Polyphosphate anions increase the activity of bovine spleen cathepsin D

Vol.

89,

August

No.

4, 1979

28,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1979

POLYPHOSPHATE ANIONS INCREASE THE ACTIVITY Shoji

Watabe,

St.Yarianna Received

July

Atsushi Shigeru

Terada, Taguchi

University

School

1161-1167

OF BOVINE SPLEEN CATHEPSIN D

Tadao Ikeda, Hiroshi and Nagasumi Yago of Medicine,

Kouyama,

Kawasaki,

Japan

213

12,1979

SUMMARY: Bovine spleen cathepsin D is activated by polyphosphate anions when bovineserum albumin is used as substrate at pH 4.6. In the presence of ATP at 10 mM, the catheptic activity at this pH is enhanced as high as 17 times over the control. Similar activating effects were observed, though to varying degrees, with sodium tripolyphosphate, nucleotides, nucleotide analogues, CoA, polyU and yeast RNA. The possible mechanism and biological significance of the activation were discussed with regard to the intralysosomal polyanionic substance. About

a decade

intralysosomal substance

not

focussed

except

difficult

on its

we tried

late

effects

of

Of lysosomal candidate

to be solved

the

polyanionic

enzymes,

to be tested specimen

is

D in the

lymphoid

tissue

cytes

'distribution

easily

by using

anionic obtainable

has attracted as described

bovine compounds

is

suggested

to be

attention

there

challenged

compounds

has

hydroare in a direct

that

might

simu-

lysosome.

spleen for

cathepsin

D as the

two reasons;

on a commercial

basis

much attention by Bowers

active

then

Because

anionic

in the

of

intralysosomal

possibility

several

importance

however,

against

et al.(Z).

the

substance

we selected

with

purified

subcellular

it

before

were

knowledge,

action

by Goldstone

the

of biologically

groups

To our

direct

discussed

uptake

or carboxyl

possible

to approach

(1)

in the

substance.

a discussion

problems

way,

Phosphate

polyanionic

for

and Barrett

substance

by lysosomes. in the

lysis

Dingle

polyanionic

involved been

ago,

first,

rat

its

and second,cathepsin

because

for

first

thoracic

of

its duct

peculiar lympho-

(3).

Abbreviations : pApp, adenosine S'rdiphosphate, 3'-diphosphate; pGpp, guanosine 5'-diphosphate, 3'-diphosphate; polyU,

adenosine 5'-monophosphate, 3'-diphosphate; pppApp, guanosine 5'-monophosphate, 3'-diphosphate; pppGpp, polyuridiric acid.

3'-diphosphate; ppApp, adenosine 5'-triphosphate, 3'-diphosphate; ppGpp, guanosine 5'-triphosphate,

0006-291X/79/161161-07$01.00/0 1161

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@ I979 by Academic Press. Inc. of reproduction in anyform reserved.

Vol.

89,

No.

4,

BIOCHEMICAL

1979

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

MATERIALS AND METHODS: Bovine spleen cathepsin D (Lot 26C-8100), bovine serum albumin [Fraction V, Lot 88C-0252 and crystallized and lyophilized, Lot 98CSOSO), bovine hemoglobin (Type I, Lot 66C-8092), ovalbumin-(Grade VI, Lot 97C8050), bovine pancreatic trypsin (Type III, Lot 66C-00142), hog stomach mucosa pepsin ( Lot 18C-80803, yeast RNA (Type XI, Lot 124C-8150), sodium dextran sulfate (Lot 48C-0177), chloroquine diphosphate (Lot 95C-0387) and potassium polyvinyl sulfate (Lot 94C-0289) were obtained from Sigma Chemical Company, St. Louis, MO.; fluorescamine(Fluram) from F.Hoffmann-La Roche 8 Co., Nutley, coefficient 9.0s) from Yamasa Shoyu ComN.J.; polyU (Lot 828, sedimentation pany, Tokyo; pApp pentalithium salt (Lot 0304), ppApp hexalithium salt (Lot 0407), pppApp heptalithium salt (Lot 0509), pGpp pentalithium salt (Lot 0302), ppGpp hexalithium salt (Lot 0409), pppGpp heptalithium salt (Lot 0507) from Sanraku Ocean Company, Ltd., Tokyo; and globin from sperm whale skeletal muscle (Lot KIJ3338) of amino acid sequence analysis grade from Wako Pure Chemical Industries, Ltd., Tokyo. Yeast cytochrome c and sodium pepstatin were generous gifts from Central Research Laboratories oT Sankyo Pharmaceutical Company, Tokyo and Dr. T.Aoyagi of the Institute of Microbial Chemistry,Tokyo, respectively. All other reagents including sodium salts of adenineand guanine nucleotides were of analytical grade. Our standard incubation mixture contained in a total volume of 200 pl of 50 mM sodium acetate buffer, pH 4.6, 5 mg of bovine serum albumin (Fraction V) and other additions as specified. Reaction was started by the addition of the enzyme dissolved in water. Incubation was for 20 to 80 minutes at 37OC in duplicate. Amounts of enzyme and length of incubation time were adjusted so that a linear time-course was assured. Incubation was halted by the addition acid and the acid-soluble products were deterof 200 ~1 of 6 % trichloroacetic mined by the fluorescamine method (4). The proteolytic rate was expressed as the amount of acid-soluble products in terms of nmoles of leucylleucine per minute. One mU of enzyme activity was defined as that amount of enzyme which liberated acid-soluble products equivalent to one nmole of leucylleucine per minute when acid-denatured bovine hemoglobin was used as substrate in 0.1 M sodium lactate buffer at pH 3.6 (4). The bovine spleen cathepsin D used in the present study had a specific activity of 3.67 mU per pg of dry weight. Purity of the enzyme was 33 % as determined by assuming its molecular weight at 42,000 (5) and by titrating the enzyme activity against acid-denatured bovine hemoglobin at pH 3.6 with sodium pepstatin (6). pH-Values were carefully monitored with a glass-electrode in all experiand no change of more than 0.05 pH-unit ments before and after incubation, was observed for all data to be reported here. Incubation conditions for trypsin and pepsin were essentially the same as for cathepsin D except for different buffers used. The compounds tested did not increase solubility of protein degradation products in trichloroacetic acid nor affect procedures leading to fluorescent measurement except for metal-chelating reagents, i.e. acetyl acetone, cupferon, sodium diethyldithiocarbamate, and ammonium pyrrolidine dithiocarbamate. The latter four compounds showed a slight quenching effect on the fluorescent measurement when used in the incubation at 5 mM, and that was corrected for in calculating enzyme activity. RESULTS AND DISCUSSION: phosphate

compounds

enhance

bovine

serum

albumin

and of

other

compounds

The degree phate

groups

of

the

During

the

the

stimulation

in a given

series

stage

activity

at a pH 4.6. on the

early

of bovine

Table

rate greater

of phosphate

1162

study,

spleen

I summarizes

proteolytic becomes

of this

the

by bovine with

cathepsin effects spleen

increasing

compounds.

we found

Three

that

D against of phosphate

cathepsin number

D.

of phos-

or more phosphate

Vol.

89,

No.

4., 1979

BIOCHEMICAL

AND

BIOPHYSICAL

TABLE Effects

of

phosphates

Compounds

and against

other compounds bovine serum

RESEARCH

I on bovine albumin at

spleen pH 4.6.

Initial reaction at concentrations

tested

cathepsin

D activity

rate with compounds shown below

2mM Control None*l) Inorganic:

COMMUNICATIONS

10 mM

phosphates

lG12”4 NaH2P04

0.104(

Na4P20i Na5P3010 Na5P3010 Nucleotides CAMP AMP ADP ATP GDP CoA Nucleotide PAPP PPAPP

plus

100

and

related

ng pepstatin*3)

173)

0.120(

200)

0.078(

130)

O.lOl(

168)

0.146(

243)

0.428(

713)

0.506(

843)

0.790(1317)

O.OOO(

0)

O.OOO(

0)

compound 0.076( 127) 0.105( 175) 0.112( 187) 0.644(1073) 0.146( 243) 0.263( 438)

0.122 ( 203) 0.170( 283) 0.657(1095) 1.040(1733) 0.436( 727) 0.364( 607)

analogues 0.471( 785) 0.519( 865) 0.777(1295) 0.293( 488) 0.452( 753) 0.742(1237)

E? PPGPP Po?FzEE?eotides*4) Yeast RNA PolyU Inorganic sulfates Na2S04

0.117( 0.064(

Na2S207 Organic polysulfates*4) Sodium dextran sulfate Potassium polyvinyl sulfate Carbonates Potassium succinate Potassium tartarate Potassium citrate Disodium EDTA

195) 107)

(0.004%) (0.004%)

0.200( 0.157(

333) (0.02%) 262)(0.02%)

0.088(

147)

0.114(

190)

0.091(

152)

0.157(

262)

0.066( 0.045(

110) (0.04%) 75) (0.04%)

O.OOO( 0.008(

0.072( O.lOO( 0.115( O.lOl(

120) 167) 192) 168)

O.llO( 0.106( 0.136( 0.290

0) (0.2%) 13) (0.2%) 183) 177) 227) ( 483)

*l) The amount of enzyme used in each incubation was 7.50 mu. l 2) Values shown in the parentheses are percent changes, control value being nonna.lized as 100. *3) Since 1 ng of sodium pepstatin inhibited 0.75 mU of the enzyme, the amount of the inhibitor used here was a 10 fold excess to inhibit all of the enzyme. *4) The concentrations of polynucleotides and organic polysulfates are shown in the Parentheses.

groups

in

catheptic were

a single

molecular

activity. most

potent

Thus, in

the

species sodium stimulation.

are

very

efficient

tripolyphosphate, Sodium

1163

ATP pepstatin

in

stimulating

and

nucleotide

could

completely

the analogues abolish

Vol.

89,

No.

the

catheptic

other

4,

activity

acid

observed fact, tic

BIOCHEMICAL

1979

than

of phosphate

chloroquine,

an inhibitor

at 10 mM with of deaminase

by fluorescamine, the

Because

with

Materials

and Methods Thus,

the

may not

be interpreted

Inorganic

albumin

slightly

and third,

that

trypsin 2 to

phosphate nor

specific

the

to 4.6

with

for

the

affect

the

added.

cathep-

In addition,

the

fluorescence

especially

for

added. the

catheptic

affect

the

activity,

listed

under

enzyme

activity

as of polyphosphate

metal-chelating

the

compounds

function.

in stimulating

sulfate

effect

of

(Figure

1).

the

and potassium

catheptic

polyvinyl

most probably

toward

optimal the

except sodium

serum

borate

that

it

due to precipi-

albumin

profiles

activating

effect

pH for

degradation

acidic

side,

that

was observed

studies mucosa

as substrate no increase

were

observed

of polyanionic

D.

1164

with

pH 3.6,

pH,

was greatest

in the

using

pancreatic

bovine

pepsin

sodium

substances

range

in a pH-range acetate,

proteolytic

in

activity

at the higher

and sodium

in the

of bovine

i.e.

the maximal

tripolyphosphate

In pH-activity

buffers,

less

second,

of 4 to 9 and ho g stomach

bovine

cathepsin

the

tripolyphosphate,

in pH-activity

Thus,

that

unchanged

or sodium

change

added.

sodium

in a pH-range 7, both

of

substrate.

was shifted

essentially

As a matter

compounds

not

effective

dextran

the

conditions

four

did

enzyme activity,

: first,

of

pH 3.5

to the

Those

of their

somewhat

were

remained

from

basis

not

for

increase

in stimulating

EDTA as well

sodium

inhibitory

findings

presence

were

contrary,

of protein Other

on the

would

analogues

5 mM but

of disodium

sulfates

To the

tation

the

effect

at

did

present

Therefore,

responsible

tripolyphosphate

the

reagents. used

(7),

present,

under

not

I.

compounds.

Bl

or nucleotide

metal-chelating

at all.

were

detected

were

other

sodium

COMMUNICATIONS

in Table

were

any,

cathepsin

which,if

RESEARCH

example

as of

EDTA was effective

other

sulfate

of

nucleotides

we tested

activity.

as well

activity

disodium

D, if

or without

was not

incubations

serum

cathepsin

effects

any trace

BIOPHYSICAL

as shown by one such

proteases

activity

AND

activity

tripolyphosphate seems to be

of

sodium

Vol.

89,

No.

4,

BIOCHEMICAL

1979

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

2.0 .-c E , 1.5 a 0 ‘; E 1.0

.; 0.5 .-> :

0 1

2

3

4

5

6

7

0

PH

Figure 1. pH-Activity profiles of bovine spleen cathepsin D with bovine serum albumin as substrate in the presence (solid curve) and absence (dotted curve) of 2 mM sodium tripolyphosphate added. To adjust pH-values, 2 mJl potassium phosphate buffer was used for a pH-range between 2 and 3 (0,~ ), 2 mM sodium lactate buffer between 3 and 4 ( 0,. ), 2 mM sodium acetate buffer between 4 and 5 ( A,h ) , and 2 mM potassium phosphate buffer between 5 and 8 ( 0,m ), respectively.

Degrad.ation cytochrome shown)

of other

.E and crystallized

was .also

stimulation

enhanced

nor

hemoglobin Instead,

change

or globin th’ere

to partial

protein

substrates

(Figure

and lyophilized

bovine

at pH 4.6

by sodium

in pH-optimum

were

from

sperm

whale

was some inhibitory

precipitation

of substrate

i.e.

serum

ovalbumin, albumin

tripolyphosphate.

observed

skeletal

effect

2),

(data

However,

in these

was used

incubations,

in the

not no

when acid-denatured

muscle

proteins

yeast

bovine

as substrate. probably

presence

of

due

sodium

tripolyphosphate. With

regard

to the

vated,

we compared

Sodium

chloride

identical (data

effect not

exerted ionic

that

the

various

increased

slhown) . Figure

strength.

by their

polyphosphate

the

spleen

in Table

when potassium

or lithium

that

effects of the

activate

than other

to ionic bovine

1165

sodium

strength. spleen

of

D is ionic

actistrength.

concentrations. chlorides

tripolyphosphate

anticipated compounds

cathepsin

I in terms

at higher

contribution anions

bovine

enzyme activity

3 indicates

greater The effects

by which compounds

was observed

significantly

interpreted

mechanism

from like It

cathepsin

were used and ATP

the

disodium is

Almost

thus D first,

increase

in

EDTA may be suggested by

Vol.

89,

No.

4,

BIOCHEMICAL

1979

AND

0 025

0

2

10

,” .> .-

500

i

100

0

20

Tripotyphosphate

BIOPHYSICAL

Increase the

COMMUNICATIONS

0 0

3

(mM)

RESEARCH

0.1 in

control

ionic

0.2 strength

over

incubation

Figure 2. Effect of sodium tripolyphosphate on the activity of bovine spleen cathepsin D. For the incubation besides bovine serum albumin ( l ), 4.0 mg acid-denatured bovine hemoglobin ( m ), 5 mg ovalbumin ( @ ), 1.3 mg yeast cytochrome c ( 0 ) or 2.5 mg sperm whale globin ( Cl ) were used. The conditions were the same as for the standard incubation with bovine serum albumin. Figure 3. Effect of increasing ionic strength on the activity of bovine spleen cathepsin D with bovine serum albumin as substrate. Sodium chloride ( 0 ) was added to the standard incubation medium at concentrations that gave the values of ionic strength shown. Data for sodium ATP ( n ), sodium tripoly) and disodium EDTA ( Cl ) were taken from Table I and phosphate ( l Figure 2 and plotted together with some additional experiments. Ionic strength was calculated assuming that all of the dissociahle groups have ionized, and was expressed as the increase in ionic strength over the control which had a When sodium salt of ATP and sodium tripolyvalue of ionic strength of 0.0715. to adjust the pH-value by adding a small phosphate were used, it was necessary The increase in ionic strength due amount of acetic acid or sodium hydroxide. to these additions was also included in the calculation. increasing

the

exerting

some

between If

ionic

strength

unknown

effect

phosphate the

groups.

to the might

albumin

have

importance

biological

effect

would

be rather

close

would

on the

interaction

is

shared should , then for

Even apart

from

in regulating also

synergistic

to optimal

substance,

significance

of our results

and second,

in vivo

proteins.

polyanionic

low concentrations

substance

degradation

and some other

intralysosomal

based

polyanionic

on protein

pH of 4 to 5 (8) serum

probably The former

intralysosomal

to our findings

at relatively

lie

1166

some

with

carbonates.

exert the

effects

reported

cathepsin the

compounds

for

similar intralysosomal

D to degrade

potential such

proteolysis

in describing

by

relevance as nucleotides

in lysosomes. the

first

time

The a

BIOCHEMICAL

Vol. 89, No. 4, 1979

group lation

of activators

of

by polyphosphates

cathepsin

AND BIOPHYSICAL

D. More detailed

and inhibition

RESEARCH COMMUNICATIONS

study

by polysulfates

on mechanism is being

of

stimu-

undertaken.

ACKNOWLEDGE:MENTS : We would like to express our sincere gratitude to Dr. Fumio Sawada, Divm of Biochemistry, National Institute of Radiological Sciences, Chiba, and Prof. Shiro Horiuchi, Department of Physiology, Life Science Institute, Sophia University, Tokyo, for their valuable discussion. We are also grateful to Sr.Jean Michalec, Department of Biochemistry, Life Science Institute, Sophia University, Tokyo, for her reviewing this manuscript. REFERENCES 1. Dingle, J.T., and Barrett, A.J. (1969) Proc. Roy. Sot. B. (UK), 173, 85-93. 2. Goldstone, A., Szabo, E., and Koenig, H. (1970) Life Sci. Part II, 2, 607616. 3. Bowers, W.E. (1972) J. Exp. Med., 136, 1394-1403. 4. Yago, N., and Bowers, W.E. (1975) J. Biol. Chem., 250, 4749-4754. 5. Ferguson, J.B., Andrews, J.R., Voynick, I.M., and Fruton, J.S. (1973) J. Biol. Ch(em., 248, 6701-6708. 6. Knight, C.G., and Barrett, A.J. (1976) Biochem. J., 155, 117-125. 7. Wibo, M., and Poole, B. (1974) J. Cell Biol., 63, 430-440. 8. Ohkuma, S., and Poole, B. (1978) Proc. Natl. Acad. Sci. USA, 3, 3327-3331.

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