Proteases from mouse submaxillary gland

Vol. 36, No. 1, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PROTEASES FROM MOUSE SUBMAXILLARY GLAND Isaac

Schenkein-:?, Mary Boesman, Edward Tokarsky, Louis Fishman and Milton Levy

Dept.

Received

of Biochemistry, College of Dentistry, New York University, New York

May 28, 1969

SUMMARY Two proteases (“A” and I’D”) from mouse submaxi 1lary gland have been isolated in high purity. At pH 8.5 trD” is more negative than “A” and is also smaller. Amino acid composition differs only in 6 constituents. “D” is activated 100-3CX3°/o when acting on tosyl arginine methyl esther by tosyl arginine and all&-amino acids. Both enzymes have a strong preference for arginyl bonds. We recently of Nerve

reported

Growth

and unusual

the

which

are devoid

report

as “A”,

(Fig.

pur i ty.

We have Both

substituted

ones.

of four

isolated

arginyl

to their

it

carboxyl

enzymes in this electro-

and “Dl’ in high

of activity

groups

glands

Irvington Medicine, Medicine,

“A”

appears

is activated

of this

gel columns

toward&N,

A number of protein too

activity

designation

acrylamide

The method of their submaxillary

specific

isolation

estero-proteolytic

two of these,

esters.

the

development

Their

on analytical

and here

address:

high

“C1’, and IrDt7 relates

Enzyme “D”

EXPERIMENTAL

-::- Present

separation

arginine

bonds involving

Two thousand

Further

show a marked degree

have been studied

tible

(1,2).

“B”,

method for

very

of NGF activity.

mobi lities

1).

(NGF) with

stability

method allows

phoretic

Factor

an improved

that

subtrates the peptide

are the most suscep-

by all&-NH2 isolation

from adult

acids.

is as follows: male mice

(over

House Institute and Dept. of New York University School of New York. 156

Vol. 36, No. 1, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure I. Analytical acrylamide gel electrophoresis of the fraction from carboxy methyl column chromatography and enzymes !1A11and I’D” after preparative acrylamide gel electrophoresis.

30 gm) obtained

frozen

from Pell-Freez

thawed and homogenized with

one liter

in a Waring

of 0. 1 M phosphate

preparation

was centrifuged

precipitate

discarded.

sulfate

(adjusted

volumes

of extract.

to pH 7.4 After

added to each

15 min at O-4” dis,carded of the

and

the mixture

with

at pH 7.4.

10 min and the

of 0.2 M streptomycin

3-s’,

the

Seven ml of cold 100 ml of the

(-15’) After

precipitate

was

absolute

supernatant.

was centrifuged.

157

The

NaOH) was added to each nine

3 hr at

supernatant.

were

at maximum speed,

lO.OOOxG for

48 ml of ethanol

streptomycin

blendor buffer

One volume

removed by centrifugation. was slowly

at

Biologicals,

ethanol After

The pellet

was

added to each 100 ml 2 hr at

-1’j5”,

centri-

Vol. 36, No. 1, 1969

fugation

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

gave a gummy red precipitates.

500 ml of cold to 35”/0

water

saturation

with

24.5

gm of the solid

3-5O,

the precipitate

supernatant

by adding

original

solution. at

35’ gm of that After

of NGF (3). all

NGF nor

of water.

This

in a small

against

165 cm column of DEAE-Sephadex as in method “B1l given

column was thoroughly

of separation

Further plished

by preparative

“Fractophoratorll obtained

for

with

shown in Fig-II.

purification

the various

was lyophi

A-25

to obtain

Peak II

gel

in Ref.5. steps

of the 158

lized,

at pH 7.5. was applied

(Pharmacia) (4).

The

buffer.

the pattern

contained

Peak V contained

acrylamide

with

and exhaus-

the Tris

of enzymes “A”

described

recovered

by the manufacturer

on at 250 ml/hr

was kept was retained

mg of protein)

equilibrated

of the enzymes of interest.

rate

HCl buffer

to a 2.5~

was carried

by the method of

of water,

of the dialysate

Elution

(200-500

cm

the estero-proteolytic

eluate

M Tris

of

to a 2.5~60

red pigment

volume

a 0.05

in

unti 1 free

The flow

of the

was centridisolved

prepared

A portion

prepared

it

was applied

ce 1lulose,

ammonium

each 100 ml of

They were quantitatively

disolved

dialyzed

for

and dialyzed

fraction

but neither

2-3 column volumes the residue

1 hr at The

with

the precipitate

water

Virtually

enzymes were he Id.

tively

30 min,

isolation

by the cellulose,

by adding

After

2-4 hr in the cold

This

below 3 ml/min.

salt

in

was brought

to ammonium sulfate

to 85’/ o saturation

distilled

the

The solution

100 ml of solution.

of carboxy-methyl

Cohen for

was disolved

was removed by centrifugation.

ammonium sulfate. column

per

15,OOOxG for

100 ml of cold

stirring. respect

was brought

sulfate

fuges

with

This

a mixture

the NGF activity.

and “D” was accom-

electrophoresis Table

using

the

I shows the yie Ids

isolation.

Vol. 36, No. 1, 1969

BIOCHEMICAL

0

0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

+ I ’ 100 Fraction

+ ’ No.

I 200

Figure II. DEAE-Sephadex column chromatography text. The arrows are points of addition of 0.1 0.2 M NaCl in the buffer respectively.

described in M NaCl and

TABLE I Frac t i on

Protein

Crude homogenate 24.800 85O/o ammoni urn sulfate 3680 Carboxy methyl fraction 2362 DEAE-Sephadex peak II I’J-JI’ Preparative Electrophoresis “A” (1)

Specific activity

Esterase mg

3.65~10~

'I

2.66~10~

”

725

'

”

2.32~10~

”

1,000

”

7.2

11

1,800

n

~10~

6.9-10.4~10~ 2.6 x105

units

” ”

150 U/mg

The enzyme unit is defined as the amount of protein that hydrolyses 1 p M of TAME/min at pH 8.0 and 37’. The “no enzyme” rate under these conditions is about 0.015 pm/min and is subtracted. The reaction mixture consists of 3 ml of solution which is 0. 1 M in Kcl, 0.0053 M TAME and 0.0026 M glycine. The reason for the presence of glycine is discussed in the text.

(2)

The total esterase units of the crude homogenate reflect the combined action of a variety of enzymes in the mouse submaxi 1 lary g land (e. g. , Kallikrein) that act on the substate (TAME) that served for the definition of the enzyme unit. These various enzymes have different specific activities and this must be taken into account in the evaluation of the table of yields.

(3)

We have observed considerable variability in total protein We ascribe as well as esterase units in these preparations. it to the age of the submaxillary glands used as starting material.

159

Vol. 36, No. 1, 1969

Figure (stained

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I includes

with

described

amido black)

in the

of protein

the analytical

isolation

bands indicating

gels

of the enzymes after At

procedure.

added to the gels,

as single

acrylamide

both

a high

the

leve Is of

enzymes “A” degree

(6) last

step

100-150 pg

and “D”

ran

of purity.

TABLE II

Amino Acid

Composition

of Enzymes “A”

Residues/M ana lyt ica 1 "A 11 ,!D’, Aspartic acid Threonine Serine Glutamic acid Proline Glycine A lani ne Cysteine Va line Isoleucine Leucine Tyrosine Pheny 1 a lanine Methi onine Lys i ne Histidine Arginine Tryptophan (from U.V. absorbance) Residue wts.

Residues/M inteoers

30.0 30.9 15.4 16.3 18.2 17.8 21.8 21.9 20. 1 16.0 g::

:gi 816 9.1 5.9 25.1

9.4

13.7 11.7 27.5

and “D”

31 15 18 22

31 16 18 22

:‘;

;E 15

1; 12 27

; 6 ‘3 9

: 5 22

: 22 : 7

c 12

30,643

( “A” ) , 28,330

( “D’)

The results given are the averages of 5 analyses of each enzyme using hydrolysis times of 24, 48, and 72 hr. The M ratios were calculated using the parenthetical figures as guides. Table acid

II

hydrolysis.

ti on between

shows the amino acid There lfAf’ and “D”.

content

of

the enzymes after

is a remarkable

similarity

Significant

differences

160

of composiare

found

Vol. 36, No. 1, 1969

only

in 6 amino acids.

gives

a molecular for

28,000

Summation

of the

found

of about

30,000

for

weight

weights

and 2.8

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1rD71in good agreement

molecular

with

residue-weights

enzyme “A”

the estimates

based on the observed

S20 values

and

of their of 3. 1 S

S respectively.

Rabbit Sephadex to

BIOCHEMICAL

antiserum

to the material

column was prepared.

Enzymes “A”

be immuno-electrophoretically

genitally

related

activity

of both

(7).

in peak II

of the DEAE-

and rrD’7 were found

pure and distinct,

The antiserum

enzymes.

It

abolished

does not

affect

but antithe enzymatic

the

action

of

trypsin. TABLE III

Hydrolyzed

Not hydrolyzed

Tosyl arginine methyl ester Benzoyl arginine ethyl ester POlyarginine Cyt.ochrome “Ct’ Lys 0 zyme Arginine and lysine rich histones Protamine sulfate

Table assayed

III

with

set at pH 8.0 of enzymatic hydrolysis methyl

ester

shows the various the enzymes.

ment of alkali

Leucine ethyl ester Lysine methyl ester Po ly lys i ne Arginyl-g lycyl-g lycine Alanyl glycine Glycyl alanine Glycyl pro line Glycyl leucine Valyl glycine p-nitro-phenyl acetate Glycyl-Glycyl a lanine

uptake

Hydrolysis

using

or by form01 activities

Form01 titrations

titration

solution

(Radiometer

by measureCopenhagen)

For rapid

in color

produced

of p-toluene

that 161

have been

(9).

in the presence indicated

that

was followed

a pH stat

the change

of an alkaline (TAME)

substrates

of phenol

scanning by

sulfonyl

arginine

red was used.

polyarginine

(M.W.

about

Vol. 36, No. 1, 1969

150,000)

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was rapidly

peptides,

with

lysine

hydrolyzed

some release

(M.W.

150-200,000) of protamine

histones

correlated

these

proteins

Addition lysis form01

well

titer

arginine, attacked

with

while

lysine

with rich

or

with

The extent

lysine

rich

content

C-terminal histones

and “D” or a mixture

consistent

poly-

at a 11.

the arginine

peptides

to the

enzymes “A”

and tri-arginine

and of arginine

and yielded

of trypsin with

of free was not

of hydrolysis also

to di-

the additional

of

arginine.

after

hydro-

of both,

gave a

splitting

of the

lys ine bonds. Figure

III

shows the

enzymes “A’, and lIDlIe

reaction

mixture,

significantly

This

the

activation

examination that

all

product

of the effect amino acids of rate

Several

of the curve

of p-tosyl

Simi lar

rate. of the

reaction,

amino acids.

are activators

to varying

of hydrolysis acids

range

tested

were as effective

for

arginine

to

in a

additon

of

has no effect.

of the hydrolysis

of free

D-amino

and phenylalanine)

nature

of TAME by

at the same time

initial

by a product

Enhancements 3oo”/o -

resulting

other

of hydrolysis

upon addition

enhanced

methanol,

course

The sigmoid

enzyme “D1’ is abolished the

time

led to the It

was found

degrees.

between

(alanine,

10Q”/~

and

glutamic

acid,

as the corresponding

L-isomers. Comparison

of sodium acetate,

indicated

that

only

activated

by either

The Km’s for determined 6.6x lo-* the values

for M/l

latter

amino acids the

both at 37’.

obtained

the

hydrolysis

enzymes.

acetamide,

activated. or tosyl

Enzyme “A”

is not

arginine.

of TAME at pH 8.0 were For enzyme “A”

For enzyme “D1’ (activated range

and glycine

from 2.4 162

to 3. 1x10-*

the

value with

M/l

is glycine)

at 37’,

Vol. 36, No. 1, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure III. Time course of action of 2.3x 1o-5 M) a. Enzyme “A’* with b. Enzyme lrDtf without activator. tosyl arginine initially present.

because

of some uncertainty

weaver-Burke of these

plots.

trypsin

shows that

appear

less

protamine reaches

active

various

from 500/, activity

100°/,.

versus

times

the extent

activities with

though

of hydrolysis

reached

that

of They

more active.

as substrates

as that

Cue’,

of the Cu”

enzyme activity

esters

protein

Fetf”, to

TAME and similar

with

eventually

by trypsin.

on enzyme “D1’ have been done with

At concentrations

agents.

1x1o-5 M of

specific

with

studies

of Line-

of the

are from 2-15

same point

Inhibition

interpretation

they

or polyarginine the

in the

A comparison

enzymes towards

of enzymes on TAME. (3 ml or without tosyl arginine. c. Enzyme *‘D” with 1.2~10~~

Zn”,

ranging Cd’+ the

EDTA can restore inhibited

enzyme.

EDTA concentration 163

from

1x10-’

M to

enzyme was inhibited up to 85o/o A plot

the

of return

at fixed

of

TAME and

M

Vol. 36, No. 1, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CU++ concentration

showed a sigmoid

(0.5’ to 3.0’/0)

had no effect

TAME or of protamine solution lyzed

inhibits

the higher

DISCUSSION

There

al.

(10)

the

as a result

of urea,

changes

embryos cultured

was not

More

synthesis

of two estero-proteases

bond specificity

group

different

with

proteins

are

The characteristics tinguish studies

them clearly as well

seems to point than

“A”

acid

residues

it

protein

from

showed scant activity

of androgens

and

on the

bio-

the mouse submaxillary by mercaptoethanol.

have not clear

or kinetic

been reported whether

the

and for

same or

involved. of the from

to a structural

as if

caused

Calissano

substrates

is not

Attardi

Proteolytic

inactivated

enzymes reported

trypsin.

as a comparison

and differs

but

recently,

from

characteristics of enzymes,

gland.

in muscle tissue

the effect

These enzymes were both

either

was

one of which

casein.

reported.

have studied

physico-chemical

hydro-

on the presence

--in vitro,

on denatured

(11)

Since

reports

on two enzymes,

Angeletti

gland.

substrate

have been earlier

activity other

of

M) in the substrate

The amount of

dedifferentiative

proteolytic

of hydrolysis

in the mouse submaxillary

11 day old chick

for

(b-10

acting

have reported

demonstrable

Urea

Mercaptoethanol

the urea concentration.

of estero-proteases et

rate

enzyme.

when enzyme stopped

sma1 ler,

on the

sulfate.

the

curve.

limited

Immune-electrophoretic

of their

amino acid

relatedness.

in composition proteolysis

on here dis-

“D”

by relatively converted

content

is smaller few amino “Al1 to rrD1t.

ACKNOWLEDGMENT The authors are grateful to Dr. R.C. Warner, Department of Biochemistry, N.Y.U. School of Medicine for the determinations of the Svedberg values. This work was supported by USPHS Grants nos. NB 07370 and AM 09873.

164

Vol. 36, No. 1, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. 2.

;: 5. 6. 7.

0. 9. 10.

11.

Schenkein, I., Levy, M., Bueker, E.D. and Tokarsky, E., Science, I-& 640, 1968. Schenkein, I., Bueker, E.D. and Levy, M., Conference on Hormones in Deve lopment. 1968, Nottingham, England (in press). Cohen, S., Proc. Nat. Acad. Sci. (US), 4, 302, 1960. Pharmacia Fine Chemicals Inc. Sephadex ion exchangers, undated. Schenkein, I., Levy, M., and Weis, P., Analytical Biochem.,

25,

387,

Davz, B. J. PP. 321, Scheidegger,

1,

103,

1968.

and Ornstein,

4049 1964. J. J.,

Inter.

L., Arch.

1955.

Ann.

N.Y.

Allergy

Acad.

Sci.,

121,

Appl.

Immunol.,

Baines, N.,G., Baird, J.B. and Elmore, D.T., Biochemical J., 90, 470, 1964. Levy, M., IN: Methods in Enzymology, (S.P. Volowick and N.O. Kaplan, Eds. ), vol. 2, 454, 1957. A,ttardi, D.G., Schlessinger, M. J. and Schlessinger, S., Science, B, 1253, 1968. Calissano, P. and Angeletti, P.U., Biochem. Biophys. Acta, 156, 51, 1968.

165