THE JOURNAL OF BIOLOGICAL CHEMISTTRY Vol. 242, No. 8, Issue of April 25, pp. 1903-1911, 1967

Printed in U.S.A.

Control of Hemoglobin Synthesis in the Cultured

Chick Blastoderm*

(Received for publication, September 8, 1966)

RICHARD D. LEVERE AND S. GRANICK

From the Department of Medicine, State University of New York, Downstate Medical Center, New York, New York 11203, and The Rockefeller University, New York, New York 10021

SUMMARY

The early, de-embryonated chick blastoderm, cultured in vitro, closely simulates a phased culture of erythroid cells. This fact permits the study of hemoglobin synthesis from colorless erythroblast precursor cells to fully hemoglobinated erythrocytes.

When b-aminolevulinic acid is added to colorless blasto- derms, thereby by-passing &aminolevulinic acid synthetase, copious amounts of porphyrins, and presumably of heme, are formed. This result indicates that in the erythroid precursor cells of the early blastoderms heme synthesis is limited by the activity of &aminolevulinic acid synthetase.

The addition of Caminolevulinate also results in an in- crease in globin synthesis and hemoglobin formation. The enhancing effect of b-aminolevulinic acid on hemoglobin formation is not abolished by actinomycin D but is prevented by puromycin. This result suggests that &aminolevulinic acid forms heme which stimulates globin synthesis at the ribosome level. It is conjectured that heme may be neces- sary for the appropriate folding of the globin polypeptide in the completion of its synthesis.

The hypothesis is proposed that the formation of d-amino- vulinic acid synthetase, which is under repressor control, is the limiting and controlling reaction in the formation of hemo- globin. Such a hypothesis would explain the fact that no free globin is formed and that monomeric globin and heme are formed in a 1:l ratio.

The almost complete elucidation, over the past two decades, of the heme biosynthetic chain has provided a unique model for the study of the control mechanisms which operate to govern such a synthetic pathway. The present study was undertaken to investigate the temporal appearance of the enzymes of this biosynthetic chain in relation to progressive erythroid matura- tion and to study the interrelationships between heme and globin synthesis. A preliminary report has been published on some aspects of this study (1).

The first enzyme in the heme biosynthetic chain, &amino- levulinic acid synthetase (see Fig. l), condenses glycine and

* This work was supported in part by United States Public Health Service Grants AM 09838 and GM 04922.

succinate to form &aminolevulinic acid. This enzyme has been shown by Granick (2, 3) to be rate-limiting for the entire bio- synthetic chain of heme in liver cells. One of the specific questions asked in the present study was whether this enzyme also limits heme synthesis in erythroid precuS_sor cells. The second question was, in what manner is the regulation of heme synthesis related to globin formation so that they are normally produced in a 1: 1 ratio?

The chick blastoderm, cultured in vitro, provides an excellent system for the study of these problems. Unlike bone marrow, the blastoderm is completely devoid of myeloid and lymphoid elements. The hematopoietic tissue of the early blastoderm starts with colorless cells at a stage prior to initiation of hemo- globin synthesis and these differentiate in 24 to 48 hours into fully developed erythrocytes. Therefore, this tissue may be regarded as a phased culture of erythroid cells and its differ- entiation studied with respect to time.

In vivo, the blastoderm is free of hemoglobin until the embryo is at the five- to six-somite stage of development (4). At this time hemoglobin appears in the area pellucida and surrounding area opaca in a horseshoe-like pattern just lateral and posterior to the developing embryo. O’Brien (5) has shown that when young blastoderms, i.e. prior to the fifth to sixth somite stage, are de-embryonated and grown on a simple glucose agar medium there is inhibition of cell migration and maturation in all cells except the developing hematopoietic mesoderm. This hema- topoietic mesoderm is arranged in clusters of syncytial tissue called blood islands, and is found in the horseshoe-shaped area where hemoglobin will eventually appear. The cells of the blood islands are comparable to the hemocytoblasts of mam- malian bone marrow. Sabin (6) showed that these cells have the potential of developing into either the primitive line of erythrocytes or the endothelial lining cells of the extraembryonic blood vessels. When the blastoderm is cultured in vitro, the endothelial cells form only poorly defined tubules within which are found the nucleated erythrocytes.

Hemoglobin first appears in the cultured, de-embryonated blastoderm after 14 to 20 hours of incubation. The time of the initial appearance of hemoglobin is directly related to the age of the blastoderm at the onset of incubation. Blastoderms closer to the five-to six-somite stage develop hemoglobin earlier than blastoderms cultured at earlier stages. Maximum hemoglobin production is reached after 36 to 48 hours of incubation in vitro. Because the maturation from precursor cell to definitive eryth-

1903

by guest on August 1, 2020

http://ww

w.jbc.org/

Dow

nloaded from

1904 Hemoglobin Synthesis in Chick Blastoderm Vol. 242, No. 8

Glycine + pyridoxal-P + succinyl-CoA

4 &AL synthetase COOH-G-l,-CHZ-CO-CH,NH,

d-amino levulinic acid (dAL) CCOH

COOH ?H,

901~.pyrryl methane Pa iy +-PBG ?

Uroporphyrinogen III (UROGEN)

1 UROGENASE

?f’ % CH3

Coproporphyrinogen III (C~~XOGKN)

1 COPROGEN oxidase + 0,

Vi 9 C%

HS$yQ;

W-@;+,

Protoporphyrin-9 (PROTO)

1 Fe++

FIG. 1. The general scheme of biosynthesis of heme. AC, CH&OOH; Pr, -CH,CX-COOH; Vi, -CH-CH2. To form porphobilinogen, 2 moles of b-aminolevulinic acid are required.

rocyte takes less than 2 days, the cultured blastoderm simulates a phased culture of erythroid cells.

Early hematopoietic cells may also be obtained by the culture in vitro of cells obtained from blastoderms dissociated by trypsin. The vast majority (7) of these cultures will yield erythroid cells to the exclusion of other cell types. This system, like the culture of the whole de-embryonated blastoderms, also allows for the study of the early stages of erythroid development. However, the poor yield of mature erythrocytes and the early dedifferentia- tion of these cells prevents this technique from offering any advantage over the whole blastoderm system.

MATERIALS AND METHODS

All embryos used were of the White Leghorn breed, obtained from Shamrock Farms, New Jersey. Upon arrival the eggs were incubated at 37” for the desired period of time (usually 20 to 22 hours).

The yolk of each egg was placed in chick Ringer’s solution (8) and the embryonic disc was removed with a circular cut through the vitelline membrane employing fine dissecting scis- sors. This and the succeeding steps are graphically outlined in Fig. 2. The blastoderms were dissected free of the vitelline membrane with a ball-tipped glass needle and then washed free

of any adhering yolk by gently sucking the blastoderms in and out of a wide mouthed pipette. Following this procedure, the embryos were removed from the blastoderms under a dissecting microscope, utilizing a glass needle and a fine tipped suction pipette. The de-embryonated blastoderms were then divided into symmetrical halves by an anterioposterior cut made with iridectomy scissors. One-half of each blastoderm was placed in a small Petri dish containing an agar gel medium composed of 1 y0 agar in Earle’s solution (9). Additions of various chemicals were made to the test and control media as indicated by the experiment. Prior to placing the blastoderms on the agar, the halves were washed in liquid medium of the same composition as the agar medium. The Petri dishes were then incubated at 37” in an atmosphere of 5% COZ-95% air for the desired period of time. After incubation, the amount of porphyrins or hemo- globin in the test and control halves was determined.

Estimation of Porphyrins in Blastoderms-Porphyrins were determined qualitatively by examining the unfixed blastoderms with a Zeiss Ultraphot fluorescence microscope. The presence of orange-red fluoresence is indicative of the presence of in- creased amounts of uroporphyrin and coproporphyrin.

Semiquantitative Determination of Heme knd Hemoglobin- Heme and hemoglobin were stained by a benzidine peroxidase stain (10) and the reaction then quantitated macroscopically and microscopically. The blastoderm halves were washed with 0.9% NaCl to free them of the agar and then were placed in the staining sloution for 3 min. The staining solution was made as

follows: (a) 100 mg of 0-dianisidine (3,3r-dimethoxy benzidine), (b) 70 ml of 95% ethanol, (c) 10 ml of 1.5 M acetate buffer, pH 4.7, (d) 18 ml of water, and (e) 2 ml of Hz02 (30%) added just before use.

Following removal from the above stain, the blastoderms were washed with distilled water. They were then dehydrated in dioxane, cleared in xylol, and finally mounted on glass slides. Each specimen was examined for the presence of brownish pink color indicative of heme or hemoglobin or both. The degree of the reaction was graded according to the following schema. Trace: stained erythrocytes visible microscopically only; 1 +, positive reaction just perceptible with the naked eye; 2+, few scattered areas of faintly positive reaction; 3 f, reticular pattern

to positive areas, cells deeply stained; 4 +, intense and extensive

reaction confined to area pellucida; 5+, same as for 4f with positive reaction peripherally in area opaca.

Quantitative Determination for Hemoglobin-The minute

FIG. 2. Schematic representation of technique used for handling blastoderms. See text for detailed description of method.

by guest on August 1, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of April 25, 1967 R. D. Levere and S. Grand 1905

amount of hemoglobin present in each blastoderm half was quantitated by spectrophotometry. Following incubation, the blastoderm halves were washed free of the agar with 0.9% NaCl and placed in thick walled serum hematocrit tubes (Clay-Adams) with particular attention paid to exclude excess 0.9% NaCl. At this point, if time was not available, the blastoderms were frozen at -20” and kept at this temperature until used. Then, in order to hemolyze the erythrocytes, 0.2 ml of a solution of 1 y0 digitonin in phosphate buffer, pH 7.4 (previously boiled to clear the solution), was added to each serum tube and the blastoderm ground in this solution with a nylon rod. When the blastoderm was finely suspended, 0.3 ml of phosphate buffer, pH 7.4, was added and the contents of the tube were mixed with a fine aluminium wire. The tubes were then spun for 5 min at 12,000 rpm in a Clay-Adams microhematocrit centrifuge. A thin, superficial lipid layer which appeared at this point was removed with a small piece of filter paper. Then as much as possible of the underlying clear hemolysate was removed with a 20-gauge needle and a graduated l-ml syringe. Care was taken not to disturb the sediment. This volume of hemolysate (approxi- mately 0.42 ml), was measured in the syringe, diluted to a constant volume with 0.05 M phosphate buffer (pH 7.4), and divided into two equal parts. Each part was placed in one of a pair of matched semimicro 4-mm wide cuvettes with a l-cm optical path. The cuvettes were covered with Teflon caps held down by Scotch tape. Each cap had two holes, one of which was used to pass gas into the cuvette by insertion of a 23-gauge needle. Each cuvette was gassed with Nz to remove the oxygen. Solid sodium dithionite (1 mg) was added to both cuvettes to convert the hemoglobin to ferrous hemoglobin. After ob- taining a base-line difference spectrum of reduced hemoglobin

OD

/ 4:: 4y ]

400 420 440 460 mtL

FIG. 3. Difference spectrum of ferrous hemoglobin with re- spect to CO-ferrolls hemoglobin measured between 418 and 433 rn#. Correcting for change in optical density of 0.002 in the base- line there is a net change in optical density of 0.010 corresponding to 1.13 pg of hemoglobin per ml.

OD 418

0.04

0.03 0.040 A OD

0.02

0.0 I

418 4433 I I I I

400 425 450 d5

m/J L,

FIG. 4. Difference spectrum of ferrous hemoglobin with re- spect to CO-ferrous hemoglobin measured between 418 and 433 mp. There is a net change in optical density of 0.037 correspond- ing to 4.20 rg of hemoglobin per ml.

FIG. 5. Fluorescence photograph of a blood island after incuba- tion of blastoderm with 1 mM &aminolevulinic acid-HCl for 2 hours. Red fluorescence is recorded as white. The intense fluores- cence is indicative of copious amounts of porphyrins. x 400.

by guest on August 1, 2020

http://ww

w.jbc.org/

Dow

nloaded from

1906 Hemoglobin Synthesis in Chick Blastoderm Vol. 242, No. 8

FIG. 6. Paired blastoderm halves stained with 0-dianisidine after 14 hours of incubation in vitro. The half on the right was incubated with 1 mM &aminolevulinic acid-HCl and gave a more intense reaction than did the control on the left (X 30)

TABLE I Effect of b-aminolevulinic acid on heme and hemoglobin synthesis in

blastoderm halves as measured by distribution and intensity of

o-dianisidine stain (after different times of incubation)”

stage

Definitive primi- tive streak

Head process

Head fold

l-2 somites

3-5 somites

-

-

6. 1

14-15 hrs

-Amino- eyliCc

3f

lf 3+ 2+

4+ 3t

3t

3t

/ Control

2+

0 Trace

0

2+ 0 1+

Trace

1618 hrs

Amino- zvulinic

acid

2+ 1-t

1+

1+

3+

2+ 2+

3+ 4t

4t 3-t

4+

4+

4+

3f

4f

Control

Trace 0

0 0 1+ lf

Trace

1+ 2+ 3+ 2+ 2+

1+

4+

l-t-

3+

18-20 hrs

.Amino- evulinic

acid

2-t

3f

3f 4+

3+

5+

a See :‘Materials and Methods” for method of scoring.

-

(

r

Jontrol

3f

2+ 3+ 2+

2+

Race

(X 40

with respect to reduced hemoglobin, one of the cuvettes was gassed with carbon monoxide. The amount of hemoglobin present was determined by measuring the difference spectrum between the ferrous hemoglobin and the carbon monoxide ferrous hemoglobin in the Soret region. The spectra were measured on a Gary model 14 recording spectrophotometer employing the 0.1 optical density full scale slide wire. The cuvette cell-holders had masks with slits (2-mm wide x 4 mm tall). The difference in absorption between 418 and 433 rnp was measured. The d 433 (ferrous hemoglobin) to d 418 (CO-ferrous hemoglobin) = 156,000 per hemoglobin monomer of 17,000 molecular weight1 Representative spectra are shown in Figs. 3 and 4. A change in optical density of 0.01 corresponds to 1.13 pg of hemoglobin per ml.

The amount of hemoglobin in the blastoderm hemolysate was then related to the nitrogen content of the hemolysate. Nitro- gen was determined by a micro-Kjeldahl technique.

RESULTS,

.lctivity of Enzymes of Heme Biosynthetic Chain

To determine the activity of the enzymes of the heme bio- synthetic pathway in the chick erythroid precursor cells, blasto- derm halves younger than the five somite stage were de-embryo- nated and incubated for 2 hours on media containing 1 m;: d-aminolevulinic acid-HCl. The corresponding control halves were incubated on regular media or on media containing 1 mM sodium succinate and 5 mM glycine.

1 This value was derived from unpublished curves of Dr. D. L. Drabkin for which the authors are greatly indebted.

by guest on August 1, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of April 25, 1967 R. D. Levere and X. Granick

TABLE II

Effect of &aminolevulinic acid on hemoglobin synthesis in blastoderm halves after 24 to 26 hours incubation in vitroa

Pg I pglmg nitrogen

Head process 0.91 0.36 9.9 0.34 0.11 4.0

Head fold 0.45 0.39 4.5 0.44 0.11 4.1

Head process 0.77 0.21 7.8 0.55 0.12 5.6 0.35 0.12 4.2 1.00 0.74 6.8 0.37 0.11 3.5

a See “Materials and Methods” for quantitative determination of hemoglobin. b Average: 2.7 f 0.8.

- i

-

Amount of hemoglobin per half-blastoderm incubated with Amount of hemoglobin incubated with

i-Aminolevulinic Aminoacetone Nothing added &Aminolevulinic

acid Aminoacetone Nothing added to agar acid to agar

3.8 1.3 3.8

1.1 2.7 1.2

1.4 7.5 1.5

Mio of d-aminolevulinic acid to control (amino- :&one or no additions)b

g hemoglobin/mg nitrogen

2.6 3.1 1.2

3.7 2.9 4.7

3.0 0.9 2.3

TABLE III

E$ect of b-aminolevulinic acid on hemoglobin synthesis in blastoderm halves after 27 to 29 hours incubation ik vitro”

stage

Head fold

Head fold 3 somites Head fold

Head process Head fold Definitive primitive

streak Head process Head process

1 somite Head process 1 somite

Amount of hemoglobin per half-blastoderm incubated with

&Aminolevulinic acid Aminoacetone Nothing added

to agar

1.15 0.80 1.21

1.32 0.80 0.46

0.68

0.83 0.80 1.43

0.84 1.07

Trace 0.55

Trace 0.24

0.76

0.44 0.59 1.26 0.42

0.46 0.34

0.82

Amount of hemoglobin incubated with

i-Aminolevulinic acid Aminoacetone Nothing added

to agar

10.0 8.4

8.3 4.5 14.7 8.2 15.0 16.1

7.5 4.0 4.9 5.1

8.3 4.1

9.3 7.0

13.0 7.6

10.2

10.6 Trace

4.9 Trace

2.3 -

pg/nzg nitrogen

I

a

iatio of d-aminolevulinic acid to control (amino-

c&one or no additions)b

g hemoglobin/mg nitrogen

1.2 1.8 1.8 0.9

1.9 1.0 2.0

0.9 >7.0

2.7 >7.6

4.4

a See “Materials and Methods” for quantitative determination of hemoglobin.

6 Average: 2.7 f 1.7.

Examination with the fluorescence microscope of blastoderms incubated with Saminolevulinate showed an intense red fluores- cence in the blood islands (Fig. 5) indicative of copious amounts of porphyrins and presumably of heme. No porphyrin fluores- cence was noted in the control blastoderm halves. These re- sults indicate that all of the enzymes of the heme biosynthetic chain exclusive of the first one, Le. Saminolevulinic acid synthe- tase, are present in nonlimiting amounts in these precursor cells. As in the liver, heme synthesis is limited by the activity of &aminolevulinic acid synthetase.

Effect of &Aminolevulinic acid Synthetase Activity on Hemoglobin Formation

enter the blastoderm cells and cause the formation of copious amounts of porphyrins and presumably of heme. The amount of iron is not a limiting factor. When stained with OL , cy’dipyyri- dyl the blastoderm becomes pink because of formation of the ferrous dipyridyl complex indicating a strongly positive reaction for iron. To supply heme earlier than normal, b-aminolevulinic acid was used to by-pass Saminolevulinic acid synthetase. For this experiment, test blastoderm halves were grown on agar, a medium containing 1 mM Saminolevulinic acid-HCl. The control halves were grown either on regular medium or on agar containing 1 mM aminoacetone hydrochloride. Aminoacetone

was used as a control since it is the aminoketone which, while not an intermediate in the biosynthesis of heme, most closely resembles &aminolevulinic acid (11).

Knowing that heme synthesis is limited by the first enzyme of Effect of 8-Aminolevulinic Acid as Measured by o-Dianisidine the biosynthetic chain, the next question asked was what effect Stain-A group of 30 paired blastoderm halves were incubated does this limitation have on the production of globin? As for 14 to 20 hours and then stained. A representative pair is shown by the previous experiment, 6-aminolevulinic acid can shown in Fig. 6. In 27 of the 30 experiments there was a greater

by guest on August 1, 2020

http://ww

w.jbc.org/

Dow

nloaded from

1908 Hemoglobin Synthesis in Chick Blastoderm

TABLE IV

Effect of 6-aminolevulinic acid on hemoglobin synthesis in blastoderm halves after 40 to 43 hours incubation in vitroa -

-

-

Amount of hemoglobin incubated with Amount of hemoglobin per half-blastoderm incubated with

&Aminolevulinic acid

Aminoacetone Nothing added to agar

:atio of I-aminolevulinic acid to control (amino- cetone or no additions1 b

stage

I-Aminolevulinic acid Aminoacetone Nothing added

to agar

-I- pgjmg nitroge?%

4.90 1.27 44.5 5.5 2.49 0.53 37.0 19.0 2.70 0.48 26.0 4.9

g hemogloMn/mg nitrogen

8.2

2.0 5.3

3.74 2.90 35.0 31.5 1.1

0.26 0.50 2.5 7.2 0.4

3.75 0.49

1.59 1.08 0.82 0.37

0.46

0.80 0.50

7.5

7.2 1.24

1.55 0.35 1.27

1.19

34.0

5.5 21.0 15.0 11.5

5.0 4.0

13.0 24.5

5.3 16.0 13.9

4.5 0.8 1.6 0.6

2.2 0.3

0.3

1 somite

Head fold Definitive primitive

streak Head process Definitiveprimitive

streak 1 somite Head process

Head process Head fold Head process

1 somite Head fold

a See “Methods and Methods” for quantitative determination of hemoglobin. b Average: 2.3 f 1.9.

@ . . . l .

; .

TABLE V

Effect of puromycin and &aminolevulinic acid on hemoglobin

synthesis in blastoderm halvesa

4%hr incubation 24-hr incubation

uromycin

acid

lromycin fllllM -amino-

Nothing added to

evulinic acid

agar

pg hemoglobin/mg pg hemoglobin/mg nitrogen nitmgen

I-

. stage P

I

Puromycin

f-a/ml

10

5

1

0.5

0.25

0

0

1.5

9.1

0 2.5 0 4.1

4.0 4.0

Trace 9.8

0

0 10.0

7.1

Trace 15.9

Definitive primi-

tive streak

Head process Head process Definitive primi-

tive streak

Head process Head process Head fold

Head process

Head process

Head process Head process

Head process

Head process Head process

Head Process 1 Somite Head Process

Definitive primi- tive streak

. . . . .

l

m

1.8

0

115.0

8.9

Trace

0

12.2

15.9

9.2 28.6

Trace 3.2 5.4 73.8

3.9 37.4

5.4 24.8

I I I I I I I I 1 I

26 28 30 32 34 36 38 40 42 44

ln vifro incubation time (hours)

l Single determination x Average value

FIG. 7. Effect of &aminolevulinic acid on the rate of hemo- globin formation as measured by difference spectrometry. After 24 to 30 hours of incubation, there was, on the average, 2.7 times more hemoglobin in the &aminolevulinic acid treated halves. With 40 to 44 hours of incubation, 2.3 times more hemoglobin was found in the test halves. l , single determination; X, average value.

peroxidase reaction on the halves cultured with b-aminolevulinic

acid (Table I). When the staining reaction was graded on the basis of 0.5 for trace to 5+ there was 2.1 times more heme and hemoglobin found in the test halves as compared to the controls. It should be noted that the peroxidase reaction is not specific for the heme of globin but also detects free heme and other hemo- proteins. Therefore, these experiments can only suggest that

- - a See “Materials and Methods” for quantitative determination

of hemoglobin.

by guest on August 1, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of April 25, 1967 R. D. Levere and X. Grand 1909

d-aminolevulinic acid may cause an increase in the rate of forma- tion of hemoglobin.

&Aminolevulinic Acid Effect as Measured by Difference Spec- trometry-In eight of nine blastoderm pairs incubated for 25 to 26 hours more hemoglobin was found in the &aminolevulinic acid treated halves (Table II). There was an average of 2.7 =t 0.8 times more hemoglobin in the &aminolevulinic acid- treated group. With 27 to 29 hours of incubation there was on the average 2.7 f 1.7 times more hemoglobin in the b-amino- levulinic acid-treated halves (Table III). After 40 to 43 hours of incubation (Table IV), the &aminolevulinic acid-treated halves had 2.3 f 1.9 times more hemoglobin than the controls.

The ratio of hemoglobin per mg of nitrogen for the test as compared to the control blastoderm halves in the above three groups are summarized in Fig. 7.

These observations indicate that d-aminolevulinic acid has a significant effect on the rate of hemoglobin formation in the chick blastoderm, increasing by 2 to 3 times the amount of hemoglobin formed during the first 30 hours of incubation on agar. With longer incubation the Laminolevulinic acid effect is less marked, because with progressive development &amino- levulinic acid synthetase develops spontaneously and no longer limits heme synthesis in the controls.

E$ect of Puromycin and Actinomycin D on Ability of

&Aminolevulinic Acid to Increase Rate of Hemoglobin Formation

In an attempt to define more clearly the mechanisms by which &aminolevulinic acid increased the rate of hemoglobin formation, either puromycin or actinomycin D was added to the agar medium alone or concurrently with b-aminolevulinic acid.

TABLE VI

Effect of actinomycin D alone on hemoglobin synthesis in blastoderm halves incubated in vitro for 42 hoursa

TABLE VII

Effect of actinomycin D plus &aminolevulinic acid on hemoglobin synthesis in blastoderm halves incubated in vitroa

24-hr incubation 4%hr incubation

Actino- mycin D + 1 nIY Nothing E-amino- added to le,t;ic agar

-

n

6

Actino- em?’ Nothing m-amino- added to levulinic agar

acid

stage Actinomycin I

M/ml

0.01

0.02

pg hemoglobin/wzg nitrogen

1 somite Head process Head fold

Head process 4 somites Definitive primi- 8.5 31.4

tive streak Head process Head fold Head fold

1 51.6 47.8

1 somite -50.2 80.5 -

a See “Materials and Methods” for quantitative determination of hemoglobin.

8.8

5.9 8.4

3.4 2.0

.- fig hmoglobin/mg

ltitrogen

3.2 8.23 35.5

11.4 78.5

3.9 14.9

4.5

2.5

When puromycin in concentrations from 0.5 to 10.0 pg per ml was added to the medium together with 1 mM d-aminolevulinic acid-HCl, little or no hemoglobin was formed during 24 hours of incubation (Table V). After 48 hours, trace amounts of hemoglobin did appear in the test blastoderm halves indicating that these doses of puromycin were not lethal. In the presence of added b-aminolevulinic acid, heme is not limiting for hemo- globin synthesis and if globin had been present hemoglobin would have been formed. In the presence of puromycin, which acts at the ribosome level to prevent protein synthesis, no globin was synthesized and no hemoglobin was formed. This experiment signifies that no free, detectable, globin is stored in precursor cells to await heme formation.

Wilt (12) treated chick blastoderms with actinomycin D and followed RNA synthesis by tracer uridine incorporation, and protein synthesis by tracer leucine incorporation. At a concen- tration of 2 pg per ml actinomycin D was found to inhibit RNA synthesis in 2 hours, and protein synthesis by 8 hours. It is known that high concentrations of actinomycin D not only in- hibit mRNA and rRNA synthesis but also inhibit protein synthesis (13, 14). To distinquish more clearly between in- hibition of RNA and protein synthesis much lower concentrations of the inhibitor were used in the present study. At concentra- tions of about 0.02 pg per ml, actinomycin D primarily inhibits RNA synthesis. When actinomycin D (0.02 pg per ml) was added to the agar medium supporting the blastoderm halves, the hemoglobin formed was, on the average, about one-fourth that formed in control blastoderm halves (Table VI). At 0.01 pg per ml of actinomycin D, the hemoglobin formed was one-third that of controls. At 0.005 pg per ml, no inhibition of hemoglobin formation was observed.

When 1 mM d-aminolevulinic acid was added to the agar medium together with actinomycin D (0.01 and 0.02 pg per ml) and the blastoderm halves incubated for 24 hours, hemoglobin formation was, on the average, nearly equal to the controls

- Amount of

hemoglobin per half blastoderm incubated with

Amount incubated with %a:~

:tinomycin D to

control

Actinomycin stage :

1

/.%/ml

0.02

0.01

0.005

Head process Head process 1 somite Head process

I “E

0.53 Trace 0.86 0.13

,g hemoglobilz/mg nitrogen

0.27 11.3 5.7 1.27 Trace 18.5 6.12 11.6 74.6 0.72 3.1 17.1

Head process 0.38 0.48 2.3 1.4

Head process 1.00 0.80 16.1 12.5

Head process 0.13 0.98 2.9 10.5

Head process Trace 1.14 Trace 26.0

Head process Trace 0.92 Trace 17.0

Head process 1.64 2.40 20.8 40.0

Head process 4.80 1.80 77.5 20.7 Notochord 0.78 2.38 11.8 26.2

Head process 2.70 2.08 45.0 18.2

1 somite 6.59 2.60 89.0 33.4

Head fold 2.40 7.14 38.7 82.1

0.2

0.3

1.3

a See “Materials and Methods” for quantitative determina- tion of hemoglobin.

by guest on August 1, 2020

http://ww

w.jbc.org/

Dow

nloaded from

1910 Hemoglobin Synthesis in Chick Blastoderm Vol. 242, No. 8

(Table VII). After incubation for 48 hours, the treated halves had, on the average, half of the hemoglobin of the untreated halves. In spite of the small number of blastoderms used in this experiment the data clearly show that the addition of b-aminolevulinic acid to the actinomycin-treated blastoderms appreciably enhanced the formation of hemoglobin. The data also indicate that there was a decrease in hemoglobin formation in 24 hours which became more marked at 48 hours. This decrease in hemoglobin formation in the presence of 0.02 pg per ml of actinomycin and 1 mM b-aminolevulinic acid is probably caused primarily by a relatively slow breakdown of globin messenger RNA which occurs concomitantly with an inhibition of RNA synthesis at the DNA level.

Because actinomycin D blocks messenger RNA formation presumably little messenger RNA was transcribed for globin synthesis and little messenger RNA was transcribed for the synthesis of b-aminolevulinic acid synthetase. When d-amino- levulinic acid was added in the presence of actinomycin, hemo- globin formation was enhanced. Since the puromycin experi- ment showed that no pre-formed globin was present in these cells this result suggests that globin was synthesized de no2ro. The enhanced synthesis of globin occurred presumably by the action of ribosomes and globin messenger RNA that were already present in the precursor erythroid cells. In addition, the synthesis of globin required &aminolevulinic acid or rather the conversion product heme.

DISCUSSION

The present studies indicate that hemoglobin synthesis in the chick blastoderm is triggered by the activation of the structural gene that forms messenger RNA for b-aminolevulinic acid syn- thetase. The following observations are advanced in support of this idea.

These studies indicate that in the erythroid precursor cells of the blastoderm the rate of heme synthesis is limited by the ac- tivity of the first enzyme in the heme biosynthetic chain, b-amino- levulinic acid synthetase. This finding is also true for heme synthesis in other cell types (1, 15). All of the other enzymes of the heme biosynthetic chain which serve to convert &amino- levulinic acid to heme are at nonlimiting activities in the hemato- poietic mesoderm of the chick blastoderm. In the colorless erythroid precursor cells, only small amounts of heme are synthesized, sufficient for the requirements for cytochrome production. At the time of initiation of hemoglobin synthesis much larger amounts of heme are synthesized. This is as- sociated with an increase in the levels of d-aminolevulinic acid synthetase.

When &aminolevulinic acid is added to colorless erythroid precursor cells, hemoglobin synthesis occurs earlier than in controls (Tables I to IV). The greatest differences occur at earlier times of incubation, before &aminolevulinic acid is made at an appreciable rate by the cells themselves. As b-amino- levulinic acid is rapidly converted to heme, it is presumed that heme is the substance which controls the rate of hemoglobin synthesis. This presumption is supported by the work of other investigators with the use of different hemoglobin-synthesizing systems. Hammel and Bessman (16) found that globin syn- thesis by isolated avian erythrocyte nuclei could be increased by the addition of heme. In addition, Bruns and London (17) found that hemin increased the incorporation of 14C-valine into the hemoglobin of rabbit reticulocytes.

How does heme increase the rate of hemoglobin formation? Three possibilities were considered. First, heme might stimulate the synthesis, i.e. transcription, of messenger RNA for globin monomer formation. To test this possibility, actinomycin D was given to the cells in order to block messengerRNA formation; at the same time &aminolevulinic acid was given to supply heme. In spite of the presence of actinomycin D &aminolevulinic acid stimulated the formation of hemoglobin. From this experiment it was concluded that messenger RNA for globin and ribosomal- RNA were not limiting hemoglobin synthesis and in the colorless erythroid precursor cells, messenger RNA for globin, and ribo- somes must be present. This agrees with the conclusions of Wilt (12) that the time of transcription of messenger RNA for globin is at the head fold stage, several hours before active hemoglobin synthesis starts.

Another possible way to explain the fact the heme increases the rate of hemoglobin formation is to assume the presence of pre-formed globin in the colorless erythroid precursor cells. Experiments with puromycin and &aminolevulinic acid showed that pre-formed globin was not present in detectable amounts. Puromycin blocks protein synthesis at the ribosome level. I f a pool of pre-formed globin existed the add&on of b-amino- levulinic acid leading to the formation of heme should have caused an increase in hemoglobin synthesis. In the presence of puromycin and d-aminolevulinic acid, no hemoglobin synthesis occurred (Table V), ruling out the presence of pre-formed globin. The findings of others support this conclusion. Wilt (12) observed that puromycin prevented hemoglobin formation in the chick embryo at all developmental stages. Grayzel, Horch- ner, and London (18) found that puromycin and cycloheximide blocked hemoglobin synthesis in rabbit reticulocytes in the presence or absence of added heme.

A third possibility to explain the fact that heme increases the rate of hemoglobin formation is to assume that heme is required for the appropriate three-dimensional folding of the globin polypeptide as it is synthesized on the ribosomea. Winslow and Ingram (19) have shown that the second half of the globin mono- mer is synthesized at a slower rate than the first half is. The iron of heme in the hemoglobin monomer is attached to a histidine in the middle of the chain (His 87 in the o( chain, and His 92 in the p chain). We propose the hypothesis that globin *is not released from the ribosomes unless it folds around heme to form a compact unit. This hypothesis would most simply explain the fact that no free globin is detectable in precursor erythroblasts. The fact that globin monomer is formed in a 1:l ratio with heme would be most readily explained on the basis that heme was limiting, and heme was required for globin synthesis. Grayzel et al. (18) observed that 10h4 M heme causes the aggregation of single ribosomes to polyribosomes; this may be an additional contributory effect of heme on hemoglobin biosynthesis.

The following hypothesis summarizes what is felt to be the situation in the erythroid precursor cell. In this colorless cell are present adequate amounts of ribosomal RNA and messenger RNA for globin formation. In addition, there are present all the enzymes needed to make heme at an appreciable rate except the first enzyme d-aminolevulinic acid synthetase which is at a limiting activity. The rate of synthesis of &aminolevulinic acid synthetase must be controlled by a repressor mechanism. Only when derepression occurs by the action of some unknown substance is the structural gene for &aminolevulinic acid syn- thetase stimulated to transcribe messenger RNA to form in-

by guest on August 1, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of April 25, 1967 R. D. Levere and X. Graniclc

creased amounts of b-aminolevulinic acid synthetase. The 6-aminolevulinic acid synthetase then produces 6-aminolevulinic acid which is readily converted to heme by the other nonlimiting enzymes of the heme biosynthetic chain. Heme, formed in the mitochondria, enters the cytoplasm. There it attaches to a globin monomer that is being synthesized on the polyribosomes. The monomer grows and folds into a compact unit around the heme. Then completed hemoglobin monomers detach from the ribosomes and combine to form tetrameric hemoglobin. Thus, according to this hypothesis, it is the activation of the structural gene for &aminolevulinic acid synthetase which is the major step that controls the differentiation of the pro-erythroblast into the mature erythrocyte.

REFERENCES

1. LEVERE, R. D., AND GRANICH S., Proc. Nat. Acad. Ski. Cl. S. A., 64, 134 (1965).

2. GRANICK, S., J. Biol. Chem., 238, PC2247 (1963). 3. GRANICK, S., J. Biol. Chem., 241, 1359 (1966). 4. ROMANOFF, A. L., The avian embryo, Macmillan, New York,

1960.

5. O’BRIEN, B. R. A., J. Embryol. Exp. Morphol., 9, 202 (1961). 6. SABIN, F. T., Carnegie Inst. Wash. Publications, 9, 213 (1920). 7. ZWILLING, E., Nat. Cancer Inst. Monogr., 2, (1959). 8. RUGH, R., Experimental embryology, Burgess Publishing Com-

pany, Minneapolis, 1962, p. 15. 9. PARKER, R. C., Methods of tissue culture, Ed. 3, Harper and

Row, New York, 1961, p. 57. 10. OWEN, J. A., SILBERMAN, H. J., AND GOT, C., Nature (Lon-

don), 182, 1373 (1958). 11. URATA, G., AND GRANICK, S., J. Biol. Chem., 238, 811 (1963). 12. WILT, F. H., J. Mol. Biol., 12, 331 (1965). 13. REVEL, M., HIATT, H., AND REVEL, J. P., Science (Wash.),

146, 1311 (1964). 14. REICH, E., Progr. Nucleic Acid Res., 8, 184 (1964). 15. SAILLEN, R., HeZv. Med. Acta, 30, 208 (1963). 16. HAMMEL, C. L., AND BESSMAN, S. P., J. Biol. Chem., 239,

2228 (1964). 17. BRUNS, G. P., AND LONDON, I. M., Biochem. Biophys. Res.

Commun., 18, 236 (1965). 18. GRAYZEL, A. I., H~RCHNER, P., AND LONDON, I. M., Proc.

Nut. Acad. Sci. U. S. A ., 66, 650 (1966). 19. WINSLOW, R. M., AND INGRAM, V. M., J. BioZ. Chem., 241,

1144 (1966).

by guest on August 1, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Richard D. Levere and S. GranickControl of Hemoglobin Synthesis in the Cultured Chick Blastoderm

1967, 242:1903-1911.J. Biol. Chem. 

  http://www.jbc.org/content/242/8/1903Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/242/8/1903.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on August 1, 2020

http://ww

w.jbc.org/

Dow

nloaded from