Existence and inhibition of hydrolytic enzymes attacking paramyosin in myofibrillar extracts of Mercenaria ,mercenaria

Vol. 49, No. 3, 1972

BIOCHEMCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

EXISTENCEAND INRIBITION OF HYDROLYTICENEYMESATTACKINGPARAMYOSININ MYOPIBRILLAREXTRACTSOF MERCENARIAMERCEBARIA Walter F. Stafford

III

Biochemistry Biological

and Biophysics

Sciences Group & Institute The University Storrs,

Received

September

Connecticut

proteins

of the widely

we of proteinase usually

been amply demonstrated of individually

least two hydrolytic enzymes in extracts under the usual isolation conditions. fluoride Inhibited hydrolase activities is extracted into high salt at pH 7.5 in a new nnlecular species of paraxyosin higher than material isolated by acrylamlde gel electrophoresis techdetect small differences in apparent

inhibitors

that (1,2,3).

in muscle homogenates

identifiable

Inhibited

partially

procedures, material

by phenylmethanesulfonyl

cleave paramyosin (PM) in extracts

the adductor

seryl

has

In such

proteins

of a probable

has not metallo-

proteinase

(PMSF), both of which

PM was Isolated

The

on Tris-dodecylsulfate

is the use of pepstatin during the isolation Cold Spring Harbor Syxposium on Quantitative

848

of

by two different

EDTA or PMSPor both.

was then examined by electrophoresis

0 1972 by Academic Press, Inc. of reproduction in my form reserved.

activity

of both red and white portions

in the presence or absence of either

* A recent exception to this troponin by D. J. Hartshome, Biology, (1972). Copyright AlI rights

fluoride

muscle of Mercenarla mercenaria.

obtained

contractile

by EDTA and a presumptive

because

in significant

hydrolysis

the existence

of ayofi-

probably

proteinase

(4-13),

or purified

proteinase

inhibited

the isolation

necessary*,

Although neutral

We have demonstrated

activity

during

such enzymes do not exist

been observed (9). activity

Science

06268

has not been considered

held opinion

amounts in the oyofibrll

extracts

of Materials

27,1972

: Paramyosin is attacked by at of the adductor muscle of M. mercenarla RICA inhibited and phenylmethanesulfonyl have been identified, When paramyosin the presence of O.Olg EDTA, we isolate with a molecular weight ~5,000 daltons previous procedures, A high resolution nique employing split gels was wed to sub-unit molecular weight.

brillar

Section

of Connecticut

suwlary

The general

and David A. Yphantls

gel4 of

BIOCHEMICAL

Vol. 49, No. 3, 1972

MATERIALS-Phenylmethanesulfonyl (hydroxymethyl)

fluoride

glycol

Inhibition

was made up freshly

solvents,

3 x lo-4fi.

was obtained

from Schwartz/Mann.

Tris-

Sodium

was MC and B, USP grade.

PREPARATIVEPROCEDURESProteinase

to all

RESEARCH COMMUNICATIONS

aminomethane was Trizma grade from Sigma Chemical Co.

dodecylsulfate

propylene

AND BIOPHYSICAL

for each preparation.

except the chromatographic

Use of PMSFas a seryl

with PMSF: PMSF (0.03g)

buffer,

to a final

enzyme inhibitor

Fahrney and Gold (14) and was suggested

Aliquots

in were added

concentration

was reported

of

originally

to us by A. G. Szent-Gyorgyi,

by

to whom

we are grateful. Method I, Chromatography

on Controlled

Pore Glass:

the adductor muscle was minced and washed in 0.e NaCl, 0.04g Tris, The supernatant

was applied

4' or 22'.

and a manuscript

procedure

solutions.

Details

to the procedure

EDTA in all

with 0.65

at 48,000 x g for 30 min.

after

recycling

was carried

three tiws

out either

of the procedure

at

with or without

have been presented

(15),

is in preparation.

Ethanol Precipitation:

according

of

to a 2.54 X 100 cm colusm of Corning Controlled

The entire

O.O@ EDTA in all

Method II,

NaCl, extracted

pH 7.5 for one hour and centrifuged

Pore Glass CPG-lo-1250 and PM was collected either

The red or white portion

The preparation

of Johnson et al.

of PM was carried

out also

(16) at 4' with and without

O.Ol!j

solutions.

MOLECULARUEIGRT DETERMINATIONBY TRW-DODECYLSULFATE GBL ELECTROPHORESISPolyacrylamide procedure instead

gel electrophoresis

of Shapiro et al. of phosphate.

EDTA (if

(17) using Trig-acetate

To each sample of protein

the PM had been prepared without

of 0.5tj Na2S03 were concentration unnecessary

was carried

when split

Later,

gels were used.

849

buffer, (0.3-2.0

according

to the

O.OW, pH 8.1, mg/ml),

0.2 ml O.Oly

EDTA), 0.2 ml of 10% SDS and 1.0 ml

added in the order listed.

of 3 X 10B4y was added.

essentially

Initially, however, this

The mixtures

PMSF at a final was found to be

were allowed

to react at

Vol. 49, No. 3, 1972

BIOCHEMICAL

AND BIOPHYSICAL

40' for one hour and then dialyzed in M.W. were detected acrylamide,

against

activity

When mixtures contaminating

in the presence of detergent.

(A similar

with yeast hexokinase preparation.) gel

was eliminated

patterns

ovalbumin,

At least marker.

the slope of the calibration subunits

to migrate

nearly

mobility

and M.W. of the PM subunit

yielded

material

(18)

of interpreting A silicone

rubber the

one sample in each run was dansylated

(21)

runs were performed with bovine serum weight standards

Thereafter,

longer

(22),

to determine

runs allowing

the PM

obtained

relative

by each procedure.

by method I or method II in the absence of EDTA

having the usual electrophoretic

there was a main band (B-PM) with intensity

with a M.W. of 0.94 X lo5 (23). tography

difficulty

to the end of the gel were used to determine

either

(a-PM) of lower staining

was made by Pringle

gels (19,20).

Initial

curve.

effective

1 mm into the top of each gel after

and @-PMas molecular

RESULTS-PM isolated

Small differences

of samples were run, it was

observation

by using split

had polymerized.

to serve as a fluorescent albumin,

buffer.

some samples was still

The resulting

septum (0.5 X 6.2 X 8 zzz) was inserted acrylamide

electrode

by comparing samples on the same gel (6 X 120 zzz, 4%

0.1% bis-acrylamide).

noted that hydrolytic

RESEARCH COMMUNICATIONS

a

properties

in dodecylsulfate:

M.W. of 1.00 X 10' (22) and another band representing

a few per cent

of the main band

When PM was prepared by method I with chroma-

at 22' in the absence of EDTA, the main component had a M.W. of 0.94 X

105. and small amounts of material If PM was isolated

with slightly

lower M.W.'s were also seen.

by method I at 22' in the absence of EDTA but with PMSF, two

bands were seen in roughly equal amounts with M.W.'s of 1.00 and 0.94 X 105, and the bands with slightly either

lower M.W.'s were no longer seen.

PM isolated

by

method I or method II in the presence of 0.01&J EDTA showed one band

(a-PM) with a M.W. of 1.05 X 10'. at a load of 75 pg. also yielded

a single

Lastly,

None of the lower M.W. PM bands could be seen

PM isolated

in the presence of both EDNA and PMSF

band with a M.W. of 1.05 X lo5 (not shown in figure

It should be noted that with O.OlE EDTA present

850

in either

procedure,

1).

extraction

BIOCHEMICAL

Vol. 49, No. 3,1972

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

7

1 FIGURE I

Tris-dodecylrulfate polyacrylamide gel electrophoresis patterns of various (1) Method I with EDIA, 4'/without EDTA, 4'C, (2) paramyosin preparations: Method II (1 hour extraction),with EDTA/without EDTA, (3) Method I with EDTA, 4'/without EDTA, 22'. (4) Method I, with EDTA, 4-/with PMSF, 22', (5) Method I with PMSF, 22'twithout PMSF, 22', (6) Method I with EDTA, 4'/Method II with EIYIA, (7) Method I with EDTA, 4', PM from red and white muclee. [PMSF] = 3 X lo-4E; [EDIA] = 1 X lo-2l.J. (The diffuse band on the right side of gel 63 is probably a result of air oxidation of Y-PM in SDS. The sample was run ~18 hrs Compare this to the right side of gel #5, which is the same after sulfonation. The gels are not all from the material run immediately after sulfonation.) same run.

times of up to 90 min could be used without results

are summarized in Table 1, and the split

CONCLUSION-We have shown that previously PM from the adductor the molecule.

of 5. mercenaria

gels are shm

used procedures

yield

8 -PM, found by the previous

procedures.

851

higher There

in Figure 1.

for the isolation

an enzymatically

In the presence of O.Olg EDTA, we isolate

of PM, a-PM, with a M.W. ~5,000 daltons

These

any evidence for hydrolysis.

of

degraded form of

a new molecular

species

than that of the main component, appear to be at least

two hydro-

Vol. 49, No. 3, 1972

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COhWvWNlCATlONS

Table I

Chain weights of paramyosin isolated various preparative procedures

by

INHIBITOR METHOD

EDTA

-5 M.W. x 10 & (Approx.

PMSF

I, 4O I, 2z" +

I, 2ze I, 4O

+

I, 4O II, 15 min ext'n 11, 1 hr ext'n II,

+ -

+

1 hr ext'n

1.00,

0.94

(95:5)

0.94 1.00, 1.05

0.94

(40:60)

0.94

(95:5)

0.94

(60:40)

1.05 1.00,

+

ratio)*

1.00, 1.05

* In most samples faint bands with approximately twice the M.W. of the main bands were seen. This material probably represents either incomplete eulfonation or recombination of chains after sulfonation (24).

lytic

activities

which attach

An EDTA inhibited

hydrolase

conversion

that

(5-15 min.).

hydrolyze

activity

to D-PM Is well

its

existence

of PM: it a-PM even with

is extracted

acts so rapidly

completed within

However, the presumptive

enough at 4' that preparations

PM when it

eeryl

seeam to require extraction

high salt

at pH 7.5.

at 4' when EDNAis absent

the usual extraction

hydrolaee

can be detected

into

times

appears to act slowly

by carefully

B-PM for substrate

examining the usual since it

does not

times of up to 90 min. in the presence of

EDTA. A neutral by

at 0.0015!

EDTA

required quite

proteinase

from rat skeletal

but not at O.OO&; therefore,

for complete inhibition

critical.

muscle (9) is completely the concentration

of the g. mercenaria

EDTA at an unspecified

852

concentration

enzyme activity

inhibited of EDTA may be

has been used in the

BIOCHEMICAL

Vol. 49, No. 3, 1972

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

isolation

of oyster

PM by Woods (24),

ostensibly

catalyzed

oxidation

of thiol

He found a subunit

daltons

groups.

high speed ultracentrifugation

using

is difficult

to inhibit

characterization

may also preclude

meaningful

of a-PM is currently

hydrochloride.

subunit

H.W. values determined

accuracy of absolute

Species differences

M.W. of 0.97 X lo5

in guanidine

to compare his value with our apparent

the limited

heavy metallion

It

size because of

by either

comparisons.

method.

Further

underway.

Acknowledgement This inveetigation

was supported

in part by NSF Grant #GB 13790 and

#GB 38025X and PHS Grants #GM-O0317 (to the University #GM-00265

the Marine Biological

(to

Laboratory,

of Connecticut)

and

Woods Hole, Massachusetts).

References (1)

Reuck, Little,

and Cameron, M. P., Ciba Foundation Brown and Co., Boston, Mass., 1963.

(2)

Dingle, J. T. and Bell, H. B., Lysosomes in Biology Holland Publishing Co., London, 1969.

(3)

Barrett, A. J. and Dingle, Publ. Co., N. Y., 1971.

(4)

Berlinguet,

(5)

Koszalka,

T. K. and Miller,

L. L.,

J. Biol.

Chem., 235, 665 (1960).

(6)

Koszalka,

T. R. and Miller,

L. L.,

J. Biol.

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(7)

Koexalka,

T. R., Mason, K. E., and Krol,

(8)

Pennington, R. J., in Muscle Diaaeees, (J. N. Walton, N. Canal, and G. Scarlato, eds.), p. 252, Excerpta-Medica, Amsterdam, 1970.

(9)

Kohn, R. R., Lab. Invest.,

A.V.S.,

J. T., Tissue Proteinasee,

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Fahrney,

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E.,

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BIOCHEMICAL

AND BIOPHYSICAL

(151

Stafford, W. F. III l.2, 283a (1972).

and Yphantis,

(16)

Johnson, W.,Kahn, J.,and

Szant Gyorgyi,

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Woods, E. F., Biochem. J.,

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Res. Coamwn., 39, 46 (1970).

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854

Res.