

membranes

granule Granule

in the presence

enzymes

activity

of this

membrane membrane

(Figure 2-fold

to distribute

marker

enzymes were

to distribute Basal

on an

maximum at d = 1.11

fractions

1).

such as DBH and

also

assayed

in nearly cyclase

for

identical

activity

was

by 10m4 M GMP-PNP, as reported

in the

GTP analogue.

708

found

concentration

was found

approximately

and cyclase

were

a protein

and the activity

to the other

previous!y

granule

gradient

fractions

were

accordingly

gm cm-3 ,

BIOCHEMICAL

Vol. 79, No. 3, 1977

DISTRIBUTION

OF ADENYLATE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CYCLASE

ON LYSED

CHROMAFFIN

GRANULE

MEMBRANES

DEH 14

PROTEIN

0.3

2

4

6

8

10

12

14

16

FRACTION

Figure

The coincidence that

the most

be observation peak.

DISCUSSION: intrinsic is

subcellular relationship

test

of a constant

As shown

in Figure constant

These

22

24

26

0.1

k

” -

28

NUMBER

an unambiguous organelle may prove

directly

of the

bs62 and adenylate membrane

of granule across

of adenylate

enzyme.

markers

would

the activity

cyclase

to either

adenylate

cyclase

cytochrome

gradient.

demonstrate

membrane

example

granule

of enzyme activities

the entire

clearly

DBH, cytochrome

co-equilibration

2, the ratios

granule

not

for

was a specific

for

ratio

over

results

chromaffin

peaks

the cyclase

rigorous

bS62 or DBH were

This

of activity

suggested

However,

26

FJ B 8

Distribution of adenylate cyclase, dopamine-S-hydroxylase (DBH) and cytochrome b562 in equilibrium sucrose gradient of lysed chromaffin granule membranes. Units are as described on the vertical axes. Fractions are one ml each. Analytic details are described in Methods.

1.

cyclase

18

0.2

that

is an

enzyme as are DBH and cytochrome localization

associated

to have some generality

709

of adenylate with

plasma

in light

cyclase

membranes. of recent

bS62. to a This

reports

of

Vol. 79, No. 3, 1977

BIOCHEMICAL

ENZYME

ACTIVITY

4

AND BIOPHYSKAL

RATIOS

FOR GRANULE

6

10

8

FRACTION

Figure

2.

cyclase

12

RESEARCH COMMUNICATIONS

MEMBRANES

14

16

18

NUMBER

Ratio of adenylate cyclase to dopamine-B-hydroxylase (DBH) and cytochrome b562 in the equilibrium sucrose gradient of lysed chromaffin granule membranes. The data are taken directly from enzyme activity values shown in Figure 1.

being

associated

pituitary

gland

(5).

inability

to detect

with

neurosecretory

We offer

granule

no explanation

adenylate

cyclase

for

activity

membrane Wilson

preparations

from

and Kirshner's

in chromaffin

(4)

granules

or granule

membranes. The granule

cyclase

activity

of the

fraction

enriched

fraction

we have

in association Isolated

found

with

that

represents

membranes. adenylate

granules

cholesterol

properties

also

lo-15%

In separate cyclase

of the total is

studies

equilibrated

on this

membrane

have other

plasma

membrane-like

(13)

of chromaffin

as well

on their granules

710

external

with

as sialic surface.

may be related

a

latter

at d = 1.14

plasma

(11,12),

cyclase

associated

and other

content

receptors

only

Much of the remainder

5-nucleotidase

germ agglutinin

membrane-like

medulla.

in plasma

chromaffin

such as a high wheat

adrenal

activity

markers

gm cmm3 (11).

properties, acid

(11) These

and plasma-

to the close

BIOCHEMICAL

Vol. 79, No. 3, 1977

structural during

relationship

that

granules

this

paper.

agonist, rise

role

However,

secrete

insensitive

(3,14)

adenylate

during

purified

granule

granule

contained

wet weight

from

a minimum 15,

radius

as water

(85%).

we were

eventually

enjoy

is

hypotonic

the finding vesicles

for

Since

of a relatively

(LDV)

and is

contain

(19)

splenic

(radius

DBH molecules/vesicle.

711

protein

of 3 umoles/mg contain

= 37.5

is recently of 25 and

protein

x min,

12 DBH

DBH activity

the

represents

laboratory

is

% of

20% of the

activity

that

gm cmm3);

gm and 1 mg

that

as many as 24 total

who calculated nerve

x lo-l5

specific

per granule

data,

(35%);

to values

would

in

(1.12

similar

membranes

few DBH molecules

--et al. from

4.69

~50% of the granule

might

that

a chromaffin

as protein

DBH in this

a minimum

purified

possible

physical

Assuming

for

intrinsic

cyclase

that

each mg of membrane

activity

Assuming

each granule

of Klein,

thus weighed

then

is

density

dry weight

12.5% DBH and each granule

membrane.

shock,

The presence

and nine

(17,18,19). ratio

and it

to calculate

the hydrated

x min at 37°C (16),

at most,

molecules/granule

able

their

on adenylate

x 1012 granules.

The specific

DBH/protein

CAMP with

cyclase

(1,3).

studies

also

One granule

protein,

the membrane

event

% of granule

is membrane

a measured

granule

medulla

the granule

granule

protein

by others

as is

reaction,

a 20-fold

from adrenal

included

granule

reported

cyclase

in

a cholinergic

and exhibit

The relevant

4.08

protein

medulla

synthesize

release

directly

to carbachol,

(11,14),

also

addressed

of 24 DBH molecules.

represented

nrnoles/mg

exposed

to these

protein

24-31

was not

the adrenal

on this

(100 nm);

x 1012 granules.

rats

granules

granule

core

membranes

Plasma membrane

observation

membranes,

granule

that

the --in vitro

in Reference

cyclase

or acetylcholine

is based

As an incidental

average

known

AMP (14).

chromaffin

response

summarized

is

to carbachol

cyclase

the tissue

it

cyclic

Isolated

*

of the granule

catecholamines

in medullary

20.4

and plasma

exocytosis. The physiologic

is

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

lost

upon

DBH molecules. in agreement

smaller

nm) contained

large

with

dense

between

one

Vol. 79, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLELXMENT

The authors

wish

to thank

Mrs.

Eleanor

Bruckwick

for

expert

technical

assistance.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Hoffman, P.G., Zinder, O., Nikodijevik,.O., and Pollard, H.B., J. Supramol. Struct. 4, 181-184 (1976). Zinder, O., Nikodijevik, O., Hoffman, P.G., and Pollard, H.B., J. Biol. Chem. 251, 2179-2181 (1976). Nikodijevik, O., Nikodijevik, B., Zinder, O., Yu, M-Y.W., Guroff, G., and Pollard, H.B., Proc. Nat. Acad. Sci., USAG, 771-774 (1976). Wilson, S.P., and Kirshner, N., Molec. Pharm. 13, 382-385 (1976). Bonne, D., Nicolas, P., Lauber, M., Camier, M., Tixier-Vidal, A., and Cohen, P., Eur. J. Bioch. in press (1977). Hoffman, P.G., Zinder, O., Bonner, W.M., and Pollard, H.B., Arch. of Bioch. and Biophys. 176, 375-388 (1976). Pollard, H.B., Zinder, O., Hoffman, P.G., and Nikodijevik, O., J. Biol. Chem. 251, 4544-4550 (1976). Bradford, M.M., Anal. Bioch. 2, 248-254 (1976). Nagatsu, T., and Udenfriend, S., Clin. Chem. 18, 980-983 (1972). Salomon, Y., Londos, C., and Rodbell, M., Anal. Bioch. 58, 541-548 (1974). Zinder, O., Hoffman, P.G., Bonner, W.M., and Pollard, H.B., Cell and Tissue Res. in press (1977). Smith, A.D., in The Interaction of Drugs and Subcellular Components in Animal Cells (ed. P.N. Campbell), Churchill, London, 1968, pp. 239-294. Meyer, D.I., and Burger, M.M., Bioph. Bioch. Acta. 443, 428-436 (1976). Guidotti, A., and Costa, E., J. Pharm. Exptl. Ther. 189, 665-675 (1974). Pollard, H.B., Zinder, O., and Hoffman, P.G., in Data Book on Cell Biology (eds. P.L. Altman and D.D. Katz), Fed. Am. Sot. Exp. Biol., Bethesda, 1976, pp. 359-362. Walker, G.A., Kon, H., and Lovenberg, W., Bioch. Biophys. Acta. 482, 309-322 (1977). S., Biochem. Biophys. Res. Comm. Rush, R.A., Kindler, S.H., and Udenfriend, 61, 38-44 (1974). cones, T., Skotland, T., and Flatmark, T., Eur. J. Bioch. 1, 525-533 (1976). Klein, R.L., Kirksey, D.F., Rush, R.A., and Goldstein, M., J. Neurochem. 8, 81-86 (1977).

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