High molecular weight complex formation of rat liver lysyl-tRNA synthetase reduces enzyme lability to thermal inactivation

Vol. 106, No. 1, 1982 May 14, 1982

BIOCHEMICAL

AND EIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 44-47

HIGH MOLECULAR WEIGHT COMPLEX FORMATION OF RAT LIVER LYSYL-tRNA REDUCES ENZYMF. LABILITY TO THERMAL INACTIVATION

SYNrHETASF

Chi V. Dang Department of Cell Biology and Anatomy The Johns Hopkins University School of Medicine Baltimore, Maryland 212n5 Received

March

22,

1982

Eukaryotic aminoacyl-tRNA synthetases tend to occur in high molecular weight multienzyme complexes in contrast to the prokaryotic counterparts. The functional significance of complex formation is not understood but stabilization of enzyme activities in the complex(es) has been suggested as one of many possible functions. We provide evidence that the 245 complex lysyl-tRNA synthetase activity is more resistant to thermal inactivation than the 6S free lysyl-tRNA synthetase activity at 30°, 37', and 43'C.

Introduction The aminoacyl-tRNA from amino

acid,

biosynthesis as high lo6

tRNA,

(1).

for

occurring

that

(2,

is presently

formation Recently

is it

molecular

has been suggested

We have reported 24s complex

(3)

and its

complex

for tend

weights

appear (11)

than

ubiquitous

250,000 in

and Drosophila

complexes

significance

thermal

(8)

chromatography of aminoacyl-

of these

advantage

formation

to occur

less

and affinity

from

protein

up to over

weights

yeast

activities

HMW com-

of complex inactivation.

may provide

inter-

(16).

isolation

disassembly

and characterization by hydrophobic

OOOS-291X/82/090044-U4$01.00/0 Copyright A II rrghls

molecular

One possible

that

in vivo -__ the

molecular

are multienzyme

understood.

acids

synthetases

from conventional

of enzyme

stabilization

from

The functional

5, 10).

stabilization

with

types

Evidence

not

with

of aminoacyl-tRNA

amino

The HMW complexes

4).

these HMW complexes

tRNA synthetases plexes

enzymes

in many cell (8).

activated

(HMW) complexes

see Ref.

the formation

aminoacyl-tRNA

to prokaryotic

to human placenta indicate

and ATP providing

weight

a review

eukaryotes

catalyze

The eukaryotic

molecular

in contrast

(l-18,

synthetases

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

44

of a rat chromatography

liver to yield

Vol. 106, No. 1, 1982 free

6S enzymes

suggesting

that

lability

BIOCHEMICAL

(3,

We report

5).

complex

kinetics

in this

formation

of enzyme activity

activation

AND BIOPHYSICAL communication

of aminoacyl-tRNA

to thermal

and Methods

Materials:

Sources

for

materials

are

in vitro -__

synthetases

inactivation

evidence reduces

by comparing

of the 6s and 245 lysyl-tRNA

Materials

RESEARCH COMMUNICATIONS

synthetase

described

the

in-

activity.

previously

(2,

3, 5).

Enzyme Preparation and Assay: The rat liver 24s lysyl-tRNA synthetase was prepared in the presence of phenylmethane sulfonyl fluoride (PMSF) as described by Bang and Yang (3). The 6s lysyl-tR.NA synthetase was prepared by chromatography of the 24s complex on a diaminooctyl-Sepharose 4B column and centrifugation in a sucrose gradient as described (3, 5). Enzyme assays were according to (3) in presence of 3 mg/ml of bovine serum albumin which protects the enzyme activity completely at 37'C for at least 10 minutes. One unit of enzyme activity is that amount catalyzing the formation of 1 nmole of aminoacyl-tRNA in 1 minute at 37'C. Thermal Inactivation of Synthetase Activities: Pooled 245 lysyl-tRNA synthetase was diluted to give comparable concentration of enzyme activity (2-4 units/ml) as the pooled 6S enzyme. The 24s and 6s enzymes (1 ml each) were dialyzed in 1 liter of buffer (50 mM Tris-HCl (pH 7.5 at 25OC), 2 mM dithiothritol, 25 mM KCl, 5 mM MgC12, 0.2 mM PMSF) containing 10% glycerol at 4'C for 6 hours to assure identical buffer conditions. Aliquots (0.2 ml) of hS and 24s enz mes were incubated in 10 x 75 mm glass test tubes in water baths at 30°C, 37 8 C, and 43OC. Aliquots of 25 ~1 were removed at appropriate times and immediately assayed at 37'C for 5 minutes in presence of 3 mg/ml BSA. Results

and Discussion

The 24s lysyl-tRNA lysyl-tRNA

synthetase

Increasing

lability

increasing

temperatures,

rates

synthetase activity

of the

creased

rate

of

at 30°,

at 30'

of the

6s Lys-tRNA

first-order

at the

three

temperatures.

to be a residual

The results resistant and 43'C. inactivation The nature

show that

to thermal

The curvilinear suggest of this

synthetase

than

semilog

at the

the 6S

lA,

B, C).

is activity

activity

synthetase

plots

is not 45

understood,

in-

activity. appears

different activity

to the but

The

to he

synthetase

inactivates temperatures.

is more at 30°,

of the 24s Lys-tRYA in contrast

has similar

is a markedly

synthetase

three

seen with

the 6S enzyme activity

activity

activity

activity

there

than

(Figs.

The 24s Lys-tRNA

the 24s Lys-tRNA

a residual residual

At 43'C

activity

inactivation

labile

synthetase

of the 24s Lys-tRNA

rate

appears

synthetase

and 37'C.

inactivation

less

and 43'C

the 245 lys-tRNA

inactivation

to what

is

37',

6s Lys-tRNA

but

of inactivation

activity

37',

synthetase 6S enzyme. it

may result

Vol. 106, No. 1, 1982

BIOCHEMICAL

AND BIOPHYSICAL

Time

Fig.

1 Thermal

(minutes)

inactivation

of 24s (-0-O-j

and 6s (-0-O-j

activity

at 30°C (panel

A), 37'C (panel

synthetase (panel

RESEARCH COMMUNICATIONS

C).

The ordinate

of initial

activity

represents

remaining

the logarithm

after

a certain

lysyl-tRNA B), and 43'C of

percent

the

incubation

time

(abscissa).

from

the equilibrium

between

24s enzyme preparation. enzyme activity free

This

of the dimeric exchange

complex

with

(5,

of other

stabilization

has been Leu-tRNA

by Ile,

molecular

stabilization

inactivation

experiments

for

the

(17).

in the complexed

than

thermal

the

inactivation

The possibility

of

enzymes may now be more definitely purified

specific

interactions.

Chinese

in-

aminoacyl-tRNA

synthetase

Arg,

Tie,

and Leu (16). in the

Gln,

stability

Glu,

for

concept

of intermolecular

by -__ in vivo

studies.

A temperature-

hamster

This

synthetase

ovary

acid

suggests

the

complex

65 and 24s Lys-tRNA 46

(GHO) cell

amino

mutant

leucine

synthetase

has

and synergistically

occurrence

in vivo. -~

Leu,

the Lys-tRNA

This

by the homologous

with

for

(3) may provide

synthetase

Val,

the

inactivation

synthetase

synthetases

substantiated

to be protected

protected

may represent

to thermal

of highly

in the 24s complex by intermolecular

been shown

and free

synthetase

10).

synthetase

sensitive

Lys-tRNA

has been reported

the availability

The presence

activity

germ leucyl-tRYA

complexed

and free

resistant

phenomenon

preparation

Met and Pro

is more

wheat

between

vestigated

The residual

which

enzyme.

complexed

of inter-

The -___ in vitro in this

com-

BIOCHEMICAL

Vol. 106, No. 1, 1982 munication

show in vitro

formation.

The similar

activity

at 30'

previously summary,

observed

stabilization rates

of

with

of

in this

of synthetase inactivation

and a faster the rabbit

among the many possible

we demonstrate lability

and 37'C

AND BIOPHYSICAL

communication

the enzyme activities

rate

activity

by complex

of the 24s Lys-tRNA of inactivation

reticulocyte functions

RESEARCH COMMUNICATIONS

at 43OC have been

16s complex

(13).

of HMW aminoacyl-tRNA

that

complex

to thermal

formation

synthetase

In synthetases,

decreases

the

this

was

inactivation.

Acknowledgements The author thanks partially performed.

Dr.

D.C.H.

Yang,

in whose

lahoratory

work

References 1. 3. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Schimmel, P.R. and Sb'll, D. (1979) Ann. Rev. Biochem. 48, 601-648. Biochem. Biophys. Res. Commun. Dar%, C.V. and Yang, D.C.H. (1978) 80, 709-714. J. Biol. Chem. 254, 5350-5356. Da%, C.V. and Yang, D.C.H. (1979) Dang, C.V. and Yang, D.C.H. (1982) Int. 3. Biochem., in press. Johnson, D.L., Dang, C.V. and Yang, D.C.H. (1980) J. Biol. Chem. 255, 4362-4366. Johnson, D.L. and Yang, D.C.H. (1981) Proc. Nat. Acad. Sci., U.S.A. 78, 4059-4062. Bandyopadhyay, A.K. and Deutscher, M.P. (1971) J. Mol. Biol. 60, 113-122. Denney, R.M. (1977) Arch. Biochem. Biophys. 183, 157-167. Glinski, R.L., Gainey, P.C., Mawhinney, T.P., and Hilderman, R.H. (1979) Biochem. Biophys. Res. Commun. 88, 1052-1061. Keller-man, O., Brevet, A., Tonetti, H., and Wailer, J.-P. (1979) Eur. J. Biochem. 99, 541-550. Dimitrijevic, L. (1977) FEBS Lett. 79, 37-41. Shafer, S.J., Olexa, S. and Hillman, R. (1976) Insect. Biochem. 6, 405-411. Som, K. and Hardesty, B. (1975) Arch. Biochem. Biophys. 166, 507-517. Ussery, M.A., Tanaka, W.K. and Hardesty, B. (1977) Eur. J. Biochem. 72, 491-500. Vennegoor, C. and Bloemendal, H. (1972) Eur. .I. Biochem. 26, 462-473. Molnar, S.J. and Rauth, A.M. (1979) J. Cell.Physiol. 98, 315-326. Carias, J.R., Mouricout, M., Quintard, B., Thomas, J.C. and Julien, R. (1978) Eur. J. Biochem. 87, 583-590. Ritter, P.O., Enger, M.D. and Hampel, A.E. (1979) Biochim. Biophys. Acta 562, 377-385.

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