Enzymatic methylations, II in vitro inhibition of tRNA and protein methylation by nicotinamide and isonicotinic acid hydrazide: Activation of a S-adenosylmethionine-splitting enzyme in rat liver

Vol. 48, No. 1, 1972

BIOCHEMICAL

ENZYMATIC IN VITRO

INHIBITION

NICOTINAMIDE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

METHYLATIONS,

OF tRNA AND PROTEIN

AND ISONICOTINIC

H. J.

Gross

May 12,

been

and D. Wildenauer

In

our

or "mixed

preceeding

mechanisms This

31,

BRD

and nicotinamide

analogs

tRNA methylation

in

type"

has been

paper

describes

inhibition

we could

the

fractions

of a weakly

splitting

enzyme,

and isonicotinic

exclude

acid

vitro,

partial

is

an assay

and a

these

free

inhibited

at all

MATERIALS

AND METHODS:

rat

postulated

methylase

of

by nicotinamide

(INH).

Nicotinamide

of the

SAM-splitting

from

enzyme.

developed

methylase

activity.

enzyme

which

were not

or INH.

(3)

Copyright 0 19 72 by Academic Press, Inc. AN rights of reproduction in any form reserved.

may

has been

SAM-splitting

by nicotinamide

(SAM)-

Hydroxylapatite chromatography and assay for rat methylases were performed as previously described thin-layer chromatography (TLC) plates (cellulose ref.

(1,2).

inhibition

liver

activated

system

separation

fractions

in

strongly

hydrazide

Methylase

+I:

(2) have

S-adenosylmethionine

as a coenzyme

enzyme

the

detection

active,

which

function

For this allowed

Biochemie,

(3).

report

possibly

fiir 2, Goethestr.

(1)

shown to inhibit

competitive

ACTIVATION

1972

nicotinamide

Recently,

BY

ENZYME IN RAT LIVER.

Max-Planck-Institut D-8 Miinchen

METHYLATION

ACID HYDRAZIDE:

OF A S-ADENOSYLMETHIONINE-SPLITTING

Received

II+

58

liver (3,4). Precoated F) were

BIOCHEMICAL

Vol. 48, No. 1, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

obtained from Merck AG Darmstadt. The following solvents were used for TLC: A (n-butanol/acetic B (isopropanol/88 % formic acid/ acid/H20 -60/15/25 - v/v/v); H20 - 70/10/20 - v/v/v) and C (isopropanol/conc. NH3/H20 70/10/20 - v/v/v). SAM-splitting enzyme activity and its stimulation by nicotinamide or INH was measured in two ways: 1.) 120 ~1 incubation volume contained MgC12: 1 pmole; Tris*HCl (methyl-14C) SAM (spec. act. 13.8 ci/M): pH 7.8: 10 pmoles; 1.44 mnmoles; aliquots of methylase fraction I (1.8 mg protein) containing SAM-splitting enzyme and nicotinamide (1 nmole) or INH (5 nmoles). 10 ~1 aliquots were placed on TLC plates after at 35OC, and the plates were 0, 5, 15 and 30 min. incubation developed in solvents, A, B or C. The solvent front was allowed to move 11.5 cm. For the evaluation of the thin-layer chromate= grams a half automatic scraper (5) was used. Radioactivity from the plates was measured in 10 ml toluene/cellosolve/PPO (lOOO/ 500/6 - v/v/w). 150 J.L~ incubation mixture 2.) Assay for SAM-splitting enzyme: contained a 50 ~1 aliquot from hydroxylapatite column fractions (4), all components of the methylase assay (3,4) and additional methylase III (4): 700 ng, and nicotinamide: 1 nmole (or INH: 5 poles). After one hour incubation at 35'C, 100 ~1 aliquots were placed onto paper filter disks which were processed as described by Mans and Novelli (6). RESULTS AND DISCUSSION: Recently INH)

(3) we have shown that

on the

can not

enzymatic

Our results possible a.)

authors

(3) might reactions

SAM is used

causing

action of protein

by a competitive

by other

an accompanying this

methylation

be explained

as suggested

the

of nicotinamide

(or

and tRNA in vitro

or "mixed

type"

inhibition

(1,2).

be explained

if

one of the

following

occurs: for

rapid

nicotinamide a decrease

methylation

of nicotinamide

methyltransferase in tRNA and protein

59

or INH by

(E.C.2.1.1.1.)(7), methylation;

Vol. 48, No. 1, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

distance

from orqn

(cm)

Fig. 1: Thin-layer chromatography in solvent A (11) of (methyl- 14C)SAM after incubation (details in "materials and methods") with methylase I (41, which contained SAM-splitting enzyme and INR (5 umoles). 10 ~1 aliquots were removed from the incubation mixture after 0 W, 5 x-x, 15 o--o and 30 M min., and immediately placed on TLC plates for chromatography. The same procedure was performed in the presence of nicotinamide (1 pole), and the chromatogram after 5 min. incubation is shown here-. Peak I: (methyl14c)sm (RF = 0.09); peak II: unidentified (RF = 0.26); peak III: unknown (RF = 0.56); this material is a by-product produced slowly from the substance in peak II. RF-values of nicotinamide: 0.7; INR: 0.57.

b.) of c.>

nicotinamide SAM, thus

or INR themselves again

a SAM-splitting

fractions

is activated

decreasing rat

catalyze

the

tR.NA and protein

liver

enzyme

by nicotinamide

a coenzyme.

60

present (or

rapid

breakdown

methylation; in methylase

INR)

or

needs

it

as

Vol. 48, No. 1, 1972

BIOCHEMICAL

s

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3 ooo-

2 2 oooE: z. 8 :: 1 ooo3 5 s ; 2 8 2 -

500-

E

200.

I

nxbatlon

time

(mtnl

Quantitative evaluation of thin-layer chromatograms Fig. 2: as described in "material and methods" and in fig. 1. The 14 C)SAM concentration (peak I in fig. 1) was measured (methylafter 0, 5, 15, and 30 min. in the following incubation (methyl-l4 C)SAM with or without nicotinamide mixtures: (1 pmole) or INH (5 umoles):w; (methyl-14C)SAM with protein fraction (1.8 mg, methylase I containing SAM(methyl- 14 C)SAM with nicotinamide splitting enzyme): o---o; (1 umole) and protein fraction as above (1.8 mg): w; (methyl- 14C)SAM + INH (5 pmoles) with increasing amounts of the above protein fraction: 0.3 mg x-x; 0.6 mg U; 1.2 mg w; 1.8 mgv-V.

The first

possibility

and INH upon and

(methyl-

is easily

in

solvents

were used

thin

layer

and INH have different nicotinamide

(methyl-

for

chromatography TLC (not

since

(fig.

(legend

should

separate

nicotinamide

from

rat

liver

radioactive l),

shown here).

RF values

and methyl-INH

even

Since

to fig.

if

different

nicotinamide l),

methyl-

in any of the

used.

The second nicotinamide

out,

incubation with methylase I (4) 14 C>SAM gave completely identical

products

solvents

ruled

explanation

is

or INH alone

ruled

out

does not

14C) SAM.

61

as shown in catalyze

the

fig.

2:

breakdown

of

Vol. 48, No. 1, 1972

BIOCHEMICAL

Finally, a.)

fig.

1 and especially 14 C)SAM is

(methyl-

nicotinamide

(or

splitting b.)

both

cause

under

amount

our

(or

rapid

in the

breakdown

(not

of protein

splitting

demonstrate: presence

of

I (which

contains

SAM-

and methylase

fraction

the

of SAM; SAM breakdown

shown in detail)

("methylase

enzyme

nicotinamide

I")

fraction efficient

than

explains

This

on tFWA and protein

in

the

presence

and INS depends

is a much more INS.

rate

on the

added; activator the

for

stronger

methylation

SAMeffect

as compared

of

to

(3).

These

results

indicate

SAM-splitting

In order

(4) were

the methylase

assay

III

methylase

(4).

This

and hence

fractions into contain

a more

not

(methyl-l4 shown in

should

In

TCA-insoluble splitting inhibited

those

of

with

material

the

(fig.

62

enzyme,

the

because

of this

experiment

decreased

methylation

which degree

of rapid is of

nicotinamide-dependent

fractions

by nicotinamide

of

however,

reduced

The result

TLC.

Thus all 14 C-methyl groups

fractions

be considerably

Methylase

than

SAM-splitting

by nicotinamide.

3. The fractions

enzyme.

assay

the

and methylase

SAM-splitting

contain

liver

components

contain

show incorporation

C)SAM breakdown. fig.

all

in

rat

nicotinamide

affected

material.

enzyme of

with

does not

nicotinamide-dependent

of methylation

this

suitable

incubated

III

to be assayed

TCA-insoluble

to locate

and additional

is

of a nicotinamide-dependent

chromatography

(4) we developed

fractions

enzyme

existence

of hydroxylapatite

supernatant Column

the

enzyme.

fractions

not

stable

INH)

conditions

nicotinamide

INH

2 clearly

or methylase

nicotinamide

of nicotinamide

d.)

INS)

rather

fig.

enzyme);

together c.)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

free 3).

of

this

Nicotinamide

SAMenzyme

are

is not

an

BIOCHEMICAL

Vol. 48, No. 1, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 3: Fractionation of rat liver methylase and nicotinamidedependent SAM-splitting enzyme on hydroxylapatite. A linear 175) potassium phosphate gradient from 0 (fr. 60) to 0.5 m (fr. was used (fr. vol. 3.3 ml). fMet Assay for tRNA methylase activity with substrates tRNAE coli and unfractionated E.coli tRbA M, tRNAy; coli o----o x-x. Assay for nicotinamide-dependent SAM-splitting enzyme: U; fractions 80-84 contained the SAM-splitting enzyme and were pooled and processed like methylase fractions as described Methylase fractions which were obtained free previously (3). of nicotinamide-dependent SAM-splitting enzyme were not inhibited by nicotinamide or INB (not shown here).

inhibitor and the may in splitting

of

tRNA methylase

tlnon-dialyzable fact

be identical enzyme

characterization

described of this

as stated

inhibitor with

of the

here. enzyme

by other

authors

tRNA methylase

(1,2), (8,9,10)

nicotinamide-dependent Experiments are in

for

SAMfurther

progress.

We thank Dr. S. Nishimura for a kind gift Acknowledgements: fM of tRNAE'.Eti. We are indebted to Prof. A. Butenandt, Prof. H. Dannenberg and the Deutsche Forschungsgemeinschaft (Sander,-

63

Vol. 48, No. 1, 1972

forschungsbereich C. Bucher for

BIOCHEMICAL

51) reading

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for generous support, the manuscript.

and C. Raab and

References: 1.) 2.)

3.) 4.) 5.) 6.) 7.)

8.) 9.1 10.) 11.)

R.M. Halpern, S.Q. Chaney, B.C. Halpern and R.A. Smith, Biochem. Biophys. Res. Commun. 42, 602 (1971) L. Buch, D. Streeter, R.M. Halpern, L.N. Simon, M.G. Stout and R.A. Smith, Biochemistry -11, 393 (1972) H.J. Gross and D. Wildenauer, Biochem. Biophys. Res. Commun., submitted Y. Kuchino and S. Nishimura, Biochem. Biophys. Res. Commun. 40, 306 (1970) G. Kasang, G. Gijldner and N. Weiss, J. Chromatography 59, 393 (1971) R.I. Mans and G.D. Novelli, Biochem. Biophys. Res. Commun. 3, 540 (1960) G.L. Cantoni in: P.S. Colowick and N.O. Kaplan, "Methods in Enzymology", vol. II, p. 257, Academic press, New York 1955 J.T. Murai, P. Jenkinson, R.M. Halpern and R.A. Smith, Biochem. Biophys. Res. Commun. 46, 999 (1972) S.J. Kerr, Biochemistry 9, 690 and 1577 (1970) S.J. Kerr, Proc. Natl. Acad. Sci. -68, 406 (1971) F. Schlenk and D. J. Ehninger, Arch. Biochem. Biophys. 106, 95 (1964)

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