Cytoplasmic proteases of rat liver parenchymal cells

Vol. 108, No. 3, 1982 October 15, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 1325-1330

CYTOPLASMIC PROTEASES OF RAT LIVER PARENCHYMAL CELLS George

Department of Physiology of Texas Health Science Dallas, Texas 75235

University

Received

September

N. DeMartino Center

9, 1982

Soluble extracts of isolated rat liver parenchymal cells contained One protease was a high mothree proteases with alkaline pH optima. lecular weight (Mr = 500,000) enzyme which was stimulated by ATP. The other two proteases were totally dependent on calcium for activity and One was halfdisplayed different calcium conc@ration requirements. while the other required only 10 uM ma$imally activated by 150 uM Ca for half-maximal activation. Ca INTRODUCTION Liver

parenchymal

lysosomal lar

proteins

(l-4). proteolysis

proteases

have been

is

cells

not

known

or

from

proteases to

Blood

contaminating

proteases

tamination

in the

learn

more

degradation, liver

including

Abbreviations bis-@-aminoethyl diethylaminoethyl

cell

mast

the

we have a high used:

cells

mechanisms studying

molecular

DTT, ether) cellulose.

from

In fact,

certain

other

may

in some cases authentic

ATP-stimulated

EGTA, dithiothreitol; N'-tetraacetic N,

liver

non-lysosomal been these

also

found

tissues

contain sources liver.

of non-lysosomal

cytoplasmic

non-

non-lysosomal

recently

from whole

and control

weight,

have

potential

enzymes

several

catalyze

contaminate

cells

of these

been

derived

both

of intracellu-

numerous liver,

which

represent

contain

which

of whole

tissues

reticuloendothelial

isolation

enzymes

are

types.

and other

and thus

about

the

cells,

degradation

Although

enzymes

in liver

and

of

the

in extracts

these

from

mammalian

for

unclear.

identified

whether

lysosomal

to

identity

is

solely

other

pathways

The

identified

originate

(5-8).

like

and non-lysosomal

lysosomal

it

cells,

proteases protease

nonof

con-

In order protein in rat (9)

and

ethyleneglycolacid; OE52,

0006-291X/82/191325-06$01.00/0 1325

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 108, No. 3, 1982

BIOCHEMICAL

two

calcium-dependent

was

to

proteases

determine

parenchymal

AND BIOPHYSICAL

whether

The

(10).

these

enzymes

RESEARCH COMMUNICATIONS

purpose

were

of

present

the

present

in purified

work liver

cells. MATERIALS

Preparation

of Rat 'Liver

AND METHODS

Parenchymal

Cells

Rat hepatocytes were prepared as described previously (2). Microscopic examination revealed less than 0.05% contamination by other cell types. Five ml of packed purified cells were washed three times with phosphate-buffered saline prior to homogenization. Preparation

of Soluble

Extracts

from

Rat Liver

Parenchymal

Cells

Cells were homogenized in 10 volumes of 5 mM potassium phosphate buffer, pH 7.6, containing 0.5 mM OTT, 2 mM EGTA, 2 mM EDTA using a Dounce-type homogenizer. The homogenate was centrifuged successively at 17,000xg (20 min) and 100,OODxg (90 min). The resulting supernatant was dialyzed for 3 hrs against 20 volumes of 5 mM potassium phosphate buffer, pH 7.6 containing 0.5 mM DTT, 0.5 mM EGTA, and 0.5 mM EDTA, and then added to 3 gm of diethylaminoethyl cellulose (DE52, Whatman) equilibrated with the same buffer. After 40 min, the resin was filtered and washed with buffer. Bound protein was eluted in 3 ml of buffer containing 1.2 M KCl. The eluted protein was dialyzed for 14 hrs against 50 mM Tris-HCl, pH 7.6, 8 mM KCl, 0.5 mM DTT, 0.5 mM EGTA. Sephacryl

S-300

Column

Chromatography

The dialyzed extract was chromatographed on Sephacryl S-300 (28x2.5 cm) equilibrated with 50 mM Tris-HCl, pH 7.6, 8 mM KCl, 0.5 mM DTT, 0.5 mM EGTA. The column was calibrated with proteins of known molecular weights (Figure 1). DE52 Ion-Exchange DE52 (3 g) DTT, 0.5 mM EGTA application, the ml, 0.0-0.4 M) in Protease

Column

Chromatography

was equilibrated with 50 mM Tris-HCl, pH 7.6, 0.5 mM and packed in a 1.5 cm diameter column. After sample column was eluted with a linear gradient of KC1 (200 buffer.

Assays ivity

eithe~r~~&l~'C] tides as described Protein

was determined casein or [methyl-' previously (10).

@y measuring Cl globin to ?AL!$"u$?teif

Determinations Protein

was measured

by the method

of Bradford

(11).

RESULTS Soluble alkaline casein

extracts protease

and

from activity

Cmethyl-14C]

globin.

rat

liver

against

parenchymal substrates

This 1326

activity

cells such

as

was significantly

demonstrated [methyl-14C] stimu-

Vol. 108, No. 3, 1982

8lOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNlCATlONS

1

FRACTION

NUMBER

Figure 1. Sephacryl S-300 column chromatography. Soluble extracts of the rat liver parenchymal cells were chromatographed on Sephacryl S-300 and assayed for protease activity in the presence (0) and absence (0) of 1.7 mM calcium. Insert. Molecular Weight Markers: A, Blue Dextran, Vo; 6, thyroglobulin, Mr = 660,000; C, ferritin, Mr = 440,000; 0, catalase, Mr = 240,000; E, aldolase, Mr = 158,000; F, bovine serum albumin, Mr = 68,000.

lated

when assayed

enzymes

responsible

graphed

on

(Figure

The This

stimulated (data

not

tivity

It

pH optimum was

Methods.

calcium

concentration

(Peak

greater

than

protease

fold)

was totally

dependent

7.0-8.5.

ion-exchange

procedure

resolved peak

requirements

required

500

uM calcium

and 25 pM calcium

The

to

II)

(Peak

when

peak eluted

each

I)

for only

for

activity.

to identify

extract

was

protease

eluted

corresponding

of 8.0 assayed

to

in

the

on calcium

presence

of

proteolytic

activity

into

two

was totally

dependent

on calcium,

were

different

10 pM calcium

1327

(Figure

half-maximal

for

to

However, half-maximal

a ac-

described

maximum activity.

ATP

and demonstrated

as

for

=

corresponding

chromatography

very

Mr

and was significantly

at a position

the

the

chromato-

activity

calcium-dependent

150 uM calcium

required full

of

a position

The second

Although

2).

peaks

at

In order the

a pH optimum

2.5

subjected

(Figure

enzyme

had

from

This

Two

eluted

up to

shown).

activities,

S-300.

first

(i.e.

of calcium.

these

activity

Mr = 150,000. broad

for

Sephacryl

1).

500,000.

in the presence

in peaks their

3).

One

activity the

and other

activity

Vol. 108, No. 3, 1982

BIOCHEMICAL

d-

-

IO

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

b 20

30

FRACTION

40

50

NUMBER

Figure 2. DE52 ion-exchange chromatography of calcium-dependent teolytic activity. Calcium-dependent proteolytic activity from Sephacryl S-300 column (Figure 1) was chromatographed on DE52 as cribed in Methods. Protease activity was measured in the presence and absence (0) of 1.7 mM calcium. Protein (A).

prothe des(0)

DISCUSSION The identified

present in

work cytoplasmic

demonstrates extracts

that of

three rat

liver

proteases (9,lO)

which are

also

we

present

l-

I-

i-

CALCIUM

( ,uM)

Figure 3. Effect of calcium concentration on the activity of calciumdependent proteases. Peak I and Peak II calcium-dependent protease activities from Figure 2 were dialyzed extensively against 10 mM TrisHCl, pH 7.5, 0.5 mM DTT and assayed for protease activity at the calcium concentrations indicated. Peak I (0); Peak II (0). 1328

first

Vol. 108, No. 3, 1982 in

isolated,

BIOCHEMICAL

purified

icant

because

whole

liver

liver

ginate

from

have

emphasized

mast

cell the

intracellular

proteolysis.

Non-lysosomal

proteases

proteases

proteins

with

short

(l-3).

Non-lysosomal some

degradation

of

proteins

any

of

the

only

cells

these

parative to

of these does

pathways.

detect

other here

such

may be of

degradation

required

to

learn

significance

in

it

Nevertheless,

agents

intact

cells these

related

which

to the

--in

that

they

regulation

in

(4).

in liver in

these

specific

may limit

are pre-

our

ability

identified

they

are

overall

Additional effects

as

participate

The

work

the

as well

that

can regulate

vitro

structures

proteases

because

(13,14).

of

by microinjection

the enzymes

interest

of

cells

involved

seems unlikely

in this

of

degradation

(12)

hepapotcytes.

used

particular

whether

and are

indicate

in

enzymes.

also

cells

other

abnormal

non-lysosomal

example,

and assays

by ATP or calcium,

protein

three

necessarily For

the

proteins

into

proteases

methodology

vitro

not

non-lysosomal

described --in

introduced

and

with are

origin

in the degradation

for

intracellular

specific

identification

parenchymal

pathways

findings

in the process

hepatocytes

and of proteins

proteolytic

of

in

of

shown to ori-

cellular

roles

to be responsible

half-lives

degradation

important

example,

appear

the

signif-

Such

to them roles

may play For

proteins.

been

tissues.

demonstrating

is

in extracts

recently

these

ascribing

finding

identified

have of

of

before

non-lysosomal

Our

(6)

importance

This

previously

tissues

RESEARCH COMMUNICATIONS

cells.

contamination

proteases

cellular

proteases

and other

intracellular

of

parenchymal

some other (5)

AND BIOPHYSICAL

work

are

of

and

regulated rates

of

will

be

physiologic

of proteolysis.

ACKNOWLEDGEMENTS This Institute thank

Diane

work

was

of

Arthritis,

Doach for

supported

by Grant

Diabetes, typing

l-R23-AM29829

Digestive

the manuscript. 1329

and

from Kidney

the

National

Diseases.

I

Vol. 108, No. 3, 1982

8lOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES 1.

Amenta,

JS and Brother,

2.

Neff, NT, DeMartino, 439-458.

3.

Knowles,

SE and Ballard

4.

Bigelow,

S,

5.

Katunuma, Chichibu; 37-50.

6.

Bird, (1980)

7.

Woodbury, Neurath,

8.

Woodbury, 2785-2789.

9.

DeMartino,

GN and Goldberg,

10.

DeMartino,

GN (1981)

11.

Bradford,

12.

Wildenthal, K, Wakeland, JR, Biophys Res Comm -96: 793-798.

13.

Goldberg,

AL and St.

14.

Kameyama,

T and Etlinger,

Hough,

SC (1981)

GN and Goldberg, FJ (1976)

R, and

N, Kaminami, K, Hamaguchi,

JWC, Carter, Fed Proc 2:

Life

Sci 28: -

1195-1208.

AL (1979)

J Cell

Biochem

Rechsteiner,

RE, Brooks,

MM (1976)

GM and

Arch Anal

Lagunoff,

AL (1979) Biochem Biochem

John,

RM and N,

-25:

83-89.

-211:

K, -52:

Spanier,

Lagunoff,

D (1978)

J Biol

Biophys

AM D and

PNAS (USA)

Chem -254:

-75;

3712-3715.

253-257.

-72: 253-257.

Ord,

JM and Stull,

AC (1976) JD (1979)

1330

Cell

E, Banno, Y, Suzuki, T (1975) Eur J Biochem

RG, Everett, M, Sanda, Y, Katumuma, HA (1978) PNAS (USA) -75: 5311-5313. RG, Gruzenski,

101: -

609-617.

M (1981)

E, Kobayashi, Y and Katsunuma,

JH, Triemer, 20-25.

J -156:

Physiol

JT (1980)

Ann Rev Biochem Nature

-279:

344-346.

-45:

Biochem 747-803.