The effect of hemin on the synthesis of globin

The effect of hemin on the synthesis of globin

Vol. 18, No. 2, 1965 BIOCHEMICAL AND BlOPHYSlCAt RESEARCHCOMMUNICATIONS THE EFFECT OF HEMIN ON THE SYNTHESIS OF GLOBIN Gail P. Bruns Department of...

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Vol. 18, No. 2, 1965

BIOCHEMICAL AND BlOPHYSlCAt RESEARCHCOMMUNICATIONS

THE EFFECT OF HEMIN ON THE SYNTHESIS OF GLOBIN Gail

P. Bruns

Department of Medicine, and Bronx Municipal

ReceivedNovember

studies

rabbit

reticulocytes

1958),

rabbit

of

two

of

synthetic

cobalt

or purine (Morel1

could

of

a mechanism

synthesis

of

the

(Karibian

may serve

to

which

the

are

summarized of

approximately

marked

and London) coordinate for (1)

briefly:

inhibition

10

-4

in

al,

1958

are

or by the

or by

incubation

rates

and Kassenaar the

in et

normal

agents,

but

the

coordination

and in

evidence

the

proposed

for

of

the

glycine

the

accompanyin

a mechanism

of

The

regulation

hemin

reticulocytes of

question

processes.

mechanism

addition

1957).

of

here

two biosynthetic

al,

a

parallelism

reported

236

the

addition

to be explored.

utilization

al,

influences,

1951)

the

on the

in

remained

provide

et

and dog reticu-

by various

that

M to rabbit the

1958)

physiologic

globin

and Morel1

in

The two mechanisms,

by various

which

basis

et

al,

normal

heme and of

al,

vitro.

indicated

be disturbed

The studies paper

et

heme and globin

1956

ribosides

effects

for

of

affected

(Richmond

Such differential rates

College of Medicine New York, New York

a parallelism

in

be differentially

medium

in

et

indicated

processes

or lead

hypotonic

synthesis

(Morel1

have

by irradiation

e.g.,

Einstein Center,

and Borsook,

marrow

1957)

could

however,

on the (Kruh

bone

(Nizet,

the

Albert Hospital

M. London

18, 1964

Prior

locytes

and Irving

in

which findings may be

a concentration

in

vitro,

there

for

the

synthesis

is

a of

8lOCHEMlCAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

Vol. 18, No. 2, 1965

the

heme;

principal

are

concerned

(2)

under

site

with

these

an increase

the

the

of

(Nutritional

water

and were

repeatedly

ml of media 1

acids,

L-Valine-Cl4

flasks

flasks

the

and

which

acid

(ALA);

results

in

newly

formed

can increase

of

the

flushed

incubated

at

the

at

with

1500

The cells

were

lysed

twice

x g for

1 volume

punc-

plasma cells

was in

supplemental

plasma,

glucose

84 mg%, and NaHC03 0.01 isotopic

substrate.

M.

Incu-

5 minutes,

stop-

shaking.

15 minutes,

45 minutes

packed

95% O2 - 5% CO2 for

with

20-25%

by cardiac

with

20% rabbit

of hemoglobin,

x g for

with

ml of

iron

distilled of

and the

medium

was the

37O C without

isolation

washed

20,000

0.15

84 mg%, streptomycin

were

obtained

15 minutes

medium,

on "low

a hematocrit

40% Eagle's

labeled)

maintained and deionized

Blood

x g for

40% NC1 culture

were

at

reactions

hemin

into

were

to maintain

contained

consisting

cells

fuged

-valine

own formation

15-20%.

1500

(uniformly

For

the

of

at

Incubation

centrifuged

14

of

Corporation)

bled

count

was centrifuged

and

addition

rabbits

Biochemical

100 mg%, penicillin

pered

of C

New Zealand

and a reticulocyte

bation

the

deltaaminolevulinic

the

its

among

globin.

diet"

removed.

is

of

incorporation

Methods:

aminr,

formation

Heme can inhibit

synthesis

3.4

inhibition

same conditions,

in

hemoglobin.

ture

of

the the

4 volumes of

distilled

at

4O C.

cell

media of

were

isotonic water

were

removed

and

sodium

chloride

and were

centri-

The supernatant

TL alanine, .0007M; arginine HCl, .00016M; aspartic histidine HCl, .0009M; lysine, .0008M; methionine, phenylalanine, .00052M; proline, .00053M; serine, threonine, .0005M; tryptophane, .OOOlW; tyrosine, leucine, .OOlM; cysteine, .00015M 237

suspensions

acid, .OOlM; .OOOlW; .0006lM;. .00026~:

Vol. 18, No. 2,1965

solution

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNlCATlONS

was applied

Verona1

buffer

optical

density

to

starch

pH 8.6.

of

The hemoglobin

spot

were

Hemoglobin

formation

added

hemin

I 5Xl65M

is

I

was eluted

0.05

M

and the

as a function in

1

I x10-4

in

determined.

shown

Figure

1.

I

2xX)-4

CONCENTRATION

1.

electrophoresis

and radioactivity

Results: concentration

block

of At

the

a concen-

I

3x10-w

4x10-h

OF HEMIN

The effect of various concentrations of added hemin on the formation of globin. The cell suspension was incubated for 30 minutes at 37O C with added hemin; the control was incubated without hemin. At 30 minutes, 1-Valine-C14, 12 mumoles, 8.4 x lo7 cpm/umole was added and the incubation was continued for 2 hours. Results are expressed as the radioactivity of the globin of the experimental samples relative to the control which is assigned a value of 100.

tration

as low

as 5 x low5

M there

is

a considerable

Cl4 -valine

incorporation.

The increased

hemoglobin

in

of

the

presence

out

a 24-hour

period

very

rapidly

and is

incubation were

(Fig.

incubated

Cl?-valine;

2b). with

in

other

of

added

incubation

readily In hemin

observed these for

experiments

formation

hemin (Fig.

is

in

238

which

first

the prior hemin

in

labeled through-

The effect the

experiments,

of

demonstrable

2a).

during

30 minutes

increase

hour

occurs of

reticulocytes

to

the

addition

and C14 -valine

of

BlOCHEMlCAL

Vol. 18, No. 2,1965

2

4

6

AND BIOPHYSICAL

6

10

RESEARCH COMMUNICATIONS

I

20

"16

12

24

26

TIME IN HOURS

$400-

b

E” l.200‘E p

~~-

1 P

800-

I” +

600-

& :

400-

a8

200I IO

2.

were

I 20

I I 30 40 TIME IN MIN.

I 50

I 60

The effect of added hemin on globin formation. After incubation for 30 minutes at 37O C with added hemin (L x 10'4 M) or without it (control) l-Valine-C14, 12 mumoles, 8.4 x lo7 cpm/umole was added and the incubation was continued for various periods of time, (a) hours, (b) minutes.

added

observed

formation

of protein

was

'in 15 minutes. Previous

laboratory

enhanced

simultaneously,

(Kassenaar

studies et al,

by Kruh 1957) 239

and Borsook have

(1956)

demonstrated

and from enhanced

this forma.

Vol. 18, No. 2,1965

BIOCHEMICAL

tion

of hemoglobin

iron

and of hemin

effects

on the

was observed question

on the

iron

in

cells

by

diet

from

the

of

hemin

have peptide

of the

newly

(a)

the

if

would

lead

synthesis: 204

(b)

of

enhancing

study.

effects

of hemoglobin

in

The on the

its

rendered

cells

of hemin

are observed

The stimulatory

iron-deficient effects

with

possible

formation

of hemin

normal

or elevated

the

of hemin chains are

subunits

to tetramers less

hemin

would

the

increase

may be a combination

240

of the

rate of

the

than in

conversion of the

the

tetramer,

effects

formatio

exert of

a feedsubunits

subunits

of polypeptide the

poly-

conversion

increased

should

concentration in the

l

the

may

of hemoglobin;

stable

of hemoglobin

synthesis,

there

(1)

may promote

be reflected

of polypeptide diminish

are:

by which

on the biosynthesis

of heme would if

mechanisms

of globin

effect addition

to a secondary (c)

effect

have been

of hemoglobin

effect

tetramer

that

polypeptide

subunits

inhibition

to the

the

formed

of hemoglobin; back

the

(2)

a stabilizing

together.

most marked

formation

or absent

accelerating

chains;

the

The principal

increases

a primary

added

increase

iron.

Discussion: added

were

of

stimulatory

or no further

stimulatory

phlebotomy.

may be minimal

concentrations

maximal

or by virtue

that

animals

and extensive

their

heme is under

be noted

increasing

concentrations

its

independently of

At

little

and iron

exerts

RESEARCH COMMUNICATIONS

iron.

produced

hemin

iron

should

derived

of

of globin,

synthesis

in

iron

singly

when both

It

addition

synthesis

of globin

effect

or of

which

of whether

synthesis

or of

on the

AND BIOPHYSICAL

and chain

of 2(a)

and

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

Vol. 18, No. 2, 1965

A major enhancing

question

effect

studies

of

proteins

in

on the

that

addition

globin the

concerned

of hemin

indicate

formation

is

the while

the

reticulocyte

cubated

in protein

in

the

is

of

The inhibitory coupled

with

provides

test.

It

(3)

is

and

(2)

currently

being

the

the

inhibition

stimulation

papers.

If could

by globin, mechanism

the

the

raises

subunits

been

might

in-

of globin of

the

to experimental

speculative

its

system

be more

sensitive

and

(4)

own synthesis.

and considered

processes

formation

possibilities

by globin

described

to a dual

own

of heme on the

of

syn-

as a working

by heme of its

it

and fourth

contribute which

inhibition

or its

third

nuclei

formation

subjected

effect

have

independently

own synthesis

is presented

of heme synthesis

by globin two processes

the

stimulatory

In addition,

of other

a schema of regulation

the

increased

erythrocyte

of heme on its

schema

(1)

in

bulk

observed

avian

increasing

for

are

found

of regulation, than

the The

in

these

to be operative, by heme and

regulation

by either

alone.

This work of the Office

the

Preliminary

results

of the

of

hemin.

This

includes:

of globin.

grant

basis

which

synthesis

they

in

of hemoglobin.

hypothesis

first

effect

a partial

thesis

of

its

of globin.

have

in

effect

specificity

stimulated.

(1964)

added

the

of hemin

not

synthesis

presence

synthesis

synthesis

Hannnel and Bessman an increase

with

was supported by USPHS grant of Naval Research, Nonr-4094. 241

HE+02803

and a

Vol. 18, No. 2, 1965

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES

Hammel, C. and Bessman, S., Fed. Proc. 23, 317 (1964) Karibian, D. and London, 1-M. (acc-mpanying paper) H. and London, I.M., J. Biol. Chem. 22p, 423, Kassenaar, A., Morell, (1957) Kruh, J. and Borsook, H., J. Biol. Chem. 220, 905 (1956) Morell, H., Savoie, J.C. and London, I.M., J. Biol. Chem. 233, 923, (1958) Nizet, A., Bull. sot. chim. biol. 39, 265 (1957) Richmond, J.E., Altman, K.I. and Salomon, K., J. Biol. Chem. 190, 817 (1951)

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