Ribosome shifts in the cold cell-free lysate during analysis of maturing reticulocyte ribosomes

Vol. 30, No. 6. 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RIBOSOFJE SI!IFTS

IN THE COLD CELL-FREE

LYSATE DURIM

MJALYSIS OF MTURING

RETICULOCYTE RI BOSOMES David

Danon*

Section

and Li.a Cividalli

of Biological

The Weizmann

Ultrastructure,

Institute

of Science,

Rehovoth,

Received

February

20, 1968

The progressive tion

loss

mammalian

of

and total somes,

RNA,

1965,

the total

ribosome

and quadrisomes

proportion

1965,

labelled

with

from which showing

blood

disnlacement

of ribosomes

the experimental Blood This

procedure

that

follows

avoided nhenyl

The increasing locytes tries

F!aifa,

using

(specific withdrawal

of blood.

10 to 15ml of packed in

saline

splenectomized

*

also

that

reported

there

The Patrick

were

Iron cells

reticulocytes rabbits

for

(Flowacki

body

from

donor

in which

they

analysis,

one cluster

E. German,

and

rabbits

were

matured

and

The results in mature

to another

of this

in

cell

was obtained

formation

and

lysate

(Rifkind Vodel in

the

cell

and Danon 1965,

more resistant

Ribosomes

by daily

and damage to the

a Fragiligraph

(SOma

Imferon)

from blood into

sequestration Professor

study,

reticulocytes

during

here.

treatment

trans.fused

to minimize

is

1967).

of osmotically

Israel).

were

claiming

Rifkind

population

D2 (Elran,

were

rich

in

a normal

labelled host

of Biological

717

Indus-

labelled

reticulocytes that

cells,

Ultrastructure.

by

phosphate before

simultaneously. rabbit

of transfused

1965).

of reticu-

Electronic

reticulocytes

was injected

bleedings, membranes

the donor rabbit intravenously with 2-3mc of P 32 labelled activity 50 to 90 Curies per rnp phosphate) 24 to 40 hours

injecting

pended

ribosomes

taken

hydrazine

was followed

results

40% reticulocytes

Heinz

proportion

Ltd.,

of hexaribosomes Danon et al.

is presented

about

ribosomal

the proportion

of polyribosomes

procedure,

containing

in

that of single et al. 1964,

to host

from

a reduction

matura-

falls,

young

were

proportion

with

during

while Pifkind

and De Bellis

samples

synthesis

content

and transfused

a reduced

associated

contradictory

experiments

P32

protein

reduced, et al. 1963,

of single

Burka

In the wresent

for

is

(Marks Somewhat

1965) .

no increased

canacity is

increases

Rowley,

Gillette

of the

reticulocytes As

pentasomes

and diplosomes

Israel

About resus-

was either or irradiated

Vol. 30, No. 6. 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with SSOr, to prevent erythropoiesis, 24 to 48 hours before transfusion. some experiments, about 20 to 30ml of blood were withdrawn from the host

In

rabbit before transfusing the reticulocyte-rich oacked cells. Ribosomesuspensions were prepared as previously described (Marks et al. 1963) and usually stored overnight in plastic centrifuge tubes in i.ce buckets placed in a cold room. Sucrose gradient analysis of the ribosomes was carried out as nreviously described (Varks et al. 1963). In order to compare the sedimentation pattern of ribosomes before and after reticulocyte maturation, ribosomes were prepared and characterized from two blood samples. The first sample of blood, taken from the host rabbit 40 to 60 minutes after transfusion was designated “young reticulocytes blood”. The second blood sample taken 20 to 24 hours after transfusion was designated “matured reticulocytes blood”. In all the experiments, the proportion of slowly sedimenting ribosomes, that matured in i.e. single and double ribosomes, increased in reticulocytes the circulation of the host rabbit. The total ribosome content per volume of cells decreased to about 10-E% of the initial amount within 24 hours of transfusion to a splenectomized host rabbit,

indicating

the transfused reticulocytes. The specific was reduced after 24 hours to approximately

remarkable maturation of

activity of the ribosomes (CPM/OD) one third of the value noted about

an hour after transfusion, However, radioactivity distribution along the gradient was similar to the optical density pattern. A representative experiment is depicted in Fig.1. The reduction in specific third of its initial value over the sametime interval,

activity to about one indicated dilution of

the labelled ribosomes by the ones newly formed in the host rabbit, as confirmed by analysis of the sedimentation natterns of ribosomes derived from reticulocytes transfused to irradiated rabbits (Pig.2). In general these sedimentation patterns were similar to those obtained when normal splenectomized rabbits were transfused. However, with the irradiated hosts, the swecific activity of the ribosomes showedno simificant difference between early and matured samples, This, incidentally, indicates successful depression of marrow activity by the relatively low radiation dose. The unexpected finding of uniform labelling along the gradient, following maturation in vivo, rather than the anticipated low activity in the polyribosome region and much higher activity in the slowly sedimenting region is not sufficiently explained by admixture of newly formed unlabelled reticuloConsidering that new reticulocytes are formed in the non-irradiated cytes. animals, the presence of practically uniform radioactivity could be due to liberation of P32 from components of the blood other than RNA, or through destruction of labelled cells and liberation of the P32, followed by reincorporation of the isotope by newly formed reticulocytes in the host animal.

718

Vol. 30, No. 6. 1968

8lOCHEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CPM

I

I

I

5

I

10

I

I

I5 20 25 number of tube

Fig. I

I

I

30

35

I

I

----o---

I

I

5

IO

r4

I

/

I

I

15 20 25 number of tube

ti5

Fig.2

from reticulocytes of a host smle1 - Sedimentation patterns of ribosomes nectomized rabbit to which P32 labelled reticulocytes were transfused, taken 40 minutes after transfusion (top) and 24 hours after transfusion (bottom). Note the reduced proportion of nolyrihosames after 24 hours and the close similarity of the radioactivity profile with that of the optica. density pattern both before and after maturation. Fig.

patterns of ribosomes from reticulocytes taken from an 2 - Sedimentation irradiated host rabbit after transfusion. Top - 40 minutes after transfusion, bottom - 24 hours after transfusion, Note that in this exneriment, in which the host rabbit was irradiated, the radioactivity per O.D. unit remained practically unchanged after maturation; in the experiment described in in which the host rabbit was splenectomized but not irradiated, the Fig.1, radioactivity per O.D. unit was reduced after maturation hy a factor of three.

Fig,

possibility

Another

ribosomes, could

that

the

from

one polysome

when unlabelled

come into

whether

the

signed.

direct

or the

“Labelled

.voung

mature SO

The mixed representative

that cells

distribution

of activity

to another

during

is

second

reticulocyte-rich

is

depicted

ratio

in Fig.3.

It

correct,

was de-

in vitro

with

1:9 washed

ribosomes Pattern

shows

of

cellular origin at clarifying

of

and unlabelled

The sedimentation

719

is

was mixed

in a volume

of labelled

together.

explanation blood”

blood”

units

lysed

experiment

hypothetical

due to dis-

the Preparation

and lahelled rihosomes of different An in vitro experiment aimed

reticulocyte

the O.D. were

even

contact.

first

Qnlabelled cells,

is

of ribosomes

placement

that

were obtained

radioactivity

packed similar. in

a is

Vol.

30,

No.

6. 1968

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

distributed almost uniformly along the gradient. The general specific activity of the ribosomes derived from the mixed cells lysed together was practically that which would be expected in a dilution of the radioactive rihosomes with ltcoldq’ ones However, the anticipated radioactivity distribution curve i.e. l

activity following the same pattern as that of the labelled ribosomes only, di d not appear. Similar results were obtained with “labelled matured reticulocyte blood” mixed with “unlabelled young reticulocyte-rich blood”. Tt was concluded that ribosomes are displaced from one cluster to another. Migration of rihosomes between polyribosomes and the monosome no01 may occur if protein synthesis takes place in the hemolysate. JJnlabelled ribosomes

could then interchange

places,

distribution of radioactivity, Protein synthesis at low temperatures Ported (Goodman and Rich 1963). However, temperature was kept very system by adding “optimal” Experiments takes place

resulting

in a practically

in a cell-free in the present

system experiments

uniform has

been

re-

the

low, and no measures were taken to activate the concentrations of enzymes, ions and substrate.

were performed to determine whether incorporation of amino acids between the time cells are lysed and the time the gradient frac-

tions are precipitated with TCA. Puromycin was added to the hemolysate in two exneriments in order to ascertain whether cessation of protein synthesis will affect the distribution results of this experiment

of radioactivity are given in Fip,

incorporation does occur. The experiments reported

here confirm

along the gradient. The 4. and show that amino acid the observation

that

as reticulo-

cytes mature in the circulation, the degree of aggregation of the ribosomes in clusters decreases, while total content in ribosomes is markedly reduced. They indicate, however, on the one hand, that ribosomes can be displaced from one cluster in the formation

to another, and of polyribosomes

that even

single ribosomes may participate again during the experimental procedure at

low temJ7erature. On the other hand, ribosomes originating from a “young” polyribosome can be attached in proximity to a single l*matured” ribosome to form a diplosome or be, at the moment of fractionation on the gradient, a single particle, explaining the radioactivity found in the 805 and 1105 region of the gradient after mixture with unlabelled matured rihosome population. The profile of a sucrose gradient of reticulocyte ribosomes seems to represent an instant image of a dynamic process that continues during its preparation. Furthermore, a gradient obtained from reticulocytes pooled from several rabbits does not necessarily represent the arithmetic average of all the individual gradients.

720

Vol. 30, No. 6. 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

b.D. 100

Fig.

Changes in sedimentation patterns P3* labelled reticulocyte-rich rabbit in which unlabelled reticulocytes of labelled ribosomes from reticulocyte-rich tern of ribosomes prepared from a mixture the same blood as above and nine volumes in which unlabelled transfused reticulocytes ribosomes of the labelled sample were of unlabelled ones. Fig.

number of tube

4

3 -

of ribosomes after mixing in vitro blood with unlabelled blood om matured, Ton - sedimentation pattern blood. Bottom - sedimentation natof one volume of packed cells from of packed cells from the host blood matured for 24 hours, so that approximately equal amount to the

Amino acids incorporation during the nreparation of ribosomes and their sedimentation in the sucrose gradient. Top - sedimentation pattern of ribosomes from reticulocyte-ri.ch hlood and the radioactivity nattern of Cl4 labelled amino acids in hot TCA precipitates. The labelled amino acids were introduced immediately after the osmotic hemolysis of the red cells. Rottom - a sample of the same hemolysate as above to which puromycin was added immediately after the additi.on of Cl4 labelled amino acids. Fig.

4 -

The reduction culocytes

is

in degree

apparently

not

of aggregation

of ribosomes

in maturing

due to dilution

of ribosomes

and the conseouent

reduced probability of a ribosome encountering a messenger since migration of ribosomes took place even in the diluted may, therefore, ribosomes keep

is

ribosomes

RNA molecule

be concluded due to the together as it

that

reduced

the

reduced

capacity

or to a reduction

reaches

the end of its

721

degree

in life

the length span,

RNA molecule hemolysate,

of aggregation

of the messenger

reti-

of

RNA molecule of the

It

messenger

to

Vol. 30, No. 6. 1968

Statistical

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

analysis of the phenomenon(Per1 1964) has indicated,

that

the reduction in the number of rihosomes ner cluster very probably takes place by “dropping off” of single ribosomes. *ether all single ribosomes in a mature reticulocyte are capable of recycling or whether the very old ones lose this capacity is a subject for further study. It may offer another explanation for the degradation of polysomes. REFERENCES Burka E.R. & De Bellis R.H. Nature 213, 724, (1967). Danon D., Zehavi-Willner T. 6 BermanG.R. Proc.Natl.Acad.Sci. 54, 873, (1965). Glowacki E.R. & Millette R.L. J.E!ol.Biol. 11, 116, (1965). GoodmanH.M. & Rich A. Nature 199, 318, (1963). Marks P.A., Rifkind R.A. Fi Danon D, Proc.Natl.Acad.Sci. 2, 336, (1963). Per1 W. J.Cell Biol. 22, 611, (1964). Rifkind R.A., Danon D, TMarks P.A. J.Cell Biol. 22, 599, (1964). Rifkind R.A. 6 Danon D. Blood 25, 885, (1965). Ri fkind R.A. Blood 26, 433, (1965). Rowley P.T. Nature E, 244 (1965).

722