The effect of limited proteolysis on enzymatic, binding and immunological properties of ligandin

Vol. 104, No. 2, 1982 January 29, 1982

THE

BIOCHEMICAL

Pages

EFFECT

OF LIMITED

AND Madhu Liver

Bharyava, Center, Albert

November

PROTEOLYSIS

IMMUNOLOGICAL

M.

Research

Received

RESEARCH COMMUNICATIONS

AND BIOPHYSICAL

9,

ON ENZYMATIC,

PROPERTIES Naohito

BINDING

OF LIGANDIN

Ohmi

and

Department of Einstein College Bronx, New York

533-540

Irwin

M.

Medicine and of Medicine, 10461

Arias Biochemistry,

1981

SUMMARY: The peptide mixture obtained from controlled proteolytic digestion of ligandin with proteinase K or subtilisin retained 40% of glutathione-S-transferase and steroid isomerase activities, immunoloyical reactivity and lower affinity bilirubin binding but binding at the primary site was abolished. When these limited proteolytic digests, which had no intact ligandin as determined by SDS gel electrophoresis, were subjected to Sephadex G-75 column chromatography, 40-50% of the peptide fragments were recoverd in fractions where intact ligandin The results suggest that intact ligandin is not required for eluted. enzymatic activities, binding of bilirubin at the secondary site, or steroid isomerase and glutathione-S-transferase immunological reactivity; activities are modulated in a parallel manner and may be mediated by the region of the protein, and primary and secondary binding sites for same bilirubin are distinct and independent, despite nicks introduced by proteolysis in ligandin's subunits, some of the fragments remain associated under non-denaturing conditions and the susceptibility of the two subunits to the proteases is different.

INTRODUCTION: binds

Ligandin

bilirubin,

variety

both

of

involved

of To

abundant

two have

of

and

between multiple

structural functions,

liver

cytosol

exhibits

25K

and

Liver

liyandin

22K.

Although

activity, bilirubin domains we

high

occur

subjected

of

on the

the

Copyrighr 0 1982 by Academic Press. .-I II r/gh IS of reproduction rn any ,form

Inc. reserved.

533

affinity

22K

protein

liyandin

Abbreviations: GSH, reduced glutathione; SGP, Streptomyces protease; PK, proteinase K; SL, subtilisin. The counter?ons for phosphate and barbital buffers ano hydrochloride for Tris. 0006-291X/82/020533-08$01.00/0

ana

ylutathione-s-

(3).

weights

that

steroids

activities

molecular

and

rat

carcinoyens,

glutathione-S-transferase

distinguish its

(l),

isomerase of

sulfobromophthalein

in

anions

steroid subunits

protein azodye

oryanic and

subunits

binding (5).

other (2)

consists

an

sulfobromophthalein,

of

transferase

is

subunit that

are

to

limited

are

griseus sodium

a

Vol. 104, No. 2, 1982 proteolytic

digestion.

ligandin's

binding,

BIOCHEMICAL These enzymatic

AND BIOPHYSICAL digests and

were

RESEARCH COMMUNICATIONS used

immunological

to

determine properties

whether are

interrelated.

MATERIAL AND METHODS: Acrylamide was obtained from Bio Rad and used without further purification. Subtilisin, proteinase K and Streptorryces griseus protease were obtained from Sigma. S. aureus obtained from Miles Laboratories. The steroid A - ~#dr~~~~~~"~,~~~ dione, used as a substrate for the steroid isomerase assay, was a yift from Dr. Paul T. Talalay. Preparation of Ligandjn: Ligandin was isolated from adult male rat liver and its purity determined by methods described earlier (6,7). Proteolytic Digestions: buffer, pH 7.4) was incubated at protease (SGP, final concentration 10 vg/ml), and subtilisin SL; incubated under identical conditions in the 10 ug/ml). Ligandin was absence of protease as a control for all proteolysis experiments. Aliquots were withdrawn from SDSpolyacrylamide Gel Electrophoresis: analyzed by SDS-polyacrylamide proteolytic digests at several intervals, SDS-polyacrylamide gel electrophroesis was electrophoresis. gel usiny 15% acrylamide in a performed in a slab gel apparatus discontinuous tris-glycine system described earlier (4). Gels were stained in a solution containing 0.2% Coomassie blue in 20% methanol 10% acetic acid and destained by diffusiun in 20% methanol - 10% acetic acid. GSH-S-transferase was Assay of Glutathione-S-transferase Activity: in absorbance at 343 nm with GSH and lassayed by measuring changes chloro-2,4-dinitrobenzene as substrates (6). a5-androstene-3,17-dione Steroid Isomerase Assay: Isomerization of was measured at 248 nm in tris-phosphate buffer pH 8.5 containing GSH and dithiothreitol as described by'Benson et al. (3). Determination of Immunoreactivity: It was not possible to perform Ouchterlony double diffusion with the limited proteolytic digests because all proteases interfered with antiyen-antibody complex formation. Therefore, prior to incubation with monospecific antiliyandin were separated from the peptides by electrophoresis. 1% > protease Immunoelectrophoresis of ligandin and its proteolytic digests was performed for 2 hours in barbital buffer pH 8.5, IL =.Ol, at 50V (8). Under these conditions, ligandin migrated twoard cathode while the proteases migrated towards anode (data not shown). After electrophoresis, monospecific anti-rat liver IyG was added and incubated at 4'C for 48 hrs. After incubation, plates were washed, stained with 0.2% Amido Black and destained in 7% acetic acid (7). Binding of Bilirubin to Peptide Fragments: A Cary model 60 spectropolarimeter with a 6001 circular dichroism attachment was used to detect 'bilirubin bindiny. Measurements were rnade in a 1 cm liyht path cell at 25'C. To ligandin (500 ug/ml), or mixtures of proteolytic fragments generated from it, small increments of 0.02 M bilirubin were added to obtain CD spectra in the 550-300 nm range (6). Sephadex G-75 Column Chromatography of Proteolytic Digests of Ligandin: Ligandin or the mixture of peptides obtained by limited proteolytic digestion with PK or SL were 'applied to 0.5 x 20 cm Sephadex G-75 columns equilibrated with 0.01 M phosphate buffer, pH 7.4. Elution was performed with the same buffer, and 1 ml fractions were collected. Every fraction was assayed catalytically for ylutathione-S-transferase and steroid isomerase activities and for irnmunoloyical reactivity by Ouchterlony double diffusion method (8). Protein concentrations were estimated by the method of Lowry et al. (9) using bovine serum albumin as a standard. 534

BIOCHEMICAL

Vol. 104, No. 2. 1982

AND BIOPHYSICAL

PROTEINASE

K

5’ GRISEUS

RESEARCH COMMUNICATIONS

10’

15’

40’

60’

PROTEASE

5’

15’

IO’

20’

40’

60’

SUBTILISIN

60’

Fig.

1

10’

20’

40’

Kinetic study of digestion of liyandin: Ligandin at a concentration 1 my/ml was incubated with SGP (lOug/ml, ginal concentration) or PK (20 ug/ml) or SL (10 uy/ml) at 37 C. at indicated time intervals (from 5 Samples were withdrawn min-60 min) and analyzed by Tris ylycine SDS-polyacrylamide gel electrophoresis. See text for details. The sample in the first lane 06 each ye1 contains liyandin which was incubated for 1 hr at 37 C in the absence of protease.

RESULTS: Analysis

of

Proteolytic

polyacrylamide

gel

limited

proteolysis

subunits

were

min

digestion

electrophoretic

not

Digests

of

electrophoresis of

with pattern

SL

patterns

ligandin

detected

after or when

Ligandin:

with 40

SGP.

min No

the 535

SL, of

Fig. of

PK and digestion

further

chanye

digestion

was

1.

peptides SGP. with was continued

shows

SDS-

obtained Intact

by

liyandin

PK or observed for

after in 4 hr

60 the at

Vol. 104, No.

BIOCHEMICAL

2, 1982

Table

1:

Effect

of

Limited

AND BIOPHYSICAL Proteolysis

Isomerase Protease

Treatment

RESEARCH COMMUNICATIONS

on GSH-S-transferase

Activities

of

GSH-S-Transferase

Steroid

moles/min/mg/Protein None

and

Isomerase

moles/min/mg/Protein

11

1.0

Proteinase

K

2.8

S. Griseus

Protease

0

Subtilisin

Steroid

Ligandin

.26 0

4.1

.36

Ligandin was incubated at 37'C with or without protease for GO min and then assayed for the indicated catalytic activity. GSH-Stransferase was assayed by measuring the change in absorbance at 343 nm with reduced GSH and 1-chloro-2,4-dinitrobenzene as substrates (6). Steroid isomerase was assayed by the change in absorbanse at 248 nm at pH 8.5 in the presence of dithiothreitol and GSH with A androstene3,17-dione as a substrate (3).

37Oc.

Since

most

determinations

were

proteolysis those

is used

for

SDS rapidly

however,

the

22K

discrete

frayments

observed.

Lower method.

GSH-

S-transferase

Digests:

by

the

and

was

prior or was

to SL not

and

PK than

was

more

mixture

of

of

digestion inmunoreactive

The

25K

subunit

was

the

22K

altered

these of

In

of

have

both

by

of

ligandin

by

incubation

ligandin

was

from

With

SGP,

cases,

5-8

5-12K

escaped

were

detection

of

catalytic

generated

and

all

Activites

of

analysis

subunit.

weight may

activities

not

for

degree

by

Proteolytic

activities PI/, and

SL

were at

were (Table

I),

abolished

37'C

for

by 60

min

protease. of

removed

incubation

40%

peptides

catalytic were

used

Isomerase

immunological the

susceptible.

peptides

dnd

and period,

molecular

Steroid 25%

but

time

preparations

weiyht

Reactivity

protease

this

in

molecular

I),

Immunological

Sl

subunit

the

catalytic

electrophoresis. by

Both

absence

within

ranyiny

respectively. (Table

binding,

in

Approximately

retained

in

done

gel

more

this

the

identical

digested

SGP

of

from with

retained

Proteolytic the

Digests

proteolytic

antibody,

the

(Fig.

2).

diyests peptide

immunological

of

536

is

not

by

mixture

reactivity, It

Liyandin: electrophoresis obtained

while possible

When

to

that ascertain

from from

PK SCP from

Vol.

104,

No.

BIOCHEMICAL

2. 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

SGP

Fig.

2

these

Inmunoelectrophoresis of ligandin and its proteolytic digests: Immunoelectrophoresis of ligandin (L) and its sixty minute proteolytic digests was performed in barbital buffer, pH 8.6, = 0.01 at 80V. After incubation with anti-rat liver liyandin IgG for 48 hr at 4'C plates were washed, stained with 0.2% Amido Black and destained in 7% acetic acid. 0 and - indicate the origin and cathode respectively.

data

if

the

immunoprecipitin

with

the

of

abolished

ligandin

the

results

probably

distinct

Catalytic

Activities

of

4,

ligandin

elutes also Fractions

at

40-50x

contains 8-14

that

the

with

immunoreactivity,

the

peptide

but

mixture

the is

primary

than

the

low

Column Immunological

peptides

generated

ligandin

steroid

isomerase

were

irrmunoreactive

binding by

binding

associate on

proteolytic

and

531

is and

is

binding. and

Determination As

proteolytic

themselves

into

can

be

seen

digestion

This

chromatogrdphy.

antibody

of

a protein

GSH-S-transferase with

3).

bilirubin action

limited

column

while (Fig.

for

Reactivities: by

sites.

retained

site

affinity

SGP

undetectable,

was

Chromatography

and

of

was

binding

primary

witn

secondary

site

disrupted

SL can

Proteolysis dnd

affinity

of by

PK or

position

acidic

primary

lower

Peptides

with the

at

that

frorn

of

to

Fragments:

at

that

domain

Separation

Fig.

more

Peptide

of

suggest a

contribute

molecule.

binding

amounts

in

was

binding

SL,

located

peptides

to

bilirubin

substantial

from

more

Bilirubin

PK and

These

or

arc

native

Binding

With

one

U

that peak

activities. to

liyandin,

but

Vol. 104, No. 2, 1982

BIOCHEMICAL

400

AND BIOPHYSICAL

450

Fig.

3

4

(“m)

spectra of bilirubin Circular dichroism ligandin and its proteolytic digests: and its proteolytic -9igests in 0.01 7.0. increments of 10 M bilirubin in until saturating ellipticity values path length cell was used. ObsFrved expressed as deyrees per 10 M proteolytic fragments, calculations protein concentration.

8 12 16 20 FRACTION NUMBER Fig.

550

500

WAVELENGTH

RESEARCH COMMUNICATIONS

24

complexes with To Tiyandin (1 Wj/mt) M phosphate buffer, pH 20 mM NaOH were added were attained. A 1 cm ellipticity values dre protein. For the were based on total

-

Sephadex G-75 column chromatography of ligandin and its limited proteolytic digests: 1 mg ligandin digesteo with either PK (A) or SL (B) or undigested (C) was applied on 1 x For details of limited 20 cm Sephadex G-75 column. proteolysis, see the text. El&ion was performed with 0.1 M phosphate buffer, pH 7.4. One ml fractions were collected and aliquots assayed for protein, GSH-S-transferdse activity and steroid isomerase activjties by methods described in the text.

538

Vol. 104, No. 2, 1982 fractions

BIOCHEMICAL

15

to

catalytically

22

nor

were

AND BIOPHYSICAL

not.

Thus,

immunoloyically

the

RESEARCH COMMUNICATIONS

smaller

peptides

are

by

diyestion

the

primary

neither

reactive.

DISCUSSION: These SGP,

studies

PK

but

and

SL

retain

is

into

regions

peptide relatively

are to

observation

abolished

by

affinity

sites

independent

extent

that

they

may

high

retained

are

on

located

each

bilirubin of

ligandin and

by be

are

steroid

limited at

high

same

low

are

the

site

affinity

that of

molecule

further site The

activities is

compatible

or

overlapping

and

low

and

are

support

affinity

is site

high

(4).

isomerase

the

susceptible

sequences

the

independent

proteolysis

located

at

provide the

prolonged

binding

suggests

at

described.

protease.

binding

binding

proteases

some

bilirubin

results

the

other

domains

These

site.

after

that

same

different

other.

occurs

affinity

are

GSH-S-transferase same

the

activities

activity

the

of

site,

secondary

conditions

whereas

30-40%

that

S-transferase

PK and

the

with

activities,

of

suggesting

cleaved, action

the

specificity

peptides 37'C)

the

at

under

that

of

suggestion

the

isomerase

the

generated

at

that

SL and

catalytic

about

rapidly

resistant The

hr

at

ketosteroid

smaller

( > 6

binding

binding

known are

obtained

bilirubin

bilirubin

peptides

times

fragments

and and

degradation

incubation

that

of

little

discrete

Further

our

incapable

reactivity

Although

and

that

GSH-S-transferase

immunological

used,

are

indicate

for

and

GSH-

observation

are

altered

with

the

regions

to thesis of

the

protein. The

results

also

the

peptide

fragments

and

mimic

activities

exhibits bind

nicks

at

this

that, to

at also

the be

native and

high due

despite

proteolytic

reassemble

activities

bilirubin could

tend of

catalytic

binding

show

under

to

This

immunological site.

conformational

site. 539

in

ligandin,

non-denaturiny

ligandin.

affinity

nicks

conditions

nicked

derivative

reactivity The

loss

changes

but of due

"high to

does affinity"

proteolytic

not

Vol. 104. No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENTS: This work was supported by grants CA 24842, AM 17702 and AM 2019 from the National Institutes of Health. Authors are grateful to Manya Shafran for excellent technical assistance.

REFERENCES: 1.

Litwack,

G.,

Ketterer,

B. and Arias,

I.M.

(1971)

Nature

234,

466-

467. 2.

Habig, W., and Jakoby,

Pabst, M., W. (1974)

Fleischner, G., Gatmaitan, Proc. Natl. Acad. Sci.,

Z., Arias, U.S.A. 2,

I.M. 3879-

3882. 3. 4. 5. 6. 7. 8. 9.

Benson, A.M., Talalay, P., Keen, J.H. and Jakoby, W. (1977) Proc. Natl. Acad. Sci., U.S.A. 74, 158-162. Bhargava, M.M., Listowsfi, I. and Arias, I.M. (1978) J. Biol. Chern. 253, 4116-4119. Bhargava, M.M., Ohmi, N., Listowsky, I. and Arias, I.M. (1980) J. Biol. Chem. 255, 718-723. Bhargava, M.M., Listowsky, I. and Arias, I.M. (1978) J. Biol. Chem. 253, 4112-4115. Arias, I.M., Ohmi, N. and Bhargava, M.M. (1979) Trans. Assoc. Amer. Phys. XCII, 113-120. Fleischner, G., Kamisaka, K., Gatmaitan, Z. and Arias, I.M. (1976) in Glutathione: Metabolism and Function (Arias, I.M. and Jakoby, W.B., eds.) pp. 259-266, Raven Press, N-Y. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275.

540