Post-translational modification of human erythrocyte pyruvate kinase

Vol. 74, No. 4, 1977

BIOCHEMICAL

POST-TRANSLATIONAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MODIFICATION

OF HUMAN ERYTHROCYTE PYRUVATE KINASE

John A. Badwey and E. W. Westhead Department of Biochemistry University of Massachusetts Amherst, Massachusetts 01002 Received

December

15,l976 Abstract

Upon storage, partially purified human erythrocyte pyruvate kinase (ATP: pyruvate-phosphotransferase, E.C. 2.7.1.40) from normal individuals was found to undergo a spontaneous oxidation to a form which displayed markedly reduced activity. This modified form of the enzyme exhibited kinetic patterns similar to those frequently reported for the enzyme in cases of nonspherocytic hemolytic anemia. The data are discussed in relation to the recently proposed theory that post-translational modification of pyruvate kinase is responsible for the abnormal kinetic patterns frequently encountered for this enzyme in the disease state. [Van Berkel, T. J. C., Koster, J. F., Kruyt, J. K. and Staal, G. E. J. (1973) Biochim. Biophys. --Acta 321, 496-5021. Introduction Staal

and co-workers

from normal

inidividuals

sulfhydryl

groups

an increased (e.g.

for

pyruvate

two cases

questioned only

individuals In this absence

of GSSG.

form

same properties

proposed

defect,

resulting cellular

of the normal

are

and

commonly

hemolytic

that

anemia

such aberrations

in

from modification levels

and co-workers

of

and fructose-1,6-P2

of PK-deficient

(1)

patterns. disulfide

with

the disorder.

(8)

of GSSG.

of the In apparent

subsequently

The overall

kinetic reported

P-enolpyruvate, PK, pyruvate

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

and co-workers of this

in erythrocytes

evidence

can be oxidized to those

Blume

the etiology

this

we present

kinase

are similar

Abbreviations: 1,6-bisphosphate; Copyright Ali righls

paper,

form by oxidation

reported

on

incubation of the partially puri--in vitro resulted in the conversion of the abnormal

of GSSG in of

the

kinase

in which

to normal

levels

pyruvate

The modified

cases

Staal

erythrocyte

P-enolpyruvate

by increased

mercaptoethanol

afflicted

pyruvate

in air

groups

the role

normal

cyte

in certain

of PK-deficiency

patterns

that

to an inactive

Since

and co-workers

proposition,

enzyme with

kinetic

for

may be a secondary

of this

reported

glutathione.

affinity (1).

enzyme Staal

sulfhydryl

support fied

this 2-7),

kinase

enzyme's

oxidized

decreased

thermolability

Ref.

have

can be converted

with

enzyme, displayed observed

(1)

which

--in vitro patterns far

shows

(9),

disease

that

displayed

the GSSG modified

they

observed

from

normal by the

several

human erythro-

rates

in the

enzyme oxidized

enzyme.

phosphoenolpyruvate; fructose-1,6-P*, kinase; DTT, dithiothreitol. 1326

have

since

obtained

at appreciable

however,

Sulfhydryl fructose-

ISSN

0006-291X

Vol. 74, No. 4, 1977

BIOCHEMICAL

oxidation

may therefore

transitions

frequently

and --in vivo (14). paper has appeared

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

be responsible noted

for

for

this

A preliminary

and kinetic

report

10-13) in this

(15).

Distilled, deionized supplied by a Millipore

Assay

the instability

(e.g. Ref. --in vitro on some of the data presented

Materials

The sources

both

enzyme both

water Milli-Q

of all

other

and Methods

was used throughout reagent grade water materials

all experiments system.

are described

elsewhere

and was (16).

Procedure

Pyruvate kinase activities were measured by a modification of the coupled assay procedure proposed by B&her and Pfleiderer (17). The reaction mixture of 0.7 ml contained in addition to the PK to be assayed: 3.7 mM ADP, 4.3 mM P-enolpyruvate, 0.31 mM NADH, 5.7 mM MgC12, 570 mM KCl, 127 mM Tris-Cl (pH 7.4) and 25 units of lactate dehydrogenase. Variations from this are detailed in the corresponding figure legends. The disappearance of NADH was followed on a Gilford 240 spectrophotometer. Temperature was maintained at 32'C. The velocity was obtained from the slope of the linear, fast phase of the reaction progress curve (16). A un$t of activity is defined as that quantity of enzyme which forms 1 umole NAD /min. under these conditions. Specific activity is units/mg of protein. Protein concentrations were determined by the method of Lowry --et al. (18) using bovine serum albumin for standard curve determinations. Partial

Purification

of Pyruvate

Kinase

All of the glassware utilized in purifying and storing the enzyme was acid washed. The removal of leukocytes from the blood samples and partial purification of the enzyme was similar to the procedures described by Chern --et al. (19) up to and including their second ammonium sulfate fractionation step (Step 2). The product from Step 2 was taken up in a minimum volume of 20 mM HEPES buffer (pH 7.2) containing 150 mM KCl, 7 mM MgC12, and 2 mM EDTA (Buffer C) and stored at 4"C, normally under a swab soaked with toluene to prevent microbial growth. Omission of toluene did not alter the appearance of the kinetic transitions The specific activities of these preparations which are to be described. and yields of 30-60% were generally achieved. varied from 0.8 to 1.8 units/mg. Since controls showed these samples to be free of phosphatases and other substances which could affect the assay system, this degree of purification was considered adequate for the present investigation. Prior to use in any of the subsequent experiments, enzyme samples were desalted on a Sephadex G-25 (fine) column (1.0 x 35 cm) equilibrated with buffer C. Results Freshly

prepared

moidal

saturation

mately

2.5 mM (Fig.

of this

enzyme,

hyperbola

(%

DTT to all kinetic

solutions

enzyme were

from normal erythrocytes displayed for P-enolpyruvate with (nH = 1.2-1.4)

1, A & B).

(20,21) = 1.0)

patterns.

enzyme

curve

Fructose-1,6-P2,

converted with

this

a greatly

curve

reduced

utilized

in purifying

The kinetic

constants

the same regardless

of whether

1327

(Fig.

a positive allosteric effector 1) to a normal rectangular

KM (0.33

mM).

the enzyme did reported

a slightly siga K. 5 of approxi

above

the blood

cells

Addition not

for

alter freshly

utilized

of 30 mM these prepared were

BIOCHEMICAL

Vol. 74, No. 4, 1977

.g \ : 0 x

0.3

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-

0.2-

: 0.1 -

3 4 PEP (mhl)

5

6

-0.4

-0.2

f-A-1 C

0

I

0.2 0.4 r I I

I

0.6 I

I

0.8 1.0 1.2 I I I I 19

1.4 I

16.. 14.. 12..

tn (PEP],

-

I,

-4

-2

”

IO-

0

2

C-s,-+)

mM

Figure

4

6

I / [PEP],

8

IO

12

14

(mM)-’

1

The P-enolpyruvate (PEP) saturation curves for the freshly isolated the 48 hr. enzyme sample (A) and the 48 hr. enzyme sample incuenzyme (0), The closed symbols bated in buffer C plus 50 mM DTT for 10 hrs. at 4'C (u). (A, 0,s) correspond to the samples described above assayed in the presence of . The concentration of ADP was 4.4 mM and all other con2.0 mM fructose-1,6-P ditions are those of 8 he standard assay mixture described under Methods. Figure lB, Hill plots of the data shown in 1A. The Hill plot for the 48 hr. (A) was not constructed since no enzyme sample assayed without fructose-1,6-P2 demonstrable V value was obtained in the saturation plot and the shape of Figure lC, double this curve pre?%des extrapolation to a theoretical V reciprocal plots of the data shown in 1B for curves o!%?fined in the presence of fructose-1,6-P . The symbols of Figures B and C correspond to the samples described in Figu?e A.

freshly drawn trose solution In contrast, changes

in

its

or stored for periods prior to use. partially P-enolpyruvate

of 5 days

purified

pyruvate

saturation

to 8 weeks kinasewas

pattern

1328

during

in acid-citrate-dexfound storage

to exhibit as outlined

rapid

Vol. 74, No. 4, 1977

BIOCHEMICAL

1.0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

I

I

0.6 -

0.6 2Vmox 0.4 -

0.2 -

-2

0

0.5

1.0 ADP

-I 0 I L” mP,. KIM

1.5

2.0

CmM)

Figure

2

Normalized ADP saturation curves for the freshly isolated (0) and 48 hr. enzyme sample (A). Velocities at different substrate concentrations are expressed as the percent of maximum velocity (v/V ). The V values were determined from double reciprocal plots. The ins%?&! shows thEa& plots of the data. The assay concentration of P-enolpyruvate was 4.3 mM and all other assay conditions are those of the standard assay mixture described under Methods.

under

Methods.

aging

is

An example

shown

in Figure

reduced

catalytic

played

an elevated

mediary

plateau

modified which

higher

enzyme assayed (OOC) nor

changes

during

lize

human erythrocyte Incubating

occurred. the

the kinetic

that

extraneous used

metal

but

kinse

did

in buffer

observed

than

changes unlikely

observed because

1329

in

1C). rate

stabilargely

prepared of sulfhydryl

storage. from

C)

at which

50 mM DTT (4°C)

acid-washed

the storage

Neither (Buffer

(3,22).

the freshly

during

1B)

freshly

to partially

an oxidation

have resulted

and 2.0 mM EDTA was present

the

this

Fig. for

medium

previously for

that

converted observed

agents

dis-

an inter-

(Fig.

retard

C plus

originally

demonstrated

by DTT could is

they

exhibited

(nH = 1.0,

storage

has been noted

48 hr enzyme sample

contaminants

hyperbola

of reducing

upon

examined,

of fructose-1,6-P2

storage

properties

sample

to contain

the KM and V,,,

The ability

pyruvate

the reactivation

throughout

both

observed

Fructose-1,6-P*

rectangular

of 1.0 mM DTT to the

This enzyme (Fig. 1, A, B, & C). groups was involved in the kinetic bility

This

and appeared

in the presence addition

modification

for

curve

concentrations

curve.

to a normal

the

the

restored

P-enolpyruvate

values

freezing

these

P-enolpyruvate

in the saturation curve

prepared prevented

at all

KC.5 for region

saturation

1 and a 48 hr enzyme sample.

activity

saturation displayed

of one such modified

its

The possichelating

glassware medium.

was

BIOCHEMICAL

Vol. 74, No. 4, 1977

The ADP saturation samples

are

curves

shown in Figure

of maximum achievable was not

changed

the

oxidized

vs.

0.52

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for

both

the freshly

2, plotted

activity

upon aging

for

(v/V,,,)

form displayed

comparative

vs.

of the enzyme a slightly

isolated

and 48 hr enzyme

purposes

ADP.

The overall

(nH = 1.0;

Figure

decreased

s

for

as the shape

fraction

of the

2, insert)

this

curve

although

substrate

(0.37

mM

mM). Discussion

The overall

kinetic

GSSG as reported similar.

by Staal

Staal

were

entirely

dependent

storing

through

the sample of GSSG.

of potassium)

a factor

which

has kinetic

presented

the view

cases that

modification bility

in

Staal kinase

(for

et al . (8)

tional

work

will is

needed

alterations

which

shown for the

Ref.

genetic

schemes,

an internal

the

pH and

also

lead

in our work

purification

proremoved

they

show that

for

result

from post-translational

a particular see 24-26)

that

classes

data

this

These lies

elsewhere. on genetic

class

of defective

the red

cell

support

This

possi-

data

(23).

pyruvate

treatment --in vitro kinetic behaviour.

of altered

enzyme

further

based

the demonstrated the protein itself of

at

enzyme so produced

encountered

defect

milieu

normal

modifications,

2-5).

an analysis

which

(1)

selectively

the modified

enzyme to normal

to determine

produce

partial

in

the enzyme.

kinases

from

oxygen

(high

--et al. difference

may have

because

to such treatment and to determine whether is the result of genetic modifications in vate

this

frequently

(e.g.

support

restore

for

are

observed

and that

bubbling

conditions

by Staal

with

here

they

alterations

post-translational

to those

the primary

classification

kinetic

storage

significant

pyruvate

have

that

of GSSG and that

similar

received

mercaptoethanol

are

glutatione

the different

can undergo

of PK-deficiency

and that

the

utilized

changes

pH nor

One procedure

the absence

some aberrant

has also

of oxidized

or labilizes

here kinase

as reported

the kinetic

produce

that

by treatment

or by storage

that

the basis

responsible.

properties

in certain

enzyme,

stabilizes

pyruvate

in vitro,

could

is possible

either

The data erythrocyte least

It were

modified

5 days at alkaline

to those

of the

kinase

(1)

stated

we have observed similar

oxidation

employed

24 hours

Since

apparent.

(1)

enzyme for

for

absence

is not

and co-workers upon the presence

of the

absence

cedures

of pyruvate

and co-workers

neither

to a rapid

patterns

enzyme will

with Addirespond

sulfhydryl lability or due to inherited hostile

to pyru-

kinase.

Acknowledgements Supported

by Grant

No.

GM-14945

from U. S. National

1330

Institutes

of Health.

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Van Berkel, T. J. C., Koster, J. F. and Staal, G. E. J. (1975) Biochim. Biophys. Acta 321, 496-502. Sachs, J. R., Wicker, D. J., Gilcher, R. O., Conrad, M. E., and Cohen, R. J. (1968) J. Lab. Clin. Med. 72, 359-362. Paglia, D. E., Valentine, W. Il., Baughan, M. A., Miller, D. R., Reed, C. F., and McIntyre, 0. R. (1968) J. Clin. Invest. 47, 1929-1946. W. N. (1971) Blood 37, 311-315. Paglia, D. E., and Valentine, But, H., Najman, A., Columelli, S., and Cartier, P. (1972) Clin. Chim. Acta 38, 131-140. Blume, K. G., Arnold, H., LoHr, G. W., and Beutler, E. (1973) Clin. Chim. Acta 43, 443-446. Nakashima, K., Miwa, S., Oda, S., Tanaka, T., Imamura, K., and Nishina, T. (1974) Blood 43, 537-548. Van Berkel, T. J. C., Staal, G. E. J., Koster, J. F., and Nyessen, J. G. (1974) Biochim. Biophys. Acta 334, 361-367. Blume, K. G., Arnold, H., Lohr, G. W., and Scholz, G. (1974) Biochim. Biophys. Acta 370, 601-604. Weismann, U.,and T&z, 0. (1966) Nature 209, 612-613. Boivin, P., and Galand, C. (1968) Nouv. Rev. Franc, He'mat. 8, 201-208. K. W., and Haas, T. A. (1971) J. Biol. Ibsen, K. H., Schiller, Chem. 246, 1233-1240. Ibsen, K. H., and Trippet, P. (1971) Life Sciences 10, 1021. Boivin, P., Galand, C., Hakim, J., and Kahn, A. (1975) Enzyme, 294-299. Badwey, J. A., and Westhead, E. W. (1974) in Isozymes-I Molecular Structure (Markert, C. ed.) Vol. 1, pp. 509-521, Academic Press, New York, San Francisco, London. Badwey, J. A., and Westhead, E. W. (1976) J. Biol. Chem. 251, 5600-5606. Bucher, T., and Pfleiderer, G. (1955) Methods Enzymol. 1, 435-440. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Chern, C. J., Rittenberg, M. B., and Black, J. A. (1972) J. Biol. Chem. 247, 7173-7180. P. (1968) in Advances in Metabolic RegulaKoler, R. D., and Vanbellinghen, tion, ed. G. Weber (Pergammon Press 6, 127-140). Cartier, P., Najman, A., Leroux, J. P., and Temkine, H. (1968) Clin. Chim. Acta 22, 165-181. K. W., and Venn-Watson, E. A. (1968) Arch. Biochem. Ibsen, K. H., Schiller, Biophys. 128, 583-590. Zuelzer, W. W., Robinson, A. R., and Hsu, T. H. J. (1968) Blood 32, 33-48. Munro, G. F., and Miller, D. R. (1970) Biochim. Biophys. Acta 206, 87-97. Staal, G. E. J., Koster, J. F., and Nijessen, J. G. (1972) Biochim. Biophys. Acta 258, 685-687. Imamura, K., Tanaka, T., Nishina, T., Nakashima, K., and Miwa, W. (1973) J. Biochem. 74, 1165-1175.

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