BIOCHEMICAL

Vol. 75, No. 1, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TRANSFER RNA METHYLASE ACTIVITY IN NORMAl AND DYSTROPHIC CHICKEN MUSCLE Bertrum

Sheid

and Lauri

Pedrinan

Department of Pharmacology State University of New York Downstate Medical Center 450 Clarkson Avenue Brooklyn, New York 11203 Received

January

18,1977

chicken muscle had 30% higher tRNA methylase activity SUMMARY: Adult-dystrophic and 42% higher tRNA methylating capacity than normal-adult chicken muscle. Eighty percent of the tRNA methylase activity of the dystrophic muscle resulted in the synthesis of NZ-methylguanine, and 9% in the formation of N2,N2-dimethylguanine. From adult-normal muscle extracts, 33% of the tRNA methylase activity was due to the synthesis of N2-methylguanine, and 45% to the formation of N2,N2-dimethylguanine. Eight other methylated bases accounted for 5-15% of the enzyme activity in both tissues. Dialyzed and nondialyzed adult-normal muscle extracts had equivalent tRNA methylase activity. However, the dialyzed extracts synthesized 22% more N2-methylguanine and 18% less N2,N2-dimethylguanine than the nondialyzed extracts. Dialysis had no effect on the tRNA methylase activity or tRNA methylation pattern produced by adult-dystrophic muscle. Considerable tally

induced

tory

role

have

mational

that

to eukaryotic in

the major

during Other

erythroid studies

modification

of cellular

cells, and minor

investigators nucleotides

differentiation

from

it

experiments for

this

confor-

quantitative

the Morris

virus-induced

occur

in relation differences

hepatomas leukemic

and quantitative

(ll),

cells

changes (13),

during

alterations

hypothesis

lymphocytes

the tRNA

which

by coinciding

have demonstrated

qualitative

in the

has been postulated

such as those

of tRNA from

an obliga-

consequently, Numerous

studies,

To support

mitogen-stimulated

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

play

tRNA isoaccepting

be reflected

tRNA.

enzymati-

of tRNA are necessary

of murine

have demonstrated enzymes

and, (1,2).

proteins,

should

that

synthesis

biosynthesis

of these

constituent

and neoplasia,

in the modification

initial

of the individual

As a consequence

differentiation

its

modifications

integrity

in a cell's

to demonstrate

and differentiation

the enzymatic

(1,3-10).

changes

of protein

growth

and functional

species that

of tRNA after

the regulation of normal

shown

has been accumulated

modifications

in

progression

evidence

and (12).

in the tRNA

differentiating

56 ISSN

0006-291X

Vol. 75, No. 1, 1977

embryonic

rabbit

cells

etiology

(1,5,15-17).

from the

normal

ferentiation altered

BIOCHEMICAL

do occur,

bases Although

(1,5). methylases

in various

differentiating dystrophy type)

X-linked

are believed

in protein cations

muscle (the

they

synthesis

malignancies

it

of tIWA during culminate

in the

of tRNA is the

enzymatic

and viral if

diversions

growth

and/or

synthesis

of

dif-

(16,17).

of enzymes

collectively

known

investigations

maarnalian

none of these

tissues,

tissue.

Since

Duchenne

type,

to be due to aberrant

of tRNA from normal

that

normal

have been numerous

(la-20),

of chemical

has become apparent

may conceivably

modification

by a group there

data

of modification

constituents

The most prominent methylated

and in various

From these

pattern

cellular

(14),

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the most

severe

and its

variant,

maturation

the need to study and diseased MATERIALS

muscle

synthesis

as the tRNA methylases concerned studies

forms

with

has involved

the slower

growing

Becker

of alterations

the enzymatically is

the tRNA

of human muscular

as a result

tissue

of the

induced

modifi-

apparent.

AND METHODS

New Hampshire Red normal and genetically determined dystrophic chickens (12-13 months old),which have been widely used as a model for human Duchenne muscular dystrophy (21), were kindly provided by Drs. A. Stracher and E.B. McGowan, of our institute. The diagnosis of muscular dystrophy was verified by the "flip test," whereby the chicken cannot right itself after being placed on its back (21). High-speed supernatant fractions (105,000 x g; 1 hr) of superior breast muscle from the test animals were prepared as previously described (17). A portion of each fraction was lyophilized, and stored until use. Other portions were either dialyzed against cold distilled water for 24 hr before lyophilization, or treated with (NH > SO to 60% saturation before dialysis and lyophilization. Just prior to the e ft2zyme 4 assays, as many of the enzyme samples as needed were reconstituted with 1 ml of water per 0.5 g tissue. Initial experiments were performed to establish the fact that the enzyme preparations stored in this manner were 100% active for a period of at least one month. The standard reaction for tRNA methylase activity consisted of 10 umoles Tris buffer, pH 8.9; 80 pmoles of ammonium acetate; 1 umole of freshly prepared and neutralized reduced glutathione; 25 ug of E.coliB tRNA; 1 pCi of neutralized [H3]S-adenosylmethionine; (11.6 Ci/rmnole, New England Nuclear); and 50-2000 ug of protein from the 105,000 x g supernatant fraction in a total volume of 0.3 ml. After 45 min at 37OC, the reaction was terminated with 0.06 ml of 50% trichloroacetic acid and 0.02 ml of 6-N HCl. The precipitates were vigorously washed in the test tubes with 80% alcohol, 2 x with alcohol-ether (3:l) and ether, then dried in vacua. They were then dissolved in alcoholic-hyamine at 7OoC, neutralized, transferred to Bray's solution and counted. Enzyme blanks (one without tRNA and another without protein) were assayed concurrently with all of the samples, The tRNA methylation capacity was determined by the addition of increasing amounts of enzyme extract protein to the reaction mixture until no significant change in the total c3~] incorporation per 25 ug tRNA could be detected. To determine the specific activity, the proportional range of enzyme activity was utilized. Utilizing this procedure, blanks

57

Vol. 75, No. 1, 1977

Table 1.

BIOCH’EMICAL

AND BIOPHYSICAL

Transfer RNAmethylase activity breast muscle.a

Tissue

Treatment

RESEARCH COMMUNICATIONS

in normal and dystrophic-chicken

Methylation capacity (maximumamount of pmoles methyl-3H incorporated/25 ug tRNA)b

Specific activity (pmoles methyl-3H incorporated/200 pg protein/25 ug tRNA)C

Adult normal muscle Adult normal muscle

Dialysis d

0.31 0.29

0.16 0.16

Adult dystrophic Adult dystrophic

muscle muscle

Dialysis

0.44 0.46

0.21 0.20

Embryonic normal muscle Embryonic normal muscle

Dialysis

1.64 1.24

0.54 0.60

aThese data are an average of 4 separate samples of each tissue assayed in duplicate. The individual results did not differ from each other by more than 6%. bThe amount of protein for maximal incorporation 1000-1250 ug for all of the samples. 'The proportional

range for enzyme activity

dDialyzed and ammoniumsulfate-treated enzyme activity.

of (methyl-3H) was between

was 175-225 ng protein.

muscle supernatant extracts

had the ssme

of 100-700 cpm for 50-2000 ug of muscle protein were obtained, while blanks of 1000-5000 cpm were found to be routine if the precipitates were first placed on filter paper discs before washing. Duplicate assays were reproducible within 5% of each other. The enzymatically synthesized methylated bases were identified and quantitated after two-dimensional thin-layer chromatography on microcrystalline cellulose (22). Protein was determined by the method of Lowry -et al. (23). RNAaseassays were performed by measuring the 260 nm adsorption of the acid-solubroligonucleotides formed from tRNA after incubation with the muscle protein extracts (24). RESULTSAND DISCUSSION Table 1 demonstrates an approximate 30% increase in the tRNA methylase specific

activity,

muscle extracts activity

and 42% increase in the tRNA methylation

capacity

as compared to normal adult-muscle extracts.

was not due to reduced RNAase activity -

extracts cific

Dialysis

and/or ammoniumsulfate

from normal and dystrophic

This increase in

in the dystrophic-muscle

or to an increase in the metabolism of S-adenosyl-L-methionine muscle extracts.

58

extracts,

by the normal-

treatment of the enzyme

muscle had no apparent effect

enzyme activity.

in dystrophic-

on their

spe-

2.

tr tr

25

25

tr

tr

tr

tr

Slnethyluracil

3 + 5-methylcytosine

l-methyladenine

2-methyladenine

C-methyladenine

6-dimethyladenine

1

1

3

6

9

tr

tr

tr

tr

44

36

36

132

532

392

fmoles CH3 inc0rp.l 100 pg tRNA

equals

trace

(less

The same tRNA methylation or made 60% with respect

d tr

C

than

1% of total).

pattern was obtained whether to (NH4)2S04 before dialysis.

the

Normal

184

238

197

604

2660

1980

extracts

from

produced

specimens.

tr

tr

tr

tr

from

normal

adult

by dystrophic

90-95%

tr

tr

tr

tr

2

4

3

12

45

33

Normal adult

(adult

muscle

and normal

were

tr

tr

tr

tr

3

2

4

8

27

55

was dialyzed

muscle

counts

tr

tr

tr

tr

36

24

40

80

280

572

% total incorp.

dialyzed muscleC

and embryonic)

fmoles CH3 inc0rp.l 100 ug tRNA

of all

normal

% total incorp.

embr onic muscle i:

derived

fmoles CH3 inc0rp.l 100 ug tRNA

pattern

3-5

enzyme

tr

tr

tr

tr

4

3

3

11

45

33

% total incorp.

aMethylation patterns are an average of the results obtained for recovered as methylated bases. b had no effect on the methylation Dialysis or (NH4)2SO4 treatment embryonic tissue.

tr

65

l-methylguanine

tr

135

7-methylguanine

d

205

79

% total incorp.

N2N2-dimethylguanine

1810

fmoles CH3 inc0rp.l 100 ng tRNA

by enzyme extracts

Normal nondialyzed adult muscle

patterns produced breast muscle.a

Dystrophic b adult muscle

Transfer RNA methylation and dystrophic-chicken

N'-methylguanine

Table

g v,

Y

$ 2

8

H

F : v, F

z v,

FT

z

I z n P

? .

.i

d.-

Vol. 75, No. 1, 1977

Table

BIOCHEMICAL

2 demonstrates

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

80% of the tRNA methylase

activity

of dystrophic

n

muscle

resulted

formation

in

enzyme activity

45% resulted

in the

methylated

normal

of N‘-methylguanine,

of N2,N2-dimethylguanine.

33% of the

other

the synthesis

resulted

formation

bases

in

for

tRNA methylase

muscle

as compared

increase

in protein

dystrophic

muscle

(19,20).

However,

of an exogenous

in protein

specific

proteins It

is

muscle

extracts

lating

capacity.

synthesized

may be suggested bases

small

molecular It

expression tant

with

dustrophic

the

between

and/or

activity

in

qualitative

the

an alteration

be of interest

Quantitation

in adult in

a different

besides

an

may be alterations

and nondialyzed activity

of the

in

than

as it

muscle

sequence

of 22% more mono-

did

are

if

normal-muscle on the tRNA

on normal

muscle,

synthesis

of methy-

due to dialyzable, normal

tissue.

the difference

and dystrophic

of a specific

of the different

60

guanine

in the

to determine normal

two methylated

in the

exclusively

between

extracts

had no effect

muscle

adult

the different

the nondialyzed

treatment

normal-

and tRNA methy-

synthesis

differences

present

in the base

that,

a

of tissue,

synthesize

there

2, although

in the

and dystrophic

factor(s)

to occur

two types

dialyzed

amount

(NH4)2S04

that

would

the

capacity

be considered

is known

specific

resulted

by dystrophic

normal

may at first

that

1 that

dimethylguanine

of the tRNA methylases

muscle.

of eight

(16,17).

same total

produced

weight

of course

while

and tRNA methylating

muscle,

tissue

extract

dialysis

pattern

lated

tRNA methylase

which

fact

as seen in Table

and 18% less Since

methylation

the

in Table

However,

the dialyzed

extract.

synthesis

had the same tRNA methylase

methylguanine

of the

muscle

in dystrophic

demonstrated

approximately

derivatives,

it

synthesis

of the diseased

also

muscle,

The synthesis

heterologous tRNA substrate, 2 2 ,N -dimethylguanine may imply

of N2-methylguanine:N

increase

adult-chicken

of N2-methylguanine,

activity

to normal

of the

ratio

the rest

specific

reflection

presence

synthesis

in the

muscle.

The increased

the

the

9% resulted

nondialyzed

of N2,N2-dimethylguanine.

accounted

and dystrophic

of dystrophic

In normal

while

methylated

in the

muscle

isoaccepting bases

is

concomi-

tRNA in

in nonfrac-

Vol. 75, No. 1, 1977

tionated if

BIOCHEMICAL

tl?NA may give

an altered

specific

misleading

tRNA is

results.

involved

isoaccepting

tRNA,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

It

in muscular one which

is more reasonable

dystrophy,

plays

it

a pivotal

will

role

to expect

be limited

that

to a

in muscle-cell

matura-

tion. Table

1 also

embryonic-chick adult

muscle.

tRNA methylase the

muscle This

is

activity

tRNA methylation

muscle

were

the different compared lified

shows

that

was 5 timzs

higher

than

in agreement

with

previous

in embryonic patterns

essentially

by embryonic

normal

muscle

tissues

produced

equivalent

tRNA methylation

to adult

the tRNA methylase

activity

the enzyme activity data

(25).

was not

which

However,

by normal-adult

to each other. pattern

in 15-day-old

produced

in normal

described

increased

as seen in Table

2,

and normal-embryonic These

by adult

a reflection

normal

experiments dystrophic

of muscle

disuse

imply muscle

that as

as exemp-

muscle. REFERENCES

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(8)

(9) (10) (11) (12) (13) (14) (15)

(16) (17) (19)

(20) (21) (22) (23) (24) (25)

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