Some properties of a glycoprotein isolated from adrenal cortex mitochondria

BIOCHEMICAL

Vol. 49, No. 6, 1972

SOME

PROPERTIES

OF A GLYCOPROTEIN

ADRENAL Tokuji

Kimura,

of Chemistry,

and Department Bunkyo-ku,

Kazuo

ISOLATED

FROM

MITOCHONDRlA

Mukai,

Wayne

State

of Biochemistry, Tokyo,

Received

CORTEX

Jau- Wen Chu,

Department

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Ineo

Ishizuka,

University,

Tokyo

and Tamio

Detroit,

University

Yamakawa

Michigan

School

48202,

of Medicine,

Japan

November

6,1972 Summary

A new glycoprotein has been isolated from bovine adrenal cortex mitochondria. F8;protein contains lucose yp), protein (20%), and an unidentified substance o , and binds CaZBand Mn ions with dissociation constants of 1 mM and 2.5 rng, respectively. The molecular weight was estimated to be aboutlb, 000. Introduction The

presence

by a number

of workers.

a glycoprotein

with

phospholipids.

bind

Ca2+,

Lehninger’s weight

and seem

translocation

of Ca2+

who

protein from

(or those

acid),

that

-peptide)

from

physiologically

adrenal

reported

hydrolysis

chromatography with

acetate:pyridine:acetic

The

postulated pigment,

to report

in liver

ruthenium

the presence

mitochondria,

(6), red,

which

of a new differs

glycomarkedly

by others.

was

and Methods

performed

= 6:3:1,

on Whatman

v/v).

out by the use of a Hewlett-Packard

Model

flame

with

detectors

involvement by Moore

mitochondria.

No.

0. 8 -M HC1 at 100° for 6 hours, using solvent acid:water = 5:5:1:3, v/v) and solvent

(propan-l-ol:ammonia:water ionization

been

of respiration cortex

glycoprotein

8% carbohydrates

membranes.

polysaccharide-staining we wish

and 30%

These glycoproteins respiration-dependent

with

has also

Materials Paper

than

of

5% carbohydrates

acid),

an insoluble

more

and 27% phospholipids.

stimulation

communication, previously

It contains

5) described

containing

reported

the isolation

and sialic

the mitochondrial

a specific

has been

(1, 2, 3) reported

of 42, 000.

(4,

to be involved

the Ca2+-induced

mitochondria

galactosamine,

in Ca2+-translocation

reported In this

weight group

across

liver

et al.

of 67, 000,

and sialic

of glycoprotein(s) inhibits

Sottocasa

glucosamine,

a molecular

(hexosamines,

in rat

a molecular

xylose,

(glucose, with

of glycoprotein

equipped

Copyright 0 1972 by Academic Press, Inc. AU rights of reproduction in any form reserved.

1 filter

paper

system

A (ethyl

system

B

Gas chromatography 402 gas chromatograph

glass

1678

columns

(6 ft x l/8

was with

after

carried dual

in) which

are

BIOCHEMICAL

Vol. 49, No. 6, 1972

packed

with

The

aliquot,

for

6 hours,

(7),

was

4% (w/w) either

after

or after

analyzed

to 250’.

or UCW-98-coated acid-hydrolysis

signals used

were

(Model

measured

in this

study

by the use of a Varian

were

tallized

when

Bureau

of Standards,

was Electron

124).

obtained

from

Lyxose

necessary.

Washington,

E-4

a kind

0. 8 g HCl

at 100°

programmed carried

Chemicals

spectrometer. of Dr.

140’

resonance

sources,

gift

from

out by the use of a

paramagnetic

commercial

was

(DMCS).

and trimethylsilylation

or temperatures

spectrophotometry

spectrophotometer

WAW with

re-N-acetylation, at 155’,

absorption

Chromosorb of the sample

methanolysis,

isothermally

Optical

Hitachi

OV-1

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and were

H. S. Isbell

recrysof National

D. C. Results

Glycoprotein method

Preparation:

described

suspended

in water,

minutes.

The

supernatant

passed

a Sephadex

through after

eluting

fraction

-M Tris buffer.

was

buffer,

the yellow

Sephadex removed

completely in Figure

found

column.

The

containing

Weight

30

sulfate

35% and 60% saturation slightly

yellowish

adrenodoxin

was

fractions

reductase.

column

were

The

equilibrated

with

0.01

0. 17 -M KCl, and washed well with the same was eluted with 0. 01 -M Tris buffer, pH 7.4, The fractions collected were repeatedly passed through until a single peak was obtained. Adrenodoxin could be material

procedure.

The

Determination:

profile

of the final

The molecular

16, 000,

using

proteins

Chemical

standard to a form

Properties:

gel-filtration

weight

as determined for

with

in the case

glucose

It was

of the hydrolyzate

In the case

is

of the glycoprotein

by polyacrylamide

calibration.

a molecular

of basic that

the

system

solvent major

revealed

and color

of solvent

(Rf = 0. 56) with

suggests

The

weight

A the major

B, the major

a minor

after

gel-

protein

of about

had a 45, 000 upon

spot

component

or hexuronic with

paper-chromatographic

development spot

found is

with

less

distinct

spots

were

acidic.

detected.

1679

silver

similarly

(Rf = 0. 67) migrated

to be identical This

by spraying alkaline silver nitrate, Furthermore, it was not identical On the chromatogram, one major acids.

three

alkaline

spot migrated

could be detected ninhydrin reagent. along

x g for

by ammonium

between

by the

at -ZOO.

separation that

at 49,000

to a DEAE-cellulose

by this

to polymerize

storage

fractionated

to be approximately

electrophoresis tendency

was

prepared

of the mitochondria

1.

Molecular was

centrifuged

the fractions applied

0. 30 -M KCl. G-100 column

shown

was

precipitating G-100

were

ultrasonication

pH 7. 4, containing

Then,

containing

fraction

mitochondria

After

solution

The

collected

(8).

the suspension

precipitation.

yellow

Adrenal

elsewhere

with major

nitrate to glucose.

faster

glucose.

than This

substance

but not by with any pentoses, ninhydrin-positive

hexoses, spot

a

6IOCHEMICAt

Vol. 49, No. &I972

AND BlOPHYSlCAt

RESEARCH COMAWNICATIONS

0.3 0.2 0. I : f e

0

Z-O.1 ! - 0.2

0

IO

20 FRACTION

Fig. Figure

The equilibrated

elntion with

2.

400

450

1.

500 WAVELEILGTH

Fig.

Profile of Sephadex Glycoprotein

1.

Figure

u 3

30 NIMBER

G-100

solvent was 0.01 the same buffer.

Ca2+ -Binding

Gel-Filtration

of Adrenal

600

2.

of Purified

M Tris buffer, ?%e transmittance

550 (nm)

Adrenal

The column was recorded at 260 nm.

pH 7.4. was

Glycoprotein

The sample and reference cuvettes contained: 0.10 ml of 1 m&4 murexide, 2. 50 ml of 0. 01 h4 Tris buffer, pH 7.4. Curve 1 represents the base line. To the sample cuvette, 0.40 ml of 0.02 M CaC12 was added and to the reference cuvetfe, 0.40 ml of H20 was added: Curve 2 represents the difference spectrum. Then, to the sample cuvette, 2, 4, 6 and 8pg of the adrenal glycoprotein flOOpg/ml) were added and the corresponding volumes of the buffer were added to the reference cuvette (curves 3, 4, 5, and 6, respectively).

Trimethylsilyl the major

derivatives

of the hydrolysate

spot on the paper-chromatogram The major technique.

gas-chromatographic 5.7 minute8

with

methanolyzate not identical The Table

with chemical

column

a peak

any known analyses

a retention

carbohydrates

time

similar

including

of the adrenal

from

using solvent A were examined by a component had a retension time of The trimethylsilyl

at 155O.

with

eluted

derivatives

of the

to pentoses.

It was

lyxose.

glycoprotein

are

summarized

in

I. The

broad

a OV-1

showed

or the substance

optical

maximum

Addition

of Ca2+

By difference found to bind isosbestic

absorption at 250 nm. or reduction

spectrophotometry ruthenium red,

points

spectrum There

of the glycoprotein was

no maximum

by borohydride

produced

utilizing tandem inducing a decrease

of 475 and 568 nm.

exhibited in the visible no change

a single region. in the spectrum.

cuvettes, the glycoprotein was in absorbance at 521 nm and

BOCHEMICAL

Vol. 49, No. 6,1972

AND BKXHYSICAL

RESEARCH COMMUNICATIONS

zsoo3ooozoo340036003800 Figure

3.

Electron Paramagnetic Mn2+ Complex 1 m&l

Ca2+-Binding:

The

Ca2’

ted spectrophotometrically spectrum

of free

was

cafcnfated

the protein resonanee

signals

be reversed Heat binding

SimilarIy, 2.5 rng

an extensive of free Mn 2t .

(9). from The

Mn2*

the spectrum apparent

buffer

was demonstrain Figure 2, the 2t of Ca -murexide constant

to the glycoprotein,

in Figure

and

3, Mn2+-binding

of the six electron

broadening of Mnzi-signals by the addition of excess produced

-

in the same

dissociation

was bound

As shown

of the glycoprotein

mg)

As shown

line-broadening This signals

Glycoprotein

of the glycoprotein

murexide

can be restored

to the unbroadened

denaturation

of AdrenaI

ability

of the glycoprotein.

to be I mM-constant was

produces

-binding

by using

mnrexide

by additions

the dissociation

Resonance

MnCl2 in 0.1 M Tris buffer, pH 7.4 MnC12 and adrenal glycoprotein (0.25 of 30 rng CaCQ to (b) were measured at room temperature.

labi: 1 rnE (4 : Addxon The spectra

complex

-SOUSS

considerable

could amounts

loss

to

paramagnetic partially of Ca2+.

of the kfn”-

capacity. DiscuBsion This

differs

from

the adrenal

glycoprotein Iiver protein

sialic

acid

which

is probably

isolated

mitoehondrial lacks

as carbohydrate

from

adrenal

gfycoproteins

pentoses,

hexoses

components,

cortex

mitochondria

described (except

gkeose),

but it contains

a carbohydrate.

obviously

by others.

In particular.

hexosamines,

an unidentified

Perhaps this unknown substauce with 2+ character is responsible for the Ca and Mn2* binding. The involvement of this adrena glyco.protein in the active transport by mitochondria is questionable, since the dissociation constant is fairly

1681

and

substance, acidic of Ca2+ high

Vol. 49, No. 6, 1972

BIOCHEMICAL

AND BlOPHYSlCAL

TABLE Chemical

Analyses

I

of Adrenal

Constiuents

RESEARCH COMMUNKATIONS

Glycoprotein

Methods

protein

biuret

Percentage

color

20%

glucose

2

unidentified

substance

78

nitrogen

b)

hexosamines

a)

not detectable

a)

not detectable

a)

not detectable

a)

not detectablec)

a)

not detectable

sialic

acid

muramic

acid

pentoses hexoses

other

gas-chromatographic determination. In the case of the unknown substance, the percentage was calculated from the area of the peak in comparison with standard glucose.

b)

elementary

c)

arabinose, xylose, 6-deoxyrhamnose, absent.

analysis

were

and low

affinity

ribose, ribitol,

corrected

The glycoproteins

(1 mg). 1 ,-pM

glucose

a)

The values

high

than

2-deoxyribose, 4-anhydroribitol

for

obtained

sites

for

Ca2+

with

it is possible

the adrenal

is maximally

identification protein are

We are for

providing

and Mr. supported

B.

grateful

to Dr.

us the crude Matson

for

by a Research

Grant

of Cazt

glycoprotein

by adrenal

may

play

had

constants

of

glycoprotein

partici-

mitochondria.

a role

by the presence substance

of 11 mMand the physiological

Acknowledgement J. J. Huang. Mr.

glycoprotein

their

dissociation the adrenal

groups

in determining

This idea may be supported by the fact that hydroxylation reaction utilizing extramitochondrial

stimulated

of the unknown under investigation.

that

all

in the sample.

and Lehninger’s

respective

Yet,

the mitochondrial structure. adrenal mitochondrial steroid

of ash present

by Sottocasa’s

pates

that

6-deoxyfucose, and lyxose were

the amount

and 1 mM. It is, therefore, unlikely in the energy-dependent translocation

NADPH

4. 3

help

samples.

in preparing

from

L.

the National

lt382

Kazim, We thank

this

manuscript. Institutes

CaZt

(10).

role

of the glyco-

and Mr. also

The

Mr. This

of Health

H.

P. Wang

D.

Pfeiffer

study

was

(AM-12713).

Vol. 49, No. 6, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1.

DeBernard, and Vittur,

B., F.,

2.

Sottocasa, Letters,

G. L., Sandri, 17: 100 (1971)

3.

Sottocasa, Biophys.

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4.

Lehninger,

A.

5.

Gomez-Puyou, A Gomez-Puyou, A. L., Biochem.’ Biophys. Res.

6.

Moore,

7.

Clamp, edited

8.

Kimura,

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Mela,

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Peron, (1967)

C. L.,

Pugliarelli, FEBS Letters,

Sandri, Commun.

T., L., F.,

G.,

Biochem.

and Galdwell,

Panfili,

M. T., Commun., Res.

B.,

FEBS

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42: 312 (1971)

Becker, G., and Lehninger, 47: 814 (1972) 42: 298 (1971)

R. E., in “Glycoproteins”, Amsterdam, p. 300. Chem.,

Biochim.

1683

B., B.,

Commun.,

Commun.,

Biochemistry, B. V.,

G. L.,

and DeBernard,

Res.

J. Biol.

Sottocasa,

and DeBernard,

T., and Chambers, (1972), Elsevier, K.,

G.,

E.,

Biophys.

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and Suzuki,

and Chance,

C., Sandri, 12:125 (1971)

G., Panfili, E., , 47: 808 (1972)

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J. R., Bhatti, by A. Gottschalk

M.

242:

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Part

A,