The melecular weight of the undegraded polypeptide chain of yeast hexokinase

Vol.

39,

No.

BIOCHEMICAL

1,197O

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE MOLECULAR WEIGHT OF THE UNDEGRADED POLYPEPTIDE CHAIN OF YEAST HEXOKINASE John

R. Pringle

The Biological Laboratories, Cambridge, Massachusetts,

Harvard 02138

University

Received February 6, 1970

Summary Commercial preparations of yeast hexokinase are contaminated with a trace of at least one proteolytic enzyme. This contaminant will digest the hexokinase in the presence of sodium dodecyl sulfate (SDS), and a reliable molecular weight can be obtained by SDS-polyacrylamide gel electrophoresis only when specific steps are taken to prevent this proteolysis. When appropriate precautions are taken, this technique yields a value of about 51,000 for the polypeptide chain molecular weight of yeast hexokinase. A similar value is obtained by gel filtration in 6M guanidine-HCl. Introduction A species generally

accepted

hexokinase

in the

isolation

seemed

possible

hexokinase

for

attack

in

and that

this

problem.

possibility,

the

enzyme 50,000

25,000

is of

the

ultimate

chain

with

smaller

possesses

46

illustrate

but

a sinSince,

known to be a problem. this

components

and also

reported

(6,7).

enzyme

a molecular

The experiments

of yeast

have

subunit

in spite

seems

subunit

daltons

proteases

products,

about

two laboratories

per

fact

a polypeptide

of

polypeptide

native

by yeast

breakdown

this

the

glucose

that

50,000,

circumvent

ultimate

and characterization

is

proteolytic

weight

However, that

site

in addition,

confirm

as the

indicating

binding

about

a molecular

(1,2,3,4,5).

evidence gle

with

of

(8,9),

of yeast weight

observed efforts

presented

it

of

were (4,8,9)

here

a significant

to seem to

BIOCHEMICAL

Vol. 39, No. 1, 1970

danger

attending

the

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

use of SDS-polyacrylamidea

gel

electro-

phoresis. Materials

and Methods

Yeast 6309395

hexokinase

and 63592961,

Calbiochem tion.

was purchased

(lot

Other

from

8550211,

proteins

Sigma

by Dr.

Weber.

Reagents

electrophoresis

incorporating

on a .9 x 80 cm column 6M guanidine-HCl by Fish,

and alkylated Preparatory

to

.09M tris-HCl, at

100°;

conducted -Results

ICH2COOH

alkylation, .003M

at

Gel

SDS-polyacrylamide et al

(Bio

Gel

All

before

EDTA, pH8.0,

containing

was added

to

with

essentially

as

samples

were reduced

to the were

column.

dissolved

5.5M

.14M,

and

eluting

application samples

Mann,

was performed

ASM),

protein

(111,

(10).

from

filtration

protein

2-mercaptoethanol

in

guanidine-HCl

and incubation

37O overnight.

and Discussion As expected

hexokinase If,

(12).

were supplied

and Osborn

2-mercaptoethanol),

et -- al

with

for

as "Ultrapure"

of Agarose

(without

standards

by Shapiro,

purification.

purifica-

and were kindly

of Weber

was purchased

further

further

and procedures

modifications

Guanidine-HCl

described

(lo),

(lots

and from

weight

were as described the

used without

6787660),

used as molecular

by Weber and Osborn

gel

(lot

Boehringer

and was used without

as described Klaus

from

(8,9),

tested

commercial

are contaminated

to a solution

of hexokinase

tris-HCl,

.OOlM

toethanol

are added

a The abbreviation

all

EDTA,

with in buffer

preparations

of yeast

proteolytic

activity.

(.4mg/ml

in

. 2M NaCl,

pH 7.6)

at 25O,

to 1% each,

a rapid

and extensive

used is

SDS (sodium 47

dodecyl

.03M

SDS and 2-mercap-

sulfate).

proteolysis

Vol. 39, No. 1, 1970

ensues

BIOCHEMICAL

unless

proteolysis

specific

weight

forms

steps

revealed

is

this

3min

exposure

are:

to 100°

and mercaptoethanol:

(b) inhibitor

used as described

in

addition

the

hexokinase

followed with

of

of hexokinase phoretic

about

of about the

region

both

band

under

material

the

If just

bands

none

of

are

conditions

of

that

gel) weight with

No material weight between it

the

little

of

is

con-

digestion an electro1,

is obtained.

80% of the observed

in a

(10,11,14)

of

molecular

weights

is

observed

in

of

25,000.

Since

chains

seems very

52,000

to

in

at least

links

prior

guanidine-HCl,

5, Figure

observed

(13;

solution

is applied,

stained

with

immediately initial

SDS

Methods, -an SDS solution. Each

into

gel

to a

solution fluoride

5.5M

of them

I presume

should

be

unlikely

that

can be other

than

a

represents

the

un-

this

hexokinase.

treatments at

sample

by inactivating

used,

weight

of yeast the

urea

mini-

of the

as described

20,500.

chain.

continued

hot

to a molecular

of molecular

monomer

in

to a molecular

and disulfide

polypeptide

degraded

enzyme

containing

on the

and about

non-covalent

single

tion

a major

corresponding

destroyed the

to that

Minor

32,000

any

similar

corresponding 52,000.

the

addition

rapidly

if

(by densitometry

position

of

(c)

works

sufficiently

can occur;

pattern,

protein

9.2M

presumably

pattern

this

but

through

protease

in

minutes

and alkylation

treatments

taminating

several

buffer,

by reduction

of these

In

in

then

effective

phenylmethanesulfonyl 9) for

molecular gel

following of the

The

subsequent

(a> subjection

SDS and mercaptoethanol; not

dialysis

the found

treatment

this.

of higher

in

immediately

proteolytic

to prevent

Treatments

proteolysis

the

taken

components

patterns.

mizing

the

are

by a replacement

by smaller

electrophoretic

AND BIOPHYSICAL RESEARCH COMMUNlCATfONS

(a)-(c)

25O after

the 48

is addition

applied,

but

incuba-

of SDS and mercap-

Vol. 39, No. 1, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BSA

2

I 0.3

6 04

1 0.5 06 * Mobility

OJ

08

I

Figure 1. SDS-polyacrylamide gel electrophoresis of yeast hexokinase protected (gel a) and unprotected. (gel b) against proteolysis in SDS solutiof;. Electrophoresis was From bottom to top of the gels as pictured. The "Minor Bands" in gel a are indicated by arrows. The exact form of the standard curve to the left of fumarase is not clear from these data; the two lines indicate the limits. BSA is bovine serum albumin: ATCase C and R the catalytic and regulatory chains of E. coli aspartate transcarbamylase.

toethanol to

gel

to the b, FLgure

disappearance

solution

1 (Boehringer,

of

and Calbiochem

hexokinase

the higher

enzymes)

apparently

occurs

incubation

of

only

in buffer,

lot

63093951,

molecular

is

observed.

after

the

such a hexokinase

addition solution

49

weight This

a pattern or with

a complete

components wholesale

of the in buffer

similar

(Sigma degradation

SDS, as a 13hr at O",

fol-

Vol. 39, No. 1, 1970

lowed

BIOCHEMICAL

by treatment a.

(a)

or (b),

that

of gel

Further

will

be presented

elsewhere.

Confirmation

of

by investigation

pletely (151,

should

the

polypeptide series

of

the

disulfide

an accurate

chain

peptide

the

6M com-

chains

and extensively

carboxy-

protein's

behavior

of its

individual

(12),have

shown for

correlation

weight

of hexokinase

observed

an elution

exists

and position

Methods,

between

50,000

The earlier

their

elution

2b).

a

be-

of elution

d,

misleading unit

of the the

fact

was ob-

material

electrophoreses in

Figure

was run

2a reveal

a strict

from

the

column

Again

the

hypothesis

hexokinase

corres-

has a molecular

and their that weight

supported.

this chain

that

peak

with

position

results for

the

to a molecular

arrows

1, gel

seems strongly

column,

A main

gel

at the

of yeast

63592961,

Some smaller

on electrophoresis.

due to proteolysis that

(Figure

Figure

polypeptide

on the

corresponding

taken

chain

lot

2a was obtained.

seen in

of migration polypeptide

was run

SDS-polyacrylamide

of material

same bands

(Boehringer,

position

51,000

as well.

on samples

in

of Figure

of about

I suggest

in

between

et al

molecular

filtration

lengths

that

profile

of about

of the

was obtained

to eliminate

bonds,

to

process

thought

was reduced

19 proteins

weight

mental

sample

Fish,

with

the

gel

is

Indeed,

served

rate

degradation

interactions

as described

pondence

on this

hexokinase

similar

column.

prepared

the

a pattern

during

chains.

When a sample

elution

yields

for

solvent

a function

tween polypeptide from

this

to destroy be strictly

results

behavior

non-covalent

and since

methylated

these

Since

all

still

information

of its

guanidine-HCl.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

indicating enzyme

must

of molecular

these 50

results

a smaller

funda-

surely

have been

weight

about

were obtained

50,000. by in-

BIOCHEMICAL

Vol. 39, No. 1, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4A”’ - 1 .*’

65

4 Molecular

Weight

(IO-‘)

Figure 2. Gel filtration of hexokinase in 6M guanidine-HCl. A. Elution profile. Blue Dextran and DNP-alanine were included as internal standards in each run. Small arrows across the top indicate sites from which samples for SDS-gels were taken. B. Standard Curve (from Rosenbusch & Weber, in preparation). kdist is the distribution coefficient (see reference 12); that for GluDH, glutamate dehydrogenase; hexokinase is shown by the arrow. Mb, myoglobin; ATCase, see Figure 1. vestigators cation avoid

of hexokinase, this,

countered fects.

who were

indicates

aware of the problem

of proteolytic

and who were taking

considerable

the extreme

in attempting I suggest

SDS, as noted

to avoid

also

above,

difficulty

that

the

obligates

phoretic

technique

controls

such as (a>-(c),

completely enhancement

which

modifipains

may be en-

such proteolytic of proteolysis

one who uses the SDS-gel

on a previously

untested This

above. 51

also

to

protein minimizes

efby

electro-

to perform the

pos-

Vol. 39, No. 1, 1970

BlOCHEMlCAL

sibility

that

some rare

cubation

usually

protein

employed,

(10).

These

results

of the

technique

for

ported

by others

(10,11,14).

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

will

as already

do not,

of course,

molecular

weight

not

be denatured

noted

by the

in-

by Weber and Osborn

negate

the

reliability

determinations,

as re-

Acknowledgements --The possibility that proteolysis was occurring in SDS solutions originally emerged in discussions with Dr. Rudolf Weil. I also thank Dr. J. P. Rosenbusch for the use of his column and his calibration data, and Dr. Klaus Weber and Professor Guido Guidotti for their most appreciated material and intellectual support. This work was supported by United States Public Health Service Grant HE-08893 and by National Science Foundation Grant GB-6360 (Guide Guidotti).

References A., Stellwagen, E., and Schachman, H.K., Fed. Proc., -.20, 387 (1961). 2. Kenkare, U.W., and Colowick, S.P., J. Biol. Chem., 240, 4570 (1965). J., Schulze, I.T., Gooding, R.H., Womack, F.C., and 3. Gazith, Colowick, S.P., Ann. N.Y. Acad. Sci., 151, 307 (1968). 4. Easterby, J.S., and Rosemeyer, M.ATB.S. Letters, 5, 84 (1969). N.R., Derechin, M., and Barnard, E.A., Biochem., 5. Lazarus, 7. 2390 (1968). s:, Ricard, J., Borel, M., and Got, C., Eur. J. 6. Noat, ll-, 106 (1969). Biochem., Colowick, S.P., and Womack, F.C.., J. Biol. Chem., 244, 774 7. (1969). 8. Schulze, I.T., and Colowick, S.P., J. Biol. Chem., z, 2306 (1969). 9. Lazarus, N.R., Ramel, A.H., Rustum, Y.M., and Barnard, E.A., Biochem., 5, 4003 (1966). and &born, M., J. Biol. Chem., 244, 4406 (1969). 10. Weber, K Vinuela, E., and Maizel, J.V.,iochem. 11. Shapiro,'i.L., 1.

12. 13. 14. 15.

Ramel,

Fish,~g~~~~~~~~~'~n~a~~~r~~g~~~'J. Biol. . Fahrn&D.E., and Gold, A.M., J. Am. Chem. Sot., (1963). Dunker, A.K., and Rueckert, R.R., J. Biol. Chem., 5074 (1969). Tanford, C., Advan. Protein Chem., 23, 121 (1968). --

52

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997