Effect of phosphate on multiple forms of Escherechia coli alkaline phosphatase

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

EFFECT OF PHOSPHATE ON MULTIPLE ALKALINE William

RESEARCH COMMUNICATIONS

FORMS OF __~___ ESCHERECHIA COLI

PHOSPHATASE

F. Bosron

and Bert

L. Vallee

Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, Division Medical Biology, Peter Bent Brigham Hospital, Boston, Received

August

of Mass.

1,1975

Summary Alkaline phosphatase isolated from E. coli strain C-90 can be separated by DEAE-cellulose chromatography into three forms which contain 0.1, 1.2 and 1.9 moles of bound phosphate per mole of enzyme, respectively. However, the phosphate-free enzyme prepared from a mixture of all three forms chromatographs as a single peak, while readdition of phosphate again results in the separation of three forms. Hence, the chromatographic behavior of phosphatase is consistent with a variation in phosphate content of three forms. Introduction ~--__ Large

numbers

of high

resolution,

molecular

basis

instances

(l),

have been for that

by DEAE-cellulose

that

Hence,

Signer

random

dimerization

translational the

phosphate. of phosphate

relative

of multiple

cistron

that

under

However,

composed

modifications

which

phosphatase

has been

but

grown

shown

Since,

three

forms

(6),

chain

result must Indeed,

phosphatase

reported

in many

(7)

subunits

cannot

subunits,

is

the

forms

the polypeptide

forms

the bacterium

(12-14).

into

of two identical

of these

While

obscure.

of the enzyme.

stoichiometry

mole of enzyme

for

distinct

phosphatase the

codes

the different

proportions

alkaline

per

observation

of genetically

conditions

Purified

has remained

capable

unambiguously

phosphatase

(2-5)

enzyme is

suggested

or epigenetic

that

upon the

of alkaline

a single

techniques

forms.

of the separation

the dimeric (9)

in multiple

has been established

that

further,

to separation

such multiplicity

the initial

was established

when subjected

shown to exist

chromatography

Following

shown

of enzymes,

forms

it

and,

(8).

from

arise

it

the

from posthas been

depend

primarily

tightly

bound

(Y-11).

to contain

has varied theoretically,

from 0.2

to 2 moles

a variation

in

Vol. 66, No. 2, 1975

the

amount

separation

of bound

phosphate

observed,

we have

phosphatase

forms

the bound

BIOCHEMICAL

isolated

could

form the basis

determined

from --E. coli

phosphate

from native

of this

phosphate-free

properties

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for

the chromatographic

the phosphate

content

strain

In addition,

phosphatase

C-90.

and examined

enzyme,

as well

of the

three

we have

removed

the chromatographic

as of that

to which

phosphate

was readded. Materials

and Methods

Alkaline phosphatase was isolated by osmotic shock of E. coli strain C-90 cells grown in 14 liter cultures as described previously (15). The water extract from the osmotic shock treatment was purified by batchwise absorption onto DEAEcellulose (Whatman DE-52) and eluted with 0.1 M NaCl in 10 mM imidazole-Cl, pH 7.2, followed by chromatography on a 5 x 150 cm column of Sephadex G-150 (Pharmacia, 40-120 u) in 10 mM Tris-Cl, pH 7.5. Multiple forms of phosphatase was were separated on DF.AE-cellulose as described in Figure 1. Apophosphatase prepared using 8-hydroxyquinoline-5-sulfonic acid (16). Enzymatic activity was measured with p-nitrophenyl phosphate (Sigma 104) in 1 M Tris-Cl, pH 8.0 at 25". Units of activity are defined as lhaoles of substrate hydrolyzed per minute using a p-nitrophenolate molar absorptivity of 1.68 x lo4 at 400 nm. Protein concentration was determined spectrophotometrically using Ai;8 = 7.2 and all calculations involving molarity are based on a phosphatase Conductivity was measured at room temperature molecular weight of 89,000 (15). using a Radiometer type CDM 2d meter and expressed in mMho. Phosphate analyses were performed using an ammonium molybdate reagent as suggested by Bloch and Certified phosphate standards (New England Reagent Laboratory) Schlesinger (12). were utilized for reference. All phosphatase samples were hydrolyzed for 24 hr at 120" in 6 N HCl and dried under vacuum over NaOH pellets before analysis. Results

and Discussion Alkaline

activity

of 39 units/mg

separation three

phosphatase

contains

of the three

forms

coli from E. __

purified

forms

1.2 moles

of phosphate

on DEAE-cellulose

when separated

of phosphatase,

40 to 44 units/mg

(Table

from 0.1

for

for

form

3 (Table

with

their

order

values eluting

and are consistent last

is

the observed

phosphate-free (12).

Hence,

per

However, I),

their reflect

a specific

mole of enzyme before The activities

I).

(Figure

phosphate

1, top),

contents,

approximately

of chromatographic

of the are

ranging integral the

elution:

form

most negative.

The possibility for

(Table

C-90 with

by chromatography

similar,

form 1 to 1.9

I).

strain

that

this

chromatographic enzyme.

Removal

apoenzyme

prepared

variation

in phosphate

separation

was tested

of metal

simultaneously

from pooled

810

fractions

content

could

account

by chromatographing removes of all

bound three

the phosphate

forms

BIOCHEMICAL

Vol. 66, No. 2, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I Activity

and Phosphate Before

and After

free

1, top) protein

of a lo-fold

1.2

Form 1

41

0.1

Form 2

44

1.2

Form 3

40

1.9

aBefore

chromatographic

forms

on DEAE-cellulose.

of another

the

activities,

treated

middle),

and by comparison

However,

chromatography

of the

three

Therefore,

it

separation

of alkaline

upon

charge

would

as a single Figure

three

forms appear

separated that

phosphatase

that,

peak

its

The

compared

to those

to the metals, Identical

Ninety

percent

as form

in the presence

1, bottom),

three

of the

(Figure

can be identified

in bound

for

pH 7.2.

from DEAE-cellulose

in the native

variations

by dialysis

in addition

reconstituted (Figure

into

followed

samples.

The metal-

by addition

to the apoenzyme.

1, top,

forms

restored

enzyme were

to both

of apophosphatase,

resolves

is

of enzyme.

10 mM imidazole-Cl,

was added

restored

with

activity

except

of phosphate were

per mole

phosphate-free

identically,

enzyme emerges

again

but

buffer,

of this

44 unitslmg,

phosphate,

of phosphate

of phosphatase

Zn(S0 4 ) and Mg(S0 4')

chromatographic

excess

phosphate-free

typical

of both

properties

molar

mole

separation

inactive,

excess

sample

a lo-fold

0.05

catalytically

chromatographic

Phosphate, Moles/Mole

39

molar

24 hr against

Chromatography

Nativea

contains

is

of Phosphatase

Activity, Unitsimg

Sample

(Figure

Content

enzyme

1. of

eluting

at conductivities

(Figure

1, top).

phosphate forms

1,

account

by techniques

for

the

dependent

differences.

The presence

of 1.6 to 2.1 moles

of tightly

811

bound

phosphate

per

mole of

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BlOPHYSlCAL

20 25 CONDUCTIVITY,

RESEARCH COMMUNICATIONS

3.0 mMho

Figure 1. Separation of alkaline phosphatase forms. Alkaline phosphatase, 200 mg, was applied to a 2.5 x 40 cm column of DEAE-cellulose equilibrated with 10 mM imidazole-Cl, pH 7.2 and eluted with a 4 liter linear gradient of 10 to 70 mM NaCl in the same buffer (top panel). Apophosphatase, 15 mg, after reconstitution in the absence of phosphate was chromatographed on a 1.5 x 26 cm column of DEAEcellulose using a 1.5 liter gradient of 10 to 70 mM NaCl in 10 mM imidazole-Cl, An identical 15 mg sample of apophosphatase, but now pH 7.2 (middle panel). reconstituted in the presence of phosphate, was chromatographed under the same For each chromatogram, phosphatase activity, in conditions (bottom panel). units/ml, is plotted versus the conductivity, in mMho, at elution.

purified

enzyme was reported

subsequent molar

studies

stoichiometry

explicable, Thus,

the

the observed relative

employed

for

radioactive coworkers

of g. J&L al f (11) -et -.-*

pulse

labeled

phosphatase

form

(11,17)

have

found

alkaline

phosphatase

isolated

may bear

upon the underlying

phosphate

to the enzyme.

the

forms

proportion

growth

cultures

three

by Bloch

This

different

of a mixture

cells

utilized

has shown

for

that

1 to form

3.

heterogeneity another

structural

basis

812

forms

would

of the

In

phosphate

conversion

sequence These

differential

to

of

Schlesinger

amino-terminal strain.

I).

be dependent

(6,9,10).

of inorganic

studies

(Table

by the conditions

enzyme isolation

In parallel

coli -E. -.-

of phosphate

the

the

is now

influenced

causes

Although

phosphate,

variation

is

addition

in the

bound

of these

35 S-methionine

with

(12).

amounts

which

of each present,

from

of tightly

(13,14).

contain

content

and Schlesinger

presence

has varied

phosphate

Schlesinger

J$. coli

confirmed

reported

since

upon the

fact,

have

initially

and of

observations binding

of

BIOCHEMICAL

Vol. 66, No. 2, 1975

The fact

that

chromatographically demonstrated

is now observed

forms

whose

negative

of a small,

and electrophoretically

for

approximately

the binding

AND BIOPHYSICAL

alcohol for

of Drosophila

phosphatase

stoichiometric separation

distinct

dehydrogenase

alkaline

charged

of -E. --coli.

quantities

of phosphate

on DEAE-cellulose

is

consistent

RESEARCH COMMUNICATIONS

molecule

can give

enzyme

forms

(18).

A similar

The binding gives with

rise

rise

to

has been situation

of to three

differences

enzyme in

charge.

Acknowledgements -__This

work

was supported

recipient

of Traineeship

Health,

of the Department

under

by Grants-in-Aid GM-02123,

of Health,

both

Education

GM-15003; from

W.F.B.

the National

is

the

Institutes

of

and Welfare.

References ---__ 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

C.L. (1975), Isozymes--- I, ------P-9 Molecular Structure Academic Press, Markert, New York, pp. l-9. Lazdunski, C. and Lazdunski, M. (1967), Biochim. Biophys. 147 --Acta -3 280-288. Salanitro, J.P. (1968), Ph.D. Thesis, Indiana University. Simpson, R.T. (1969), Ph.D. Thesis, Harvard University. Bosron, W.F., Kennedy, F.S. and Vallee, B.L. (1975), Biochemistry 14, -_-2275-2282. Bach, M.L., Signer, E.R., Levinthal, C. and Sizer, I.W. (1961), Fed. Proc. I____ 20, 255. Garen, A. and Garen, S. (1963), J. Mol. Biol. 1, 13-22. Rothman, F. and Byrne, R. (1963)~l~ol. 6, 330-340. Signer, E.R. (1963), Ph.D. Thesis, Massachusetts Institute of Technology. Schlesinger, M.J. and Andersen, L. (1968), Ann. N.Y. Acad 151 ._--z-.--_*Sci -3 159-170. Schlesinger, M.J., Bloch, W. and Kelley, P.M. (1975), Isozymes I, Molecular Structure, Academic Press, New York, pp. 333-342. -___ Bloch, W. and Schlesinger, M.J. (1973), J. Biol. Chem. --9 248 5794-5805. Norne, J-E., Csopak, H. and Lindman, B. (1974), Arch. Biochem. Biophys. 162 ~__ -9 552-559. Chappelet-Tordo, D., Iwatsubo, M. and Lazdunski, M. (1974), Biochemistry 2, 3754-3762. Simpson, R.T., Vallee, B.L. and Tait, G.H. (1968), Biochemistry 12, 43364342. Simpson, R.T. and Vallee, B.L. (1968), Biochemistry 12, 4343-4350. Kelley, P.M., Neumann, P.A., Shriefer, K., Cancedda, F., Schlesinger, M.J. and Bradshaw, R.A. (1973), Biochemistry 12, 3499-3502. Schwartz, M., Gerace L., O'Donnell, J. and Sofer, W. (1975), Isozymes I, Molecular Structure, Academic Press, New York, pp. 725-751.

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