Absorbance-temperature profile of ribosomes from seeds of Pinus lambertiana

Vol.

52,

No.

BIOCHEMICAL

3,1973

AND

BIOPHYSICAL

ABSORBANCE-TEMPERATURE FROM

PROFILE

SEEDS

OF

PINUS

Received

Polytechnic

April

Department Institute

19,

of

OF

COMMUNICATIONS

RIBOSOMES

LAMBERTIANA

Thong-Sung

Virginia

RESEARCH

Ko*

Biochemistry and and State University,

Nutrition Blacksburg,

Va.

24061

1973

Summary: Th e a b sor b ante (260 nm)-temperature Pinusmbertiano, in the temperature range When r-proteins in ribosomes were dansylated chloride dispersed on celite (180 molecules

profile of ribosomes from of about 25 - 60° was observed by 5-dimethylamino-1-naphthalenesulfonyl of 5-dimethylamino-1-naphthalenesulfonyl

chloride per one ribosome particle), each transition midpoint the sharpness of the transition of each phase was increased. suggests discrete cooperative transitions of the conformation its control by r-proteins.

seeds of to be biphasic.

was lowered by 2 - 3O and The result of the present work of r-RNAs in ribosomes and

Introduction Conformationol in the

stepwise

nucleic

acids

transition

through

the

elementary

On

the

other

in ribosomes

of protein

biosynthesis

proceed

intramolecular steps hand,

which

may

instead

ribosomes,

examination * Present

be

transformation,

r-proteins

may In this

indicative

address:

molecule

large

monophasic of r-proteins

of such

in the

I report steps

a treatment

Department of Microbiology, vania, Philadelphia, Penna.

result

the

School 19174

state

regulation

of such

of the

of other

segbetween

by small

effects.

conformational

changes

absorbance-temperature transitions

Differential etc.,

conformational of

in

process

interaction

be induced

in ribosomes, on the

elementary

may

of cooperative profile.

importance

transitions

by the

a polyphasic

1051

be of decisive

of a cooperative

changes

involved

paper,

words,

is influenced

AS the

of distinct

of a simple

effects

be

may

Th e conformational

In other

of the

of the

ribosomes

(1,2,3).

interactions.

or derivatization of the

within

cooperatively. segment

(4,5,6,7,8).

of ribosomes, from

usually

in r-RNA

of un individual

ments

file,

process

transitions

Medicine,

in the

detachment ond

proconformation of r-proteins

then

subsequent

transition

profile

University

of

of Pennsyl-

Vol.

52,

ribosomes mationol

No.

3, 1973

may

be employed

transition

somes

were on

selectively

celite.

were

as the

result

celite

reagent

carried

out

not

under

does

using RNA

were

prepared

serum

Schwarz

Materials

and usual

(pH

residues was

7.8),

M KCI,

as the

dialized

overnight

0.02

M

of dansyl-Cl-celite

was

used

of the

dansylation,

and

the

celite

was

carried

out

KCI,

0.005 then

as a control. test

as described

of the

be

for

the

lambertiana water

overnight.

method

method

of

(11)

dansyl

Lowry

was

--et al.

used

from

the

against

1052

in acetone 0.5%

for

gently

reaction

A system

for

composed

time

2.5 by

celite course, with

from

2%

h in the centrifugation.

of 0.025

containing

components

was

M sodium

mixture

of the

assay

ribosomes, 0.025

stirred

a buffer

of ribosomal

calorimetric

chloride,

was

determination

(9).

chloride

containing

M MgC12.

reactivity

on celite,

nsyl

mM

cold

The

previously

of Pinus

by the

dispersed

removed

0.005

can

suitable

in distilled

orcinol

M MgCl2,

in the

and

chloride-

ribosome,

etc.)

estimated

(5 ml.)

to 8.0

were

dansyl

standard.

mixture

and

the

the

seeds

soaked

Dische’s

methyl-T4C-da

Dansyl-Cl-celite

(pH

was

and

0.02

affect period,

been

Calbiochem

8.8),

supernatant

had

from

reaction

of

to ribonuclease, with

(9) f rom

standard.

RNA

r-proteins properties

sensitive

might

methods

preparation

as the

derivatized

Methods

that

(w/ v ) w h’ IC h corresponded

cacodylate

in ribo-

effects.

chloride

The

r-proteins

sedimentation

reaction

Dansyl

Inc.

confor-

chloride)

dansylation

that

ribosome:

Bioresearch

Tris-HCI

solvent

side

yeast

on the

purpose,

reagent

more

that

of the

purchased

dansyl-Cl-celite

found

without

ribosome

using

that

that

became

Calif.)

albumin

this and

temperature,

by the

of the

determination,

was

we any

Davis,

content

bovine

ribosomes

integrity

COMMUNICATIONS

of r-proteins

for

that

in ribosomes,

(buffer,

effect

work,

observed

include

conditions

Dansylation

The

not

RESEARCH

(5-dimethylamino-I-naphthalensulfonyl

Also,

of California,

Protein

cold.

whereas

of ribosome

Seeds

1 l%,

altered,

the

present

(9) we unreacted

BIOPHYSICAL

of studying

chloride

dansylation.

Ribosomes

the

dansyl

r-RNA

which

AND

In the

Previously

of the

maintenance

(10),

by

leaving

ribosomes

as methods

of ribosomes.

dansylated

dispersed

(Forest

BIOCHEMICAL

M instead

the dansyl-CI-

extent

Vol.

52,

No.

3, 1973

BIOCHEMICAL

AND

BIOPHYSICAL

Temperature,

RESEARCH

COMMUNICATIONS

OC

Figure 1. Absorbance-temperature profiles of undansylated ribosomes and dansylated ribosomes (180 molecules of dansyl chloride per ribosome particle) at 260 nm. Ribosomes were suspended in a buffer containing 1.5 mM MgC12, 0.025 M Tris-HCI (pH 7.8), 0.02 M KCI, and 0.006 M 2-mercaptoethanol. The initial absorbance, 0.5 - 0.6 were normalized to 1 .O. The profile was scanned with Gilford 2400 Spectrophotometer. The heating was 1.63O per minute. Correction for the thermal expansion of water was not done. Numbers on the curve indicate Tm values. , Donsylated ribosomes; undansylated ribosomes. -----I In the preparation of dansylated ribosomes used in the present work, the uptake of dansyl group by ribosomes increased linearly with time and reached a plateau after 2.5 h, with about 45 rq~ moles of dansyl chloride bound per mg of ribosomes, under the reaction conditions described in the text. This corresponds to 180 molecules of dansyl chlorider per ribosome particle, assuming the molecular weight of the ribosome to be 4 x 106 (12).

Absorbance-temperature ribosomes the

(180

legend

molecules

of

Figure

profiles of dansyl

at

chloride

260

nm for

normal

per

ribosome

(undansylated)

particle)

and

were

tested

dansylated

as described

1. Results

Absorbance-temperature somes

are

shown

r-protein

at

(cf.,

in the

precipitated,

and for

Figure

1.

13,14,15)

temperature

profiles

profiles

both

The

therefore normal

absorbance

or ckmsylated

experimental the and

at 260

tested.

profiles

were

for

change

r-proteins

mnge

dansylated

nm

undansylated of either

at 260 Above

plotted

ribosomes

1053

were

and normal

nm was

about

60°,

dansylated (undansylated)

negligible

with

ribosomes

started

up to below

this

biphasic

instead

ribo-

temperature. of simple

increasing to be The mono-

in

Vol.

52,

No.

BIOCHEMICAL

3, 1973

Table 1. cooperative of dansyl

Transition lengths chloride

Phase of profile

Ribosomes

AND

BIOPHYSICAL

RESEARCH

midpoint (Tm); molar enthalpy (AH) of transition (n), and ratio of cooperative parameters ( b), per ribosome particle) to undansylated ribosomes.

Tm,

OC

at the dansylated

“dansylated

Kcal mole

AH,

COMMUNICATIONS

Tm; (180

6dansylated dundansylated

“undansylated

1

Dansylated Undansylated

39 41

58 23

2.5

0.16

2

Dansylated Undansylated

52 55

26 15

1.7

0.33

phasic

profiles.

somes

lowered

phase.

The

from

Furthermore, the

transition

effect since

both

of 42%

molar for

length”,

enthalpy

where

O-

[II +[I11

in absorbance that

the

transition

n.

other

Among

transition

increased

remowl

of

the

dansylated

forms

[II]

the

this

where

and

transition leads

enthalpy

at

apparent

the large

of

slope

ribo-

in each

amounts

of proteins

contained

the

= concentration

generally

same

increases

to a characteristic

the

rate

Tm in each

constant

is:

of species.

are

can

be

hypochromic

equated

with

with

respect

gross

with

increase

phase Kapp

If the

the

to the

d In Kapp

l/T,

dansylation

was = K”

the

in the

calculated 0 = 1-Q

normalized

’

increase

quantity,

1 - 8,

denatured

species,

based then

on the at

fact

a

midpoint: (

the

1,

ribosomes

of the

things, This

case,

the

and

sharpness

AHapp.

LI],

by 2 - 3’, simply

Table

protein. the

and

1 and

’

during

ordered

58%

In this

[II

not

in Figure

and

transition,

model.

--

was

and

of transition

a two-state

(Tm)

undansylated

RNA

In cooperative “cooperative

midpoint

of dansylation

ribosomes,

composition

as indicated

ratio of molecules

AH, van’t and the

is the Hoff the

)

dT molar

enthalpy, value

dansylated

enthalpy AHapp,

of transition was

is shown

in Table

ribosomes

to be the

for

the

obtained

from

1.

taking

Also, same,

the

1054

n.AH,

A Happ F=

m =

RT2

elementary a van’t

ratios

the for

transition Hoff

values

plot; of AH,

coopemtive

process. i.e.,

In Kapp for

undansylated

lengths

n and

Thus, against

cooperati-

BIOCHEMICAL

Vol. 52, No. 3,1973

vity

parameters

Table

6

of

dansylated

and

AND BIOPHYSICAL

undansylated

RESEARCH COMMUNICATIONS

ribosomes

were

calculated

and

shown

in

1. Discussion

profile

Present

work

of the

ribosomes

transitions.

tests

in a few phasic

the

discrete

often

transition

could

Mg*,

the

profile

is obscured.

tests

at small

the

conformational

in the

hardly

be

can

see

coopemtive of the

coopemtive

the ascribe

dependent

helix-coil

in the

n and

of r-RNAs

importance

thermodynamic

the

greater

Tm of

charges

fixed

may

present

ribosomal

functions

transition

of ribosomes

occur

observation

of Mg*.

first

in the thereby

of the can

The

However,

phase

of of

transition

methods

further

poly-

be

presence

the

as relaxation detect

it is

usually

The

and

require

observation,

(17,18).

to

a two-state

smaller

or careful changes

in

sensitivity. transition

in Figure

coopemtivity

present

ribosomes

1 and,

parameter

observation

by the for

shows

the

dansylation

stability increase

of r-RNAs between

reflect

transitions

with

such

of the

temperature-dependent transition

cooperative

6 that

accordingly, in Table

r-proteins

1,

upon

control

in ribosomes.

of r-proteins

relation phosphate

the

may

on heating,

possible

decreased

Thus,

of the

be

sharpness

transition

of Mg*.

of techniques,

with

stepwise

concentration

easily

absorbance-temperature

discrete

and

spectrum

absence

it may

increased

ribosomes.

particles

occurs

employment

length

lowering

tentatively

the

In connection

of a proper

profile

transition

shows

between

(16).

the

two-state

conformational

intervals,

the

with

of the

a continuous

of ribosomes

transition

that

assumption

precursor

presence

By the

shows

of cooperative

et al.

detected

temperature

One

The

Lumry

ribosomal

only

precipitation

dansylation

step

of the

than

seeds

in accordance

discrete

rather

pine

of u temperature-induced

visualized

increased

both

kinds

behavior

polyphasic

by

that

from

validity

as described

to be mentioned

the

is each

although

further

somes

ribosomes

Presumably

transition,

the

with

in ribosomes,

Tm and

electrostatic

on nucleic

acid

1055

of r-proteins

of ribosomal

structure.

in absorbance and free molecules

apply

to the

energy (19),

within

the

equation due

riboOne

tempemtureexpressing

to interactions

might

Vol.

52,

No.

BIOCHEMICAL

3, 1973

Tm = where free

AHo,

ASS,,

energy

Since

AFdansyl

repulsions upon

to

AFdansyl

between

the

stability

of Tm

present of r-RNAs

changes

must

be

charges due

observation

to the

BIOPHYSICAL

AFhnsyl

(AH,+

of molar

enthalpy,

lowered

with

fixed

on the

decrease

demonstrates

RESEARCH

entropy, in the

helical

importance

and

transition

dansylation,

of positive the

COMMUNICATIONS

)/AS,

respectively,

r-proteins,

phosphate

of r-proteins The

are

dansylation

is negative,

dansylation

dansylation. structural

due

and

AND

as the segment charges

electrostatic of helix

to coil.

electrostatic

of r-RNA

increase

of r-proteins

by

of r-proteins

for

the

in ribosomes. References

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19.

Arnott, S., W. Fuller, A. Hodgson, and I. Prutton, Nature 220, 561 (1968). Chaung, D. and M. W. Simpson, Proc. Natl. Acad. Sci. lJS38, 1474 (1971). Schreier, M. H. and H. Nell, Proc. Natl, Acad. Sci. US, 68,805 (1971). Page, L. A., S. W. Englander, and M. V. Simpson, Biocheatry 6, 968 (1967). Sypherd, P. S. and B. S. Fansler, J. Bact. 93, 920 (1967). Sypherd, P. S., J. Mol. Biol. 56, 311 (197v Heintz, R., H. McAllister, R. ;h;linghaus, and R. Schweet, Cold Spr. Harb. Symp. Quant. Biol. 31, 633 (1966). Sherman, M. I. and K V. Simpson, Proc. Natl. Acad. Sci. US, 64, 1388 (1969). Ko, T. S., Ph.D. Thesis (1970), Va. Polytech. Inst., Blacksburg,%. N. J. Rosebrough, A. L. Fan, and R. J. Randall, J. Biol. Chem. Lowry, O.H., 193, 265 (1951). Dische, Z., in The Nucleic Acids. Edited by E. Chargaff and J. N. Davidson, Academic Press, New York, 1955, Vol. 1, p. 300. The physical and chemical properties of ribosomes, Elsvier, Petermann, M. L., New York, (1964) p. 121. Zubay, G. and M. H. F. Wilkins, J. Mol. Biol. 2, 105 (1960). Cotter, I. R., P. McPhie, and W. B. Gratzer, Nzture 216 864 (1967). -’ Cox, R. A., Biochem. J. 114, 753 (1969). Lumry, R., R. Biltonen, andJ. F. Brandts, Biopolymers 5, 997 (1966). Spirin, A. S., Molecular structure of ribonucleic acids. Reinhold Press, London (1964). Britten, R. J ., B. J. McCarthy, and R. B. Roberts, Biophys. J. 2, 83 (1962). Schildkraut, C. and S. Lifson, Biopolymers 3, 195 (1965).

1056