Hexokinase isoenzymes in human erythrocytes of adults and newborns

Vol. 3 1, No. 1, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

HEXOKINASE IYCENZYl&ES IN HUMAN .ERYTHROCYTES OF ADULTS AND NE'MBGRNS uVern.er Schrijter Department University

Received

March

5, 1968

In hemolysates tally

distinct

strated

and

lysates

these

three

to the

rat

Brown et strated was seen

bands

al.,

in

activity

B, which

a faster

Because

of their

were

designated

dyes.

Eaton into

a1.(1967)

Types

I and III

I to

(Grossbard

and newborn

7

hemo-

saw additional-

electrophoretic

types

et 6 to

band A. In

as types

specimen

been demon-

was divided

--et

liver

blood

have

migrating

Holmes

adults

each

two electrophoreti-

tetrazolium

infants,

1967).

both

with

region

inconstantly

and human

in

gels

a wide

region.

erythrocytes

of hexokinase the

of newborn

a third

adult

regions

found

subbands

ly

of

by staining

-a1.(1966)

and Bilfried Tillmann of Pediatrics, of Hamburg, Germany

migration III

in

analogy

and Schimke,l966;

were

inconstantly

infants,

whereas

type

(Holmes

e-& aJ.,

of newborns

demonII

1967). Identification the

presence

of

of hexokinase

the

the

red

was used.

could type

The higher

isoenzymes

dyes

In

blood

hexokinase

of the

Therefore,

erythrocytes

of both

distinct

be demonstrated

I and III

because

is

cells.

two electrophoretically

activity to

tetrazolium

in

a new method borns

of hexokinase

bands

constantly,

hexokinase activity

isoenzymes of newborn

92

difficult low in

in

activity this

adults

study

and new-

of hexokinase

which from

are

similar

human

erythrocytes

liver. is

Vol. 31, No. 1, 1968

located

in

BIOCHEMICAL

the

demonstrated

type

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I region.

in newborn

No type

II

isoenzyme

could

be

infants.

METHODS AND RESULTS High-voltage Pfleiderer,

1955)

identified of

the

formed the

was used.

The hexokinase

isoenzymes

sensitive

hemolysates

hexokinase

aged

of the

both

adults

(region

method

of each 1 to

isoenzymes,

intensity

in

blood

adults

in

A both

whereas

and partly

covered

B is

for

4 'l/2

divided

uncovered

the

II

hexokinase

only

occasionally,

isoenzyme as the

type

could

be demonstrated

During

first

year

breadth

of region

B diminish levels

well

decrease

of

and the

increase

Table

of

of glucose

hemoglobin

bend

B was and of

usual

3 l/2

by hemoglobin

end of

the

as the

region

B, visible

of hexokinase

SO blood

about

sa;nples. and the

5 months

These

of age

changes

F (Betke

et

al.,

correlated 1959)

A,,.

the

effect

and fructose

93

of different

on the

hours,

F so that

impress

intensity

(Fig.1).

hemoglobin

1 demonstrates

of the

the

and at

to adult

the

both

same

VJhen electrophoresis

No variant in

of life

had decreased with

I.

Region

B could

and the

The

in newborns

of newborns

two subbands

isoenzymes the

fluorescence

instead

of region

anode.

migrating

and breadth.

hours

two

A) was the

slower

hemolysates

part

the

(region

by hemoglobin.

into

leading

type

in

found

towards

band

intensity

region

was performed region

both

of 40 adults

we constantly

migrating

were

fluorescence

specimen

migrated

and newborns,

the

and

of 366 mu.

5 days,

which

faster

adults,

than

trations

using

B) showed a much more marked

wider

the

(Wieland

NADPH at a wave length

and 40 newborns,

than

electrophoresis

by a more

In

in

starch-gel

intensity

concenof the

Vol. 31, No. 1, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Hexokinase Isoenzymes Region

A

(Type III>+ ilegion (Type Hemoglobin

B I>

+ ->

Origin

3 25

295

1

+

E;3,3

24,2

5

Months 2,7 Hemoglobin F (%>

4 Starch-gel of hexokinase isoenzymes el .ectrophoresis in hemo ysates of erythrocytes from infants aged one to five months. Horizontal electrophoresis (23,5 v/cm) was carried out fog 3 l/2 hours; the cooling fluid had a temperature of -4 to -6 C. The 0,02 M sodium barbital gel buffer, pH 8,6, contained 0,001 M EDTA and 0,005 M 2-mercaptoethanol. The electrode buffer was 0,06 M sodium barbital , pH 8,6, containing 0,0017 M EDTA and 0,005 M 2-mercaptoethanol. Before lysing, the cells from heparinized venous blood were centrifuged at 600 g for 10 minutes at 4'C, and plasma and the buffy coat were removed. The cells were suspended in 0,9 per cent saline containing 0,02 M glucose, andothe suspension was centrifuged at 2000 g at 4 C. Thig procedure was repeated twice. Hemofor 10 minutes lysates were prepared at 4 C by lysing washed red cells for 6 hours with one volume of a solution containing 0,005 M EDTA, 0,005 M 2-mercaptoethanol and 0,02 M glucose, pH 7,0. A clear supernatant was prepared by centrifugation at 20 000 g for 20 the sliced gel was incubated minutes. After el%ctrophoresis, for 2 hours at 37 C with 20 ml of a reaction mixture containing 0,l M tris-HCl, pH 7,4, O,OO25 M NADP, 0,0025 M ATP, 0,005 M MgCl 0,002 M KCN, 8 I.U. glucose-6-phosphate dehydrogenase or glucose When either ATP, !:lgCl and 810005 M glucose. The hexokr 4 ase isoenzymes were omitted, no bands appeared. were identified as fluorescence of the formed NADPH at 366 q.~.

9

hexokinase high

bands.

The activity

concentraeions

hexokinase

type

of glucose, III

from

rat

of

region

a fact and human

A is which liver.

inhibited also Both

by

characterizes regions

Vol. 31, No. 1, 1968

appear liver

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

fructose

region

A is

as substrate. not

inhibited

As type by high

III

from

human

concentrations

of

fructose. Effect

of different

fructose

on the

activity

Molarity strate cubation

of subin the inmixtures

Glucose

10

Table

I.

-1

10-3 1o-4

5 x IO -4 10-5 10

of hexoKinase

Newborns Region B Region (Type I) (Type

1o-2

Fructose

concentrations

-1

1o-2 10-3 1o-4

of glucose

and

isoenzymes.

Adults Region B Region

A III)

(Type

I>

(Type

+++ +++ +++ ii+

(+I ++ ++ ++

++ ++ ++ if

(+> ++ ++ ++

+++

++

++

if

+

+

+

+

+++ +++ ++ -

ii ++ +

if ++ +

ii ++ +

A III>

In the experiments with fructose 10 I.U. phosphohexose isomerase were added to the incubation mixtures. Each concentration of both glucose and fructose was studied in two runs of electrophoresis with each three to four different blood specimens. The intensity of the bands is expressed as faint band, following: - no visible band, (+) extremely + faint band, ++ intermediate band, +++ strong band.

DISCUSSIOT\T The table activity this

clearly

of newborns region

constantly

exhibits

demonstrates is

located

a high

in hemolysates

that in

stability. of adults

95

the

region

higher

hexokinase

B. The isoenzyme It

could

and newborns

of

be demonstrated when the

Vol. 3 1, No. 1, 1968

BIOCHEMICAL

heparinized

blood

fore

lysing.

band

are

whereas

properties

showed

the

kinase

from

pared

results

similar

bands

migration,

their

types

liver. the

produced

When we used found

in

the

the the

region

type

A

III

hexo-

were pre-

that

II

the

erythrocytes

the

to

that

their

might of

of region

seen

they

are

B) of

by Holmes

et --

be an artefact,

region as Eaton

blue

rat al.

which

B by hemoglobin

B into

of tetrazolium

electroglucose

A) and I (region

isoenzyme

two

from

substrates

we assume

(region

demonstrate

erythrocytes

B (probably

of synthesis III

of

same method

division

Region

--et al.(l966),

F. we

6 to 7 subbands, region

B was an uniform

W-light.

Our data of newborn

the

be-

liver,

which

suggestion

to

interruption

absence

as the

According

of newborns

by the

mobility

stability,

The type blood

enzyme.

affinity

III

from

rats,

24'C

widespread

instable

the

identical.

in

in

us to

are

the

at

--et al.(l967).

and newborns

and their

relative

of adult

hemolysates

and human

in

and the

I hexokinase

the

to

(1967)

lead

72 hours

a more

by Holmes

and fructose

type

is

as described

phoretic

band

II

48 to

type

homogenates

adults

but

the

liver

hexokinase

also

stability of

type

for

same electrophoretic

These

is

was stored

The high

liver

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

type

of type during

that

the

nigher

hexoKinase

activity

hexokinase

isoenzyme

is

located

in

I),

This

indicates

I hexokinase

is

the

different

that

the from

regulation that

of

development.

ACKNOWLEDGUJXNTS This work was supported by a research grant to W.B. from the Deutsche Forschungsgemeinschaft,Bad Godesberg. W.T. thar.ks the Deutsche Forschungsgemeinschaft for a stipendium.

BIOCHEMICAL

Vol. 31, No. 1, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES Betke,K.,

Marti,H.R.,and

(1959).

Brown,J.,

155,

Eatonm!l.,

(1966).

Miller,D.b!l., 205 (1967).

Grossbard,L., Holmes,E.W.,Jr., Science, Wieland,Th.,

Brewer,G.J.,

Schlicht,I.,

Nature

Holloway,M.T.,and

J.Biol.Chem. Winegrad A.I., Angew.Chem.

97

1877

Leve,D.G.,

and Tashian,H.T.,

and Schimke,R.T., Malone,J.I.,

184,

Nature

Science 212,

944

m, 3546 (1966). and OsKi,F.A., 3,

257

(1955).