Vol. 64, No. 2, 1975

BIOCHEMICAL

Serum Factors

Affecting

Mizue (Zoological

Institute,

April

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cathepsin

Wiyamoto Faculty

and

Release Hiroshi

of Science,

from Lysosvmes

Terayama

University

of Tokyo,

Tokyo,

Japan)

1,1975

SUMMARY: Rat or bovine serum exhibited t,he inhibitory effect, on cathepsin release frvm rat liver lysosvmes in vitro. The inhibitory activity of serum was enhanced after dialysis. HeatedO' C, 3-5 min) serum showed the marked stimulatvry effect on the cathepsin release. It was indicated that the adult and infant, rat. or bovine sera contain a large-molecular, heat-labile inhibitory factor and a small-molecular-heat-stable stimulatvry factor. Fetal bovine serum, however, was found to be very poor in these factors. The activity of each of the serum factors was found to change characteristically within a short period after partial hepatectomy. Although biology,

tissue

the real

proliferation

mechanisms

have not yet

we have reported cell

coat

acid

that

of cell

may be responsible inhibitors

mitosis

regeneration

Adams (4)

well

been

temporary

showing

fully

disappearance

proliferation

as that

by Allison

pepstatin)

and migration

nucleic

disappearance

of them from

Copyright o I9 75 by Academic Press, Inc. All rights of reproduction in any form reserved.

acid

of cell

as detected

soon after

the deep inside

617

by the

related

since early

with

cathepsin cathepsin

periods

coat

of liver

accompanying

reports,

the

partial number

the

the report

enzyme activities

in

papers

(RNA, DNA) biosynthesis,

to our previous

a decrease

in

cell

the lysvsomal

during

of some lysosomal

showing

coat,

regeneration

injected

fl-glucuronidase) (5)

of cell

2) and that

In connection

the elevation

of tissue

In the preceding

of liver

the

subjects

seems to be intimately

(1,

the initiation

(3).

control

understood.

stain,

as the temporary

phosphatase,

one of the most attractive

the homeostatic

or retarded

acid

particles

for

and/or

inhibited

as well

DNase,

for

(leupeptin

regeneration

is

mucopolysaccharide

the initiation

liver

regeneration

by

(acid

hepatectomy

as

of lysvsvmal

to the periphery

of liver

Vol. 64, No. 2, 1975

cells within

BIOCHEMICAL

a few hours after

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

partial

hepatectow

seemto be particularly

interesting. During the course of searching for possible humoral or cytoplasmic factor(s)

responsible for the V1lysosomalactivation",

or bovine serum contain factors

affecting

we have found that rat

the permeability

of lysosomal

membranesagainst cathepsin.

MATERIALSAND -___ METHODS Lysosomeswere prepared from the liver 150 g.

A 1:8 (w/v) liver

of male, Wistar rats weighing 100-

homogenatein 20 mMTris (pH 7.2) - 0.25 M sucrese

was subjected to the successive centrifugations,

750 x -g for 10 min, 3,300 x

g for 10 min, and then 15,000 x -g for 20 min, to sediment lysosome-rich pellet, which was Once gently rinsed.

The lysosomal pellet

derived from 10 g liver

was suspendedin 20 ml of 0.25 M sucrose - 1 mMEDTA, pH 7.2 (lysosomal suspension).

Measurements of cathepsin release were carr:c?

,>ut as follWJs.

A 2.0 nit aliyuot

of the lysosomal suspension and a small volume (0.1-0.6

of test material

or 0.9s NaCl as the control

ml)

were mixed and the mixture was

incubated at 37’ C for certain periods of time with gentle shaking, followed by centrifugation

at 15,000 x g for 20 min.

The pellet

was suspendedin 2.0

ml of 0.25 M sucrose - 0.2% Triton X-100 and the suspension was kept at O0 C for 60 min, followed by centrifugation

at 15,000 x -g for 20 min.

supernatant thus obtained was used for the assay of the "residual cathepsin D activityI'.

The "total

cathepsin activity"

2.0 ml of the lysosomal suspension directly D were

with Triton.

carried out by the method of hnson (6).

618

The (unreleased)

was measured by treating Assays for cathepsin

A 0.1 ml aliquot

of supernatant

Vol. 64, No. 2, 1975

from

the

Triton-solubilized

acid-denatured incubated 1.0

solution

at 37O C for

(Total

inhibition)

material

release

control)/$Release

stimulation

and - sign

for

inhibition.

was defined

as the activity

will

The more detailed

be presented

removing

the median Newborn

(8).

in the

and adult

respectively. Science

Bovine

Research

Rockville,

and left

sera

Institute

U.S.A.)

sera were (adult and fetal

was a gift

from Dr.

for

or stimulator

from

and Anderson

and f;-week

the Mitsubishi

serum (Slow

Y. Mizuno

serum factors out by

to Higgins

from 5-day were

of the

was carried

according

prepared

bovine

: + sign

the assay

hepatectomy

and calf)

stimulation

or 10% stimulation,

for

lobes

control

as 100 x

of test

of inhibitor

1% inhibition

lateral

rat

D was defined The percent

in the

Partial

of

acid-soluble

in the presence

conditions

text.

by the addition

at 700 nm by the method

One unit

to cause

prepared

pH 3.2 and

containing

of cathepsin

as 100 x ($Release

the

respectively.

was stopped

activity.

- $R e 1 ease in

ml of freshly

buffer,

to calorimetry

activity)/Total

was defined

1.9

The supernatant

The percent

- Residual

with

M acetate

The reaction

was subjected

(7).

activity

0.18

acid.

hydrolyzates et al.

was mixed

in

60 min.

ml of 10% trichloroacetic

of Lowry

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lysosomes

hemoglobin

hemoglobin

(or

BIOCHEMICAL

old

rats,

Life

Laboratories,

in our Institute.

RESULTS AND DISCUSSION ___In Fig. liver Inhibition

l-a

lysosomes

are in

the presence

of cathepsin

more and more marked

illustrated

release

the kinetics

of cathepsin

and the absence by the

as the incubation

of adult

serum appears time

619

increases,

D release rat

to begin

from rat

serum. at 2 h, become

and finally

become

Vol. 64, No. 2, 1975

BIOCHEMICAL

0

Fig.

1

Kinetics presence

1.

2

3

AND BIOPHYSICAL

L

5

0

I

2

RESEARCH COMMUNICATIONS

3

L

5

6

of cathepsin D release from rat liver lysosones of rat serum (a) or of heated rat serum (b).

in the

(a) 0.2 ml of freshly prepared adult rat serum was added to 2.0 ml of lysosomal suspension. As the control, 0.2 ml of 0.25 M sucrose-l mM EDTA was added. (b) Adult rat serum was mixed with an equal volume of 0.9% NaCl, heated at 1OOoC for 5 min, cooled in ice-water and centrifuged. 0.6 ml of the supernatant (equivalent to 0.3 ml of the original serum) was added to 2.0 ml of the lysosomal suspension. As the control, 0.6 ml of 0.9% NaCl was added. 0 : control, l : adult rat serum, and A : heated adult rat serum.

almost

complete

appears

at 4-5

to proceed

experiments, factor.

4.5

almost

bovine

rat

and calf

serum inhibits

serum was found contain

Table sera only

of dialyzed

inhibitory

while

the release

time.

Therefore,

was adopted I,

can inhibit

assaying

cathepsin

dialysis,

which

to increase

be noted

release,

620

in the

the following

serum inhibitory

sera

as well while

the inhibitory suggesting

that

that

the

fetal

the sera

effect

the

inhibitory animals

as the

activity

the

as the age of donor factor

the control

in the

may counteract

should

in

and bovine

Furthermore,

after

It

of a stimulating

rat

the

slightly.

factor.

for

the adult

factor,

serum appears

The presence

with

to be increased

a small-molecular

molecular

linearly

h incubation

As shown in

newborn

h of incubation,

of may

of the largeactivity becomes

serum was more directly

older

BIOCHEMICAL

Vol. 64, No. 2, 1975

Table

Inhibition of cathepsin and dialyzed sera.

I.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

D release

from lysosomes

by various

sera

0.3 ml of various sera or dialyzed sera (see Fig. 2) from different donors was added to 2.0 ml of lysosomal suspension. As the control, 0.3 ml of 0.9s NaCl was added. The mixtures were incubated at 37' C for 4.5 h.

Addition

Exp. No.

$ Release

None (central)

50.7

----

Adult

36.7 36.2

27.6 28.6

34.5 36.5

32.0 28.0

rat

Newborn

serum

serum a) Bovine serum Calf serum

I

Fetal

rat

bovine

serum

None (control) Adult II

rat

serum

Dialyzed Bovine

adult

rat

serum

serum

Dialyzed

bovine

serum

None (control)

III

a)

kinetics rat

adult

Dial;rzed

newborn

Dialyzed

adult

Dialyzed

calf

Dialyzed

fetal

serum.

by observing

release

Therefore,

serum rat

serum serum a)

bovine bovine

9.1

44.4 32.5

me--

26.8 30.4 15.7

39.6 31.5 64.6

55.7

----

33.0

40.7

41.7 41.8

25.1 32.3 24.9

47.3

15.1

26.8

37.7

serum serum

effects in

of heated

effect

the following

621

In Fig.

and the

effect

of heated

and to become less in

sera.

the presence

to the inhibitory

stimulatory

1 h of incubation

prolonged.

the

In contrast the

rat

46.1

serum was used.

of cathepsin

dialyzate, after

Dialyzed

Immobilized

evidenced

'$ Inhibition

serum marked

experiments

l-b

absence

of whole

we show the

of heated

adult

serum or serum

seems to be most evident as the incubation the

stimulatory

time factor

is

BIOCHEMfCAL

Vol. 64, No. 2, 1975

Table

II.

Stimulation sera.

of cathepsin

AND BIOPHYSICAL

D release

from

RESEARCH COMMUNICATIONS

lysosomes

by various

heated

Heated sera were prepared from various sera from different donors as 0.6 ml of heated serum (equivalent to 0.3 ml of original described in Fig. l-b. serum) was added to 2.0 ml of lysosomal suspension and the mixture was incubated at 37O C for 1 h. As the control, 0.6 ml of 0.9s NaCl was added.

Addition

$ Release

8.2

None Heated

adult

Heated

newborn

Heated Heated

bovine serum a) calf serum

Heated

fetal

a)

rat

serum rat

was assayed activities

serum

bovine

Immobilized

25.9

210

21.4

161

22.9 21.4

161

179

8.7

serum

at 1 h of incubation.

except

---

6

serum was used.

of various

the sera

$ Stimulation

the

sera fetal

In Table

heated

at 100"

bovine

II

C for

serum contain

are

compared

5 min.

It

the

s'cimulatory

appears

the heat-stable

that

all

of

stimulatory

factor.

for

AS

shown in Fig.

2, almost

both

the inhibitory

factor

heated

serum within

a rational

basis

Based factors

for

in dialyzed

below

in

Table

at various

assay

III,

after

partial

higher

the initial

the

factor

hepatectomy, level

of the

after

stimulatory

reach

at around

stimulatory

factor

partial

of the rat

hepatectomy.

was found a maximal

level

partial

serum As

to increase almost

in

affording

serum factors.

activities

Z-3 h after

622

can be observed

or 140% stimulation,

relation,

times

relation

serum and the

354 inhibition

the quantitative

measured

immediately than

dose-response

on the above dose-response

were

sum?larized

a range

linear

Lfold

hepatecttomy

and

BIOCHEMICAL

Vol. 64, No. 2, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

50

1 - 30 =II: 20 f: IO LO

0

01

02

Dialyzed

Fig.

2.

or

Dose-dependent inhibition lysosomes by dialyzed rat

03

OS

heated

5erum

05

06

I ml I

or stimulation of cathepsin serum or heated rat serum.

D release

from

Various volumes of adult rat serum dialyzed against 0.9s NaCl at 4oC overnight or heated adult rat serum prepared as described in Fig. l-b were added to 2.0 ml of lysosomal suspension. The final volume was adjusted to 2.6 ml by adding 0.9% NaCl. The mixture was incubated at 37oC for 4.5 h in case of dialyzed serum or for 1 h in case of heated serum. Percent inhibition or stimulation was measured as described in Materials and Methods.

Table

III.

Change

of activities

of the

serum factors

after

partial

hepatectomy.

Serum was prepared from 2 rats at various times after partial hepatectomy. The 0 h serum was prepared from 2 sham-operated rats. Dialyzed or heated serum was prepared as described earlier (Fig. 1, Fig. 2) and used for the assay of the inhibitory factor or the stimulatory factor, respectively. In each assay, two different volumes of serum (usually 0.1 and 0.2 ml) were added to 2.0 ml of lysosomal suspension in order to check the linear dose-dependency. The mixtures were incubated for 1 h or 4.5 h to measure the stimulatory factor or the inhibit,ory fact,or, respectively.

Time after hepatectomy

decline remained

partial (h)

Activity Stimulatory

0

62

3

136

6

93

gradually unaltered

thereafter. for

the first

On the

(units/ml factor

factor 126

12-7 180

other

few hour

623

serum) Inhibitory

hand,

period,

the inhibitory followed

by the

factor gradual

BIOCHEMICAL

Vol. 64, No. 2, 1975

increase. (5,

Taking

6) as well

accomplished in this

paper

as the within

partial

early

to conclude

factors

considerations initiating

a very

showing

after

regeneration.

into

the

hepatectov that

that

stimulus

early

period

temporary

this

the apparent for

as well

partial

of the

interesting,

may be an actual

lysosomal

hepatectomy,

serum

chemical

the results

stimulatory

although

it

trigger

as the mode of actions

activation

(9) seem to be

the regeneration

after

surge

seems quite

The nature

are now under

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

factor may be still

for

of these

soon too

the liver serum

investigations. REFERENCES

1. 2. 3. 4. 2:

7. 8. 9.

Yamamoto, K., Omata, S., Ohnishi, T., and Terayama, H. (1973) Cancer Res. 33, 657-572. Ohnishi, T., Yamamoto, K., and Terayama, H. (1973) Histochemie 2, 15-20. Miyamoto, M,, and Terayama, H. (1973) Biochem. Biophys. Res. Commun. 2, 84-90. Adams, R. L. J'. (1963) Biochem. J. 87, 532-536. Allison, A. C., and Mallucci, L. (lg4) Lancet 26, 1371-1373. Anson, M. L. (1938) J. Gen. J)hysiol. 22, 79-89. N. J., Farr, A. L., and Randall, R. J. (1951) Lowry, 0. H., Rosebrough, J. Biol. Chem. 193, 265-275. Higgins, G. M., and Anderson, R. M. (1931) Arch. J'athol. 12, 186-202. Fuji&a, M., Koga, M., and Lieberman, I. (1963) J. Biol. mem. 238, 34°1-3406*

624

Serum factors affecting cathepsin release from lysosomes.

Vol. 64, No. 2, 1975 BIOCHEMICAL Serum Factors Affecting Mizue (Zoological Institute, April Received AND BIOPHYSICAL RESEARCH COMMUNICATIONS...
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