Control of heme synthesis by feedback inhibition

Vol. 18, No. 2, 1965

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMJNICATIONS

CONTROL OF HEME SYNTHESIS BY FEEDBACK INHIBITION Doris

Karibian

and Irving

Department of Medicine, Albert and Bronx Municipal Hospital

ReceivedNovember18,

al,

1958)

involved animal

provides the

cells.

regulation

the

acid

bilinogen

(PBG) was less

bacterium,

catalyzes

however,

was observed

pointed

to ALI

In the

studies

incorporation

The results feedback

rabbit

suggest inhibition

that in

here

the

conversion 243

in

from

This

enzyme

the

form

cells

site

for in

of

this

by hdn

have

These

negative

this

effects

feed-

bacterium. of hemin

into

on

heme in

been

mechanism,

of glycine

porpho-

precursor.

and ALA-4-Cl4

control

A to

The enzyme ALA

was the

(reticulocytes) a similar

processes

synthetase

synthesis

by hemin

et

shown that

intact

as the

14

of glycine-2-C

the

of ALA to

not ALA,

reported

erythrocytes

1954).

of porphyrin

synthesis

Morel1

and succinyl-coenzyme

With

synthetase

of porphyrin

have

condensation

but

of

by hemin.

(Shemin,

inhibition

synthesize

mechanism

(ALA)

inhibited

inhibited.

when glycine,

control

immature

the

study

(1962) acid

to 1950,

biosynthetic

of glycine

deltaaminolevulinic

et al,

the

and Lascelles

condensation

which

the

for

are

cells

Shemin

of this

spheroides

dehydrase

back

1950,

of deltaaminolevulinic

catalyzes

College of Medicine New York, New York

erythroid

an opportunity

Rhodopseudomonas

findings

immature

et al,

Burnham

preparations

form

of

(London

in

Einstein Center,

1964

The capacity heme --in vitro

M. London

examined. involving

to ALA,

is

Vol. 18, No. 2, 1965

operative

in

BIOCHEMICAL

these

animal

Methods: were

bled

(about

were

centrifugation.

produced

reticulocytoses

tiorsplus

one ml

containing

the

in

15-20%.

were

added

following:

0.76%

0.02

buffer,

When a lysate

distilled

adjusted

with

the

saline

lysate;

incubation.

0.8

to

cpm/umole

ml

1.0

to of

give the

The tubes

were

95% 02-5%

co2.

I

.

mg per

or ALA-4-C14,

solution 0.12

of

ml

total

(1)

hemin

umole

volume

At

the

in

KCl,

pH 7.8

of 1.2

cell

14

1.6

solution

without

a 37O water

bath

incubation,

MgC12.6H20,

they

was and

suspensions. in

mixture

cell

In hemin room were

12

suspension

or

and streptoof

umole

cpm/umole;

in

cells

and plasma

, 0.25

ml.

of

tonicity

penicillin

4 x lo5 to

solution

the

solution

glycine-2-C

prepara-

volumes

The incubation

of

end of

cell

or K2HP04 - KH2P04

1 minute,

of

cells

0.15%

equal

whole

and

bleeding

a saline

aliquots . solution'; (3)

0.5

of

8 ml of

1 ml

(4)

packed

such

0.039%

rabbits

3 days)

One ml of

as for

ml;

KOH-buffer-HCl incubated

ml of

saline

of

least

series

for

concentrated

Conditions

0.12

l-3

to

mixed

added

(2)

mycin

were

included:

at

was required,

were

tubes

top

buffer,

solution

conical

ml

lo-fold

for

NaCl,

M Tris-HCl

water

New Zealand

prolonged

of

100 mg% glucose,

and cold

the

More

plasma

pH 7.4.

Preparations. 30 ml daily

obtained

following

RESEARCH COMMUNICATIONS

cells.

Reticulocvte

repeatedly

reticulocytes

AND BIOPHYSICAL

(5)

control

8 x lo5 saline samples,

was added. air chilled

or in in

ice,

Hemin solution: (a) 1.2 mg hemin was dissolved in 0.1 ml 1N KOH and diluted with 1.0 ml freshly boiled distilled H 0 and 0.1 ml 0.08 ml 1N HCl was ad 8 ed with mix1M tris or phosphate buffer: ing and the volume was made up to 2 ml with H20; (b) for higher concentrations, 8 mg hemin was dissolved in twice the above volumes of KOH, buffer and HCl and the volume was again made The final concentration of hemin when up to 2 ml with H 0. 0.12 ml of solute -8 n (a) was present in an incubation mixture of 1.2 ml was 1 x lOa M.

244

Vol. 18, No. 2, 1965

and 10 ml of ~20)

BIOCHEMICAL

cold

was added

solution,

cell

lysate

to provide

equal

experimental

red

in

to

glacial

that

which

was added

from

pyridine-chloroform,

infinite

acetic

acid

to

duplicate

with

(Fischer,

Nearly

agreement

the

+ 5 per

% Change

from

1 vol

Hemin added

hemolysate It

to

in

the

after

treatment

was recrystallized was measured

experiments

Effect of Hemin on Utilization for Heme Synthesis in Intact

were

carried

at

out

in

cent.

I

of Glycine-2-C and Lysed Rabbit

14

of Glycine-2-C into Heme

and

samples.

radioactivity

Table

Incorporation

control

1941).

all

within

cells

had been

the

from

and its

thickness.

packed

heme as carrier.

Hemin was crystallized with

RESEARCH COMMUNICATIONS

(1 vol

unlabeled

amount

samples,

AND BIOPHYSICAL

14

Incorporation into

Control

% Change

and ALA-4-C14 Reticulocytes

14

of ALA-4-C Heme from

Control

42

21

Mean change

-48.6

-12.2

S.E.

+ 3.00

+ 4.84

Number

of

experiments

p < .OOl

Results: on the by the tions

utilization

intact

Table of

and lysed

percentage to which

I presents

no hemin

14

glycine-2-C

rabbit

change

.05>p).O2

the

effects

and ALA-4-C

reticulocytes.

from

the

245

14

values At

this

added

for

The data

control

was added.

of

hemin

heme synthesis are

obtained concentration

recorded in

as

prepara(1 x 10m4M)

Vol. 18, No. 2, 1965

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

the

on the

effect

tory

of hemin

in all

but

inhibition

of nearly

mean effect tion

of

one of

of hemin

only

12 per

nificance.

the

and in

42 experiments. cent

on the

utilization

cent

hemin

instances

was an

significant.

The

of ALA-4-C 14 was an inhibi-

and was of only

several

was inhibi-

The mean effect

and is highly

of added

14

of glycine-2-C

50 per

The effects

variable

utilization

marginal

statistical

on utilization

enhanced

sig-

of ALA were

synthesis

of heme was

observed. Figure on the

1 shows the

synthesis

effect

of heme from

of concentration

glycine

in

intact

of added rabbit

hemin

reticulocytes

d E iooe 86 1

60 fL

;

60-

Y g :

\ %

-*

-\

.N

.0

HEME *z

40-

5 :F

20-

% LI:

0

Preincubotion Incubation

I I x10-4

The effect utilization

I

I 3x10-%4

is

creased

at higher

appreciable

Figure throughout

2.

at

1 x 10

-4

M and is progressively

on the

in-

concentrations,

The effects incubation

4xlO-‘M

OF HEMIN

of various concentrations of added hemin of glycine-2-C14 for heme synthesis.

Inhibition

term

-------.

2x10-4

CONCENTRATION

1.

-5

2 hours

I 5X165M

--.- \

with HemIn 30’

of hemin

and in

a short-term

The inhibitory a 24-hour

on the

period

effect of

synthesis

incubation occurs

incubation.

rapidly

of heme in a longare presented and is

in

demonstrable

BIOCHEMICAL AND BlOPHYSlCALRESEARCHCOMMUNICATIONS

Vol. 18, No. 2, 1965

;E1400 F 5 2 1000

1000 c5

/

N \ 1200

3 1200 B

-CONTROL

P

800

E 3 600 2 =

400 200 4

2.

8

12

16 20 HOURS

Inhibitory effect for hema synthesis.

24

7.90

in

isotonic

and in Tris-NaCl

was observed inhibitory pH which

at pH 7.3-7.4 effect

the

was observed

added

range

to 8.70.

absence

The results

inhibition

heme synthesis

in

by hemin rabbit

of

hemin

was

of pH 6.35 Maximal

added

over

inhibition

by hemin which

precede

of these of

the

reticulocytes;

of ADA was variable

reactions

the

72

the

synthesis

hemin. full

to

The

range

of

was studied.

significant

of

pH 7.25 in

of hemin

Discussion:

tion

buffer,

in

1

60

of glycine-2-$*

and without

buffer

/

36 48 MINUTES

on utilization

of heme with

phosphate

I

w1

31

of hemin

The synthesis studied

28

and less appears the

studies

utilization the

therefore

formation

of glycine

effect

significant.

indicate

on the

+ Glycine

of ADA: -co2 ?o(

Pyridoxal Phosphate

247

site

among the

ADA Synthetase Succinyl-CoA

utiliza-

The principal to be found

for

-amino 4 -Keto adipic acid (=w

*

ALA

Vol. 18, No. 2, 1965

8lOCHEMlCAL AND 8lOPHYSlCAL RESEARCHCOMMUNICATIONS

These activation the

of

reactions

glycine,

condensation

of

by ALA synthetase, to

form

tion

ALA.

of

these very

It

readily;

in

form

ANA,

possible

hemin

but

the

that

rabbit

of

succinate,

the

system;

catalyzed

decarboxylation

of ARA

may inhibit

the is

activa-

available

on

The decarboxylation

of ARA occurs

but

that

it

is

possible,

of AKA to ALA.

a site

succinate,

phosphate

no information

and Lascelles

likely

and

and the

that

conversion

of Burnham

synthesis

glycine

seems unlikely,

the

seems most

activated

at present. it

activation

by a pyridoxal

or glycine,

possibilities

may influence

it

is

the

probably

to

succinate

studies

include

In

the

hemin

light

of

in Rhodopseudomonas

spheroides,

of

of

feedback

reticulocyte

is

control

at

the

level

the

heme

of ALA

synthetase.

which

the

The concentration

of

hemin

inhibitory

is

observed

extent

to which

of

binding

its

note in

that

effect the

within

free

at

of

this

a mechanism

Coupled

with

the

globin

(Bruns

in

coordination

of

the

are

order

feedback

affords

a grant

cell

concentration

Negative

the

the

for

the

stimulatory

and London),

This work the Office

of

the

unknown,

the

not

reticulocyte

cell

known.

It

is

of

10B4M,

in

Rhodopseudomonas of

regulation effect

of

control

synthesis

present

is in

the extent

relevant

to

porphyrin spheroides.

heme synthesis

by heme

of porphyrin

synthesis.

hemin

synthesis

on the

of

may participate

heme and of

globin.

was supported by USPHS grant HE-02803 of Naval Research Nonr 4094. 248

at

reticulocytes

can inhibit

mechanism of

since

and the

which

control

this

the

is

can penetrate

protoporphyrin,

a concentration

synthesis

hemin

within

and

Vol. 18, No. 2, 1965

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

BrUnB, G.P. and London, 1.X. (accompanying paper) Burnham, B.F. and Lascelles, J., Biochem. J. 87, 462 (1963) Fischer, H., Org. syntheses 2l, 53 (1941) London, 1.X., Shemin, D. and Rittenberg, D., J. Biol. Chem. 183, 749 (1950) MOrell, H., Savoie, J.C. and London, 1.X., J. Biol. Chem. 233, 923 (1958) shemin, D., The Harvey Lectures, Series 50 (1954-1955) (Academic Press Inc., New York, 1956) p. 258 Shemin, I)., London, 1.X. and Rittenberg, D., J. Biol. Chem. 183, 757 (1950)

249