A smaller molecular weight retinol binding protein in rat testis seminiferous tubules

Vol. 61, No. 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A SMALLER MOLECULAR WEIGHT RBTINOL BINDING PROTEIN IN RAT TESTIS SKMINIPBROUS TUBULES Kanwal

K. Gambhir

and Balwant

S. Abluwalia'

Departments of Medicine and Ob.-Gyn. Howard University Medical School Washington, D. C. 20059

Received

October

9, 1974 SUMMARY

In the cytosol fraction in rat testis seminiferous tubules a lower molecular weight protein of %4,800 daltons that binds retinol with high specificity has been isolated and purified by ammonium sulfate precipitation and on Sephadex column chromatography. The hexane extract of the component gave a characteristic retinol fluorescence spectrum. The amino acid composition was qualitatively similar to the retinol binding protein in the blood with the exception that cystine and cysteine were absent. It

is well

a specific

protein

such as the retinol

known

that

@BP)

testis

is

diffuses

into

retinol

is

(l-3).

not

clear.

Whether

the

tissue

and then

According

to Bashor

binding

protein

component

of %16,000

several

rat

isolated

multiple

have isolated a,800

daltons

testis

that

are

including

reported

retinol

the

in

retinol

with

high

another

(4)

with

a specific is

present

components weight

specificity.

tissue

the tissue protein tissue

in

or free needs

retinol

the cytosol

study in

a target

of

Ong and Chytil

the rabbit

lung.

protein

component

tubules

(S.T.)

The results

(5) We

of

of the rat of the

study

from were

Hoffman

purchased

Ta Roche, from

should

AND MBTBODS

-acetate

- retinyl-11,12[3H2]

' From whom reprints

Eastman

0 1974 by Academic Press, of reproduction in any form

Inc. reserved.

with All

Inc.

Kodak.

be requested.

551 Copyright All rights

binds

with

here.

All-trans

and retinol

enters

of the seminiferous

MATERIALS

mg was a gift

RBP itself

In another

protein

in combination

when RBP reaches

daltons

a low molecular

the cytosol

binds

--et al.

testis.

binding

and purified

in the blood

What happens

investigation.

tissues

carried

specific trans Purity

activity

unlabeled

of 253 uCi/ retinyl

of the compounds

acetate was

Vol. 61, No. 2, 1974

BIOCHEMICAL

assayed

on an alumina

protein

molecular

all

other Adult

male

fed and watered were

removed,

separated washed

were

S.T.

were

were

They were

and then (7)

glycol.

each flask.

The contents

a steady

experiment

the

bation,

the

any adhering

(1.3 oxidation

were

of nitrogen

testes

from

centrifuged

and protein fraction

was determined was subjected

tions

of this

7.4.

The amount

culated G-200

from column

the protein

2,

salt.

to obtain

mixing

a red After

light.

of Lowry

The fraction Sephadex

G-25

of the X-100

to remove

S.T.

were

passed

through

at 105,000

x g in

et al.

(8).

Nuclear),

The cytosol

at different

satura-

pH between

saturations

purified

by Sephadex

was used

for

B.,

Devi,

A. and Clark,

552

F. J.

Fed.

Amer.

Sot.

7 and

was Cal-

desalting

fractions.

2 Ahluwalia, 4093 (1973).

then

in a

(New England

various

(coarse)

incu-

20 minutes

on at O'C with

was further

each

in a Beckman LS-255

precipitation

for

to

fraction.

in 12 ml of acquasol

required

For

termination

x g for

added

at 37'C

KH2P04 pH 7.4,

was determined

of ammonium sulfate

were

The washed

the cytosol

by the procedure

containing in 0.5 ml of

previously2

was centrifuged

was carried

chromatography.

medium

predissolved

at 9,000

to ammonium sulfate

(9).

physiological

O.lM

Precipitation

the table

The

physiological

membrane.

with

in the aliquots after

saline.

with

as described

The supernatant

ultracentrifuge

counter

were

10 ml of 0.1% Triton

and the

scintillation

the S.T.

with

cheesecloth

The radioactivity

From each testis

twice

buffered

were testes

used.

in 0.3 M sucrose

a Beckman LS-45

and

The animals

2 ml of 0.1% pyrogallol

on the outside

centrifuge.

and

and the

in a room with

homogenized

refrigerated

study.

were

filtrate

G-200

from Pharmacia,

10 rats

were washed

radioactivity

G-75,

by decapitation,

nCi./testis)

incubated

stream

S.T.

twice

in 40 ml of Hanks'

To prevent

obtained

in this

decapsulated.

retinyl-11,12[3N2]-acetate

propylene

used

killed

and washed

incubated

were

(coarse),

grade.

rats

--ad libitum.

G-25

kit

of reagent

Sprague-Dawley

cleaned

Sephadex

determination

mechanically

purified

under

weight

chemicals

(6).

column

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fxp.

Biol.

Vol. 61, No. 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

VOLUME

Fig.

1

Chromatography on Sephadex G-200 of fraction III obtained after precipitating retinyl-11,12-3H2-acetate-labeled cytosol fraction of rat testis seminiferous tubules with 50-1002 ammonium sulfate saturation. The column (5 x 100 cm) was equilibrated with 0.05 M potassium phosphate buffer containing 0.1 M NaCl and 0.02% NaN5. The sample applied to the column contained 50-70 mg of total protein in 10-15 ml buffer. Elution was carried out with the same buffer at the flow rate of 30 ml per hour at 4-8°C.

The molecular technique

(10)

standardized

volume (V,)

molecular

The purity polyacrylamide (12).

by the addition

protein

was determined

G-75 column

(Sigma

A small

was measured

of their

Anker

insulin

void

volume

protein

of the

on a Sephadex

proteins.

to determine effluent

weight

with

ovalbumin


Chemical),

amount

(V,)

(2.6

and the values

of Ve/Vo

polymer

After

to the center

The column

and

was chromatographed

chromatography,

of the peak

were

was first

chymotrypsmogen

dextran

of the column.

corresponding

x 70 cm). RNase,

of blue

by a gel-filtration

plotted

the

of the

against

eluted

the logs

weights. of the retinol

gel

binding

electrophoresis

Polymerization

for

protein

by the

systems

polyacrylamide

of 0.1% ammonium persulfate

tetramethylethylenediamine,

component

and gels

were

553

gel

was determined

of Reisfeld electrophoresis

in the presence stained

--et al.

for

by (11)

and

was induced

of N,N,N',N'-

at least

one hour

in a

Vol. 61, No. 2, 1974

0.1% solution destaining Amino sealed

BIOCHEMICAL

of aniline

blue

in 7% acetic

acid.

acid

tube

analyses

at llO°C

black

of the

for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in 10% trichloroacetic

component

22 hours

with

were

acid

carried

out

followed

by

in an evacuated

a Beckman 120B amino

acid

analyzer.

RESULTS AND DISCUSSION The cytosol

fraction

different

saturations:

three

and 50-100X (5955

(fraction

Fraction

mg protein). M salt

fractionated

O.lM

shown in Fig.

Fractions of higher

I was found

concentration

on a Sephadex

containing is

were

to ammonium sulfate

O-15% (fractionI),

III).

DPM/mg protein)

0.01-1.0

was subjected

I

G-200

15-50%

(3597

than

to be insoluble

column

The isolation

and purifzcation

(912 DPM/ buffer

was soluble

at 4-8°C.

II)

II

a 0.05 M phosphate

NaCl and 0.02% NaN3 at pH 7.4, 1.

fraction

III

at

and III

in phosphate

fraction using

(fraction

DPM/mg protein)

specificity

at pH 7.4;

precipitation

and was buffer

The elution

procedure

with

is

pattern

summarized

TABLE I Fractionation

of Labeled

Seminiferous

Tubules1

DPM/mg Protein

Total Protein (4

Homogenate

9,776

1,026

Cytosol

4,444

Type of fraction2

0-15X

fraction

(NH4)2S04

E-50% 50-100%

Sephadex

(NH4)2S04

fraction

(NH4)2S04 G-200

II

fraction111

912 5,955 42,735

tubules uCi/testis)

obtained

after

100.0

423

3,597

column

1 The seminiferous 12-[3H ]-acetate (1.3 descri t ed in the text. 2 Fractions see text.

I

554

41.2

18

1.8

219

21.3

123

12.0

0.25-0.5

from 50 rats were labeled under the experimental differential

Percentage of Total Protein of Homogenate

centrifugation.

.025-0.05

uith retinyl-11, conditions For details

BIOCHEMICAL

Vol.61,No.2,1974

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2.

2.

Yb

1.

11

0.

t

1

3.5

I LOG

Fig.

2

I. last

protein

The hexane

extract

peak - 8.6 pg/mg

in the other

with

non-labeled

retinyl was similar

retinyl

acetate

(Fig.

acetate

corresponded

tion

with

Fig.

1 contains

peak

showed

WEIGHT

The molecular filtration

- compared

In an experiment and then

to the peak from acetate. that

band

technique

where

the

fractionated

suggest

binds

retinol

with

was found

was incubated

high

column,

with

level

isolated

results

555

1.0 pg/mg

cytosol

the highest

These

2).

than

on a G-200

of retinyl

following

that

the

incuba-

last

peak in

specificity.

applied

to be ~4,800

the

labeled

This

electrophoresis

50 to 55% of the radioactivity

(Fig.

was concentrated

upon incubation

the cytosol

of the protein

retinol less

in analyticalpolyacrylamide

almost weight

that with

The peak containing

a component

and contained

revealed

to the one obtained

1).

[3H]-retinyl

a single

of peaks

acetate

pattern

gel

MOLECULAR

protein

peaks.

elution

pH 3.5

1 5

4.5

Estimation of molecular weight by gel filtration. A portion of the purified protein was dissolved in 0.05 M potassium phosphate buffer, pH 7.4, 0.1 M NaCl and 0.02% NaN3 and chromatographed on Sephadex Elution with the same buffer was carried G-75 column (2.6 x 70). out at a flow rate of 40 ml per hour at 4-8"~. Fractions of 5 ml Elution was monitored at 230 nm. each were collected.

in Table in the

I

4.0

at to the gel.

daltons

by the

Vol. 61, No, 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

c 220

Fig.

3

Elution profile of cytosol fraction obtained after labeling seminiferous tubules with retinyl-11,12[3H2]-acetate. The Sephadex G-200 column (5 x 100 cm) was equilibrated with 0.05 M potassium phosphate buffer, pH 7.4, 0.1 M NaCl and 0.02% NaN3. Elution was carried out under the same experimental conditions as in Fig. 1.

In another with

labeled

tion

but

study retinyl

instead

column.

(Fig.

3) suggesting

binding

acetate

Two peaks

is

a part

It

of molecular

weights are

The amino and cysteine

following

subjected

weights cytosol

molecular

as to whether

of a bigger

has been reported between

incubation

that

U4,OOO

directly

~4,800 there

and U6,OOO are at least These

the lower

molecular

or there

in the rabbit and Q7,OOO

are lung

that

precipita-

on the Sephadex

weights.

molecule

of the S.T.

to ammonium sulfate

and fractionated

in the S.T.

answer

components.

specificity

was not

of different

any definitive here

fraction

of molecular

that

components

reported

the cytosol

was freeze-dried

G-200

vide

1360

observed

two retinol

results

do not

weight

pro-

component

two individual three

bind

were

components

retinol

with

high

present. acid

(Table

composition II).

of the component

The smaller

size

556

showed

and the amino

the absence acid

of cystine

composition

Vol.61,No.2,

1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE II Amino Acid Isolated from

Composition of Retinol of Rat Testis

Binding Component Seminiferous Tubules

Cytosol

Estimated Number of Residues'

Percentage Distribution'

Amino Acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine l/2 cystine Valine Methionine Isoleucine Leucine Tyroslne Phenylalanine Tryptophan

6.87 1.57 2.40 16.43 5.26 6.97 18.53 8.19 11.92 7.14 0 4.59 0.84 3.50 4.60 0.97 1.98 N.D.3

3 1 1 7

2 3 8 3 5 3 0 2 Trace 2 2 1 1 -N.D.

TOTAL4

44

1 Average

of two determinations.

2 Calculated molecule.

on the basis

of 1.0 residue

of histidine

per

3 Not determined. 4 Excluding

suggest it

that

to bind

this

ammonia

component

retinol

The function

with

The possibility

partial

degradation

Bashor

-et -'al

is analogous the tissue

that

binding

a very

and higher the lower

of a single

to steroid

have

loose

structure

which

enables

affinity.

lower

(4) postulated

retinol

might

high

of the

known.

and tryptophan.

molecular

component that

hormone

molecular

cannot

the tissue receptor

components

557

weight

components

component

be excluded

cytosol

proteins; compared

weight

retinol however

to steroid

from

is

not

might

be a

these

studies.

binding

component

the smaller

size

receptor

proteins

of

Vol. 61, No. 2, 1974

and our

findings

BIOCHEMKAL

of the presence

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of retinol

in bovine

the possibility

that

the tissue

retinol

binding

similar

of the hormone

receptor

proteins

that

to that rat

serum RBP (1)

pre-albumin interest

to form

a complex

to investigate

component(s)

in the

one tenth

the fact ponent

that for

Our study ineffective deficient

study

shows

of the binding

retinoic has shown

component (13). weight

molecular

may not It

have a role

has been

reported

and is associated

weight.

of RBP to the

It

would

with be of

tissue-binding

testis.

at least

that

compared

affinity

in the

for

testis

acid

may render

that

retinoic

in restoring rats

of %70,000

the relationship

Our preliminary only

is of Q20,OOO molecular

acrosomes 3 raises

sperm

with

retinoic

the absence this acid

the germinal

epithelium

this

acid.

This

of a specific

compound implanted

retinol

biologically locally to normal

component

has

may explain binding

com-

ineffective.

in

the testis in vitamin

was A

(l4). ACKNOWLEDGMENT

This

work

was supported

by NLH grant

#HDO6045-02.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Muto, Y. and Goodman, D. S., J. Biol. Chem. 247, 2533-2544 (1972). Invest. 47, 2025-2044 Kanai, M., Raz, A. and Goodman, D. S., J. Clin. (1968). Peterson, P. A. and Berggard, I., J. Biol. Chem. 246, 25-33 (1971). Bashor, M. M., Toft, D. 0. and Chytil, F., Proc. Nat'l. Acad. Sci. USA 70, 3483-3487 (1973). Ong, D. E. and Chytil, F., Biochem. Biophys. Res. Commun. 59, 221-229 (1974). Futterman, S. and Andrews, J. S., J. Biol. Chem. 2, 81-84 (1964). Hall, P. F., Irby, D. C. and de Krester, D. M., Endocrinology 84, 488496 (1969). Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., J. Biol. Chem. 193, 265-275 (1951). Green, A. A. and Hughes, W. L., in Methods in Enzymology, vol. 1, Colowick, S. P. and Kaplan, N. O., eds, Academic Press, pp. 67-78 (1955). Whitaker, J. R., Anal. Chem. 35, 1950-1953 (1963). Reisfeld, R. A., Lewis, U. J. and Williams, D. E., Nature 195, 281-283 (1962). Anker, H. S., Fed. Eur. Biochem. Sot. Lett. 7, 293 (1970). Sherman, M. R., Corvol, P. L. and O'Malley, B. w., J. Biol. Chem. 245, 6085-6096 (1970). Ahluwalia, B. and Bieri, J. G., J. Nutr. l&, 141-152 (1971). 3 Gambhir,

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