Characterization of messenger RNA for aldolase B in adult and fetal human liver

Vol. 104, No. 2, 1982 January 29, 1982

CHARACTERIZATION Claudine Institut

BIOCHEMICAL

RESEARCH COMMUNICATIONS Pages 369-375

OF MESSENGER RNA FOR ALDOLASE B IN ADULT AND FETAL HUMAN LIVER Gregori,

Claude

de Pathologie

Received

AND BIOPHYSICAL

November

Besmond,

moleculaire, 18,

Axe1 Kahn and Jean-Claude

24 rue

du Faubourg

Dreyfus.

Saint-Jacques,

75014

Paris

1981

High molecular weight cellular RNA was isolated from adult and fetal human liver tissue by a procedure of ethanol precipitation in concentrated guanidine-HCl solutions. About 5 mg of RNA were obtained from one gram of liver. RNA was fractionated by sucrose gradient ultracentrifugation. Aldolase B neosynthesizedin a reticulocyte lysate cell-free system under the direction of total or fractionated RNA was purified by immunoaffinity microchromatography. Messenger RNA specifying synthesis of aldolase B exhibited a sedimentation coefficient of 16s both in adult and fetal liver. This enzyme represented 1.3 % of the total neosynthesized proteins in adult liver, 0.1 % in the liver of a b-month-old fetus and less than 0.01 % in the liver of a 4.5 month-old fetus. INTRODUCTION Cell-free first

synthesis

step

for

the

preparation

of the corresponding of some inherited rification

several

The enzyme, isozymes

liver

type

an inborn

(3),

error

liver

synthesis not

yet

is

muscle

on

present

type

Hereditary

and rat

of aldolase tetal

the

(B),

by "resurgent

B has been described

mammals

or

in

369

type

man.

is

under

The

it fetal

on adult present

dis-

(C).

(4,

of

(2).

Only

responsible

Intolerance

since

pu-

synthesis

genetically

deficiency

RNA (8)

for

has allowed

kinase

brain

A has been reported

replaced

This

pyruvate

Fructose

hepatoma

is

type

the

mechanisms

methods

cell-free

form of three

: its

fructose

of aldolase

aldolase

knowledge,

liver

studying

of human tissues.

in the

(A),

for

has developed

and of human liver

to metabolize

RNA (7)

amounts

are

RNAs and characterization

be a tool

Our group

to our

(1)

of metabolism,

muscle

hepatoma,adult

time

enzymes

synthesis

of chicken

Cell-free

first

aldolase,

is able

Cell-free

but

the

small

human enzymes

messenger

may also

disorders.

RNA from

human muscle

tinct

DNA. It

metabolic

for

of neosynthesized

of specific

genomic

of total

us to obtain,

and detection

the for

5, 6).

the direction is

known that, isozymes" rat

in (9).

(10,111.

paper

0006-291X/82/020369-07$01.00/0 Cop.vrighf 0 1982 by Academrc Press, Inc. All nghrs of reproducrion in any form reserved.

Vol. 104, No. 2, 1982

BIOCHEMICAL

reports

of human aldolase

cell-free

the

synthesis

rabbit

reticulocyte

AND BIOPHYSICAL B directed

RESEARCH COMMUNICATIONS by human liver

RNA in a

system.

MATERIAL AND METHODS . Material _-_---_ Human adult liver was obtained after surgical partial hepatectomy. Human fetal liver was obtained after therapeutical abortion of 2 fetuses at 4.5 and 6 months of intra-uterine life. Glutaraldehyde-activated "Ultrogel" was supplied by Industrie Biologique Francaise ; acrylamide, bisacrylamide, sodium dodecylsulfate, No Screen and Royal-X-Omatic R-l films by Eastman Kodak ; some "C markers, 35S methionine, Econofluor, Protosol, En3 Hance autoradiography Enhancer by New England Nuclear ; methylmercuryhydroxide by Ventron-alpha Lab. Other reagents were of the highest purity grade and came from Sigma CC and from Merck. . Methods Preparation of aldolase and of specific antibodies human aldolase B was prepared from liver obtained at autopsy according to Penhoet and Rutter (12) with modifications. Isolation involved ethanol precipitation, followed by phosphocellulose chromatography and specific elution with 5 mM fructose-l-phosphate. The aldolase preparation showed only one band after sodium dodecylsulfate electrophoresis. Antibodies were produced by repeated intradermal injection into rabbits. They were monospecific and gave only one band against a liver extract on Ouchterlony plates. Specific antibodies were purified by chromatography on an aldolase B-linked agarose bead column (1). Isolation of RNA Total cellular RNA from human liver was isolated by a method of ethanol precipitation in guanidine HCl, slightly modified from Cox (13), Harding et al (14) and Deeley et al (15) and described in detail in (1) for isolation of human adult muscle RNA. For liver, some further modifications were brought to this technique : first ethanol precipitation was for 30 min at -20" C (instead of overnight) and 0.5 % (w/v) lauroyl sarcosine was added to all the solutions until the last two steps of washing in 66 % (v/v) ethanol. Sucrose gradient sedimentation 250 pg of RNA were dissolved in 250 ~1 of a 20 mM borate buffer (pH 8) containing 20 mM methylmercuryhydroxide ; this solution was incubated at room temperature for 10 min, then diluted with 250 pl of a 10 mM Tris-HCl bufand 0.1 % fer (ph 7.5) containing 100 mM NaCll 1 mM EDTA, 10 mM iodoacetate (w/v) lauroylsarcosine. After centrifugation at 10 000 x g for 1 min, this sample was deposited at the top of 11 ml tubes containing a 5-20 % (w/v) SUcrose gradient in the Tris-HCl buffer described above. The tubes were centrifuged at 20" C in a SW 41 Beckman rotor at 40 000 RPM for 5 h. 0.4 ml fractions were collected, RNA was precipitated with ethanol and used to direct cell free-synthesis. Cell free translation and electrophoresis C 11-free translation was performed in a nuclease-treated reticulocyte lysat: according to Pelham and Jackson (16). Neosynthesized aldolase B was purified by immunoaffinity microchromatography, as reported elsewhere (l), except that elution of the antigen was provoked by incubation of the immunoabsorbent in 50 pl of a 62.5 mM TrisHCl buffer (ph 6.5) containing 2 % (w/v) sodium dodecyl sulfate, 5 X (v/v) B-mercaptoethanol, 10 % (v/v) glycerol and 2 mM diisopropylphosphofluoridate at 98" C for 3 min. Analysis was made by SDS polyacrylamide gel electrophoresis according to Laemmli (17), using 14C labeled proteins as molecular weights standards (18). Gels were dried and the immunopurified polypeptides were revealed by autoradiography using Kodak NS films or by fluorography using preflashed XR-1 films (19). Specificity of the neosynthesized bands was checked by immunological competition with unlabeled aldolase (1). 370

Vol. 104, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

RESULTS The method to obtain,

of high

starting

molecular

from

weight

ratio

was about

sharp

peaks

of 18s and 28s ribosomal

which

by the could

grity

molecular

of the Fig.

vitro

is

similar

ver.

For comparison,

molecular

slab either

columns

phy starting

whose

weight

between

by the

usual

to

1:2.

RNA

polypeptides, gradient

a relatively

from

fresh,

gel

high

argues

was not

turated

by excess

immunoabsorbent

propor-

for

the

(or

inte-

A further the cell-free

system

that

the addition

size

of the

purified amount

liver

slightly)

extract.

(not

aldolase

as reported retained then

on the

elec-

preparations,

elution

as both

human li-

from

phospho-cel-

microchromatograaldolase

B pro-

14C aldolase

markers.

aldolase

B was con-

in ref.

1 and 2 : this

by immunoabsorbent was retained

poly-

previously

sa-

by a non-saturated

shown).

for with

from

Neosynthesized

weight

aldolase,

the conclusion its

of various

aldolase

pure

38 000 band as neosynthesized

unlabeled

argument

B purified

RNA,

polypeptide

has been deposited (18)

of in

by human liver

a neosynthesized

of specific

same molecular

only

mixture

or by immunoaffinity

competition

column

the

labeled

procedure

a crude

was directed

aldolase

"'C

crude

by immunological

peptide

from

of aldolase

by fructose-l-phosphate

of the

l),

synthesis

to that

gels

Identification

(fig.

1:1.8

of numerous

(l),

starting

neosynthesized

the

Since

RNA showed

SDS-polyacrylamide

microchromatography

ved to have exactly

firmed

of

of

(> 60 000 daltons)

possible,

products

with

lulose

it

by immunoaffinity

purified

synthesis

previously

allowed

RNA used.

translation

trophoresis

analysis

RNA in the ratio

weight-chains

1 shows that

to purify

gradient

directed

shown

RNA isolation

5 mg RNA. 260 : 280 absor-

about

by one dimensional

As already

of high

density

procedure

be analyzed

electrophoresis. tion

2. Sucrose

above

cellular

one gram of liver,

bance

prepared

total

that

definitive

aldolase

molecular

antiproteolytic

was synthesized

weight

agents

(1,2)

comes from did

in the

fact

not modify

the

subunit. neosynthesized

aldolase

of enzyme

synthesized 371

B appeared under

to be absolutely

such conditions

could

pure be di-

Vol. 104, No. 2. 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Mr[KD] -200

-68 -46

-18.5 -12.3

3

5

4

1. SDS-polyacrZylticle al&lase ; compaKson with

7

eZ electrophoresis of 35S labeled neosyn94 C labeled enzyme purified from adult

Figure

thesised

6

liver. Channels 1 and 10 : l'+C labeled protein markers : Myosin heavy chain (Mr = 200 000) ; Muscle phosphorylase B (Mr = 93 000) ; Bovine serum albumin ; Ovalbumin (Mr = 46 000) ; Carbonic anhydrase (Mr = 30 000) ; (Mr = 68 000) Lactoglobulin A (Mr = 18 500) ; Cytochrome C (Mr = 12 300) ; channels 2 and 6 : Aldolase B ourified from adult liver bv affinitv elution, from a phosphocellulose column, then 14C labeled ; channels 3 and-7 : 35S labeled neosynthesized aldolase B purified by immunoaffinity microchromatography (1,2) ; chanB purified from liver crude extract by affinity micronels 4 and 8 : Aldolase chromatography, then 14C labeled ; channel 5 : Eluate from a microcolumn containing 10 ~1 of "ultrogel" coupled to non immune y globulins, to which the medium containing the translation products has been applied ; channel 9 : Neosynthesized serum albumin. Cell-free synthesis was performed as described (1,2), in a total reaction mixture of 75 ~1 containing 22.5 pg total high molecular weight cellular RNA from adult liver and 1.5 m Ci [35S] methionine (specific activity > 1000 Ci/mmol). Under these conditions, radioactivity incorporation into Trichloracetic precipitable material was about 150 000 CPM per ~1 of translation mixture, with a blank without addition of exogenous RNA of about 5000 to 6000 CPM/pi. Neosynthesized albumin represented 8 % and aldolase B 1.3 % of the total neosynthesized proteins. After electrophoresis according to Laemmli (17), the gel (lo-20 %, w/v, acrylamide) was dried and exposed for 5 days to a "No Screen-l" Kodak film. rectly of

measured the

from

the

antibody-bound

TCA-precipitable

resin

column.

synthesized

under

the

direction

6-month-old

fetus

and

less

Ultracentrifugation concentration RNA for and

of of

of than RNA

It

represented

adult

liver

0.01 in

% using

sucrose

B to

be

1.3 RNA, RNA

enabled

determined

found

: it

was

% of

0.11 from

gradient

methylmercuryhydroxide

aldolase

fetal

radioactivity

in the

RNA

neofrom

a 4.5-month-old

by

coefficient

be

16s

for

both

adult

RNA.

present

RNA extracted

work from

demonstrates fresh

human

that liver

it tissue, 372

is

possible, to

isolate

starting in

from one

step

high of

DISCUSSION The

a

fetus.

denaturation

sedimentation to

eluate

proteins

% using

after

found

the

total pure

Vol. 104, No. 2, 1982

neosynthesized bent

BIOCHEMICAL

liver

aldolase

microchromatography

col?rmns

saturated

with

The subunit

molecular

shed

of 41 000 (20).

enzymes

the

liver

tissue

contrary

possibility

livers.

tonishing

to reach

Aldolase

that

it

aldolase

molecular

is

The high portance

is

amount

report

with

on free

liver

RNA : 5 mg per gram of tissue, (1 and Munich

lation,

one may conclude

study

the messenger

these

data It

disappearance

correlated that

increase

cumulation that

aldolase

with

liver

a parallel of liver

B messenger

not

weight.

asThe

to have the same

liver

of aldolase

RNA activity

increases 373

with nascent

the

high

isozymes,

aldolase

This is

pep-

yield

of

of transto

to apply

Intolerance.

was associated

B (9).

the

be sufficient

in progress

Fructose

more

gram of adult

efficiency would

Work is

development

messenger

high

im-

represents

to 100 ug per

development

B during

specific

Given

material

of the fetal

practical

% of total

and the

aldolase.

fetal

increase

of translatable

is

aldolase

was aldolase.

of Hereditary

that

aldolase

0.5

as compared

for

study

in the

ribosomes

in good agreement

about

10 mg of biopsy

RNA activity

to the molecular

that

that is

in preparation)

that

has been established

gressive

rat

et al,

it

may be of great

in liver

tides

muscle

on free

appears

Our evaluation

stating

from

of

have shown that,

molecular

antibodies

to esta-

the amount

therefore,

definitive

publi-

of liver

important

synthesized

aldolase

synthesis

ribosomes

the

aldolase.

studies.

of Oda and Omura (10)

aldolase.

than

Oda and Omura (10)

its

seen on auto-

synthesis

therefore,

enzyme and,

of neosynthesized

protein

is

smaller

cell-free

was,

used to elicit

in pathological

enzyme on

in man, and to evaluate

a cytosolic

synthesized

authentic

of neosynthesized

is mainly

as neosynthesized

1 % of total

about

success.

aldolase

preparation

weight

published

of such a work

necessary

the

immunoabsor-

No impurity

gels

none in man ; it

to serum albumin,

in rat

than

but

to specific

with

aldolase.

RESEARCH COMMUNICATIONS

of 38 000 was somewhat

have been

in animals,

pure

electrophoretic

weight

papers

blish

pure

from

They bind

and compete

unlabeled

prepared

Several

subunits.

columns

radiographs

values

AND BIOPHYSICAL

work

with

a pro-

A and C, demonstrates

due to increasing

RNA : a functional more than

10 fold

assay

acshows

between

Vol. 104, No. 2, 1982 4.5

and 6 months

latter

fetal

BIOCHEMICAL

ofintra-uterine

stage

is

that

expected

does not

contain

for

that

it

and that the

in a cell-free

RNA. The yield could it

presence

aldolase

of active

the

B synthesis

during accumulation

ger

messenger

sedimente

in sucrose

protein,

which

gradient

between

under

liver

the

this

preparation

aldolase

liver

RNA specifying as a165

RNA (16s)

that

this

RNA

liver

is

material

deficiencies.

such

for

Increase

of human liver

aldolase

liver

of a CDNA probe,

biopsy

of translatable

aldo-

of human liver

RNA in adult

needle

development in the

signifies

the direction

messenger for

B messenger

and characterize

of analyzing

fetal

10 fold

sequences.

to isolate

point

of mRNA in

due to increasing RNA. Finally,

aldolase

system

possibility

and action

for

aldolase

be a good starting opens

more than

3' or 5' untranslated

we have been able

neosynthesized

total

found

a 40 000 dalton

extensive

then

RESEARCH COMMUNICATIONS

life.

coefficient

In conclusion, lase

life,

and the adult

The sedimentation

AND BIOPHYSICAL

is

shown to be

specific

B synthesis

of

messen-

was shown

to

species.

ACKNOWLEDGEMENTS Our thanks are due to Dr Fanny Schapira, who conducted extensive on aldolase and fructose intolerance, and helped us with suggestions cussions. We thank Mrs C. Brunner for typing the manuscript.

work and dis-

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Il., Daegelen, D. and Dreyfus, J.C. (1981) Eur. J. -.Kahn, A., Cottreau, Biochem., 116, 7-12. mJ.,-Simon, M.P., Dreyfus, J.C. and Kahn, A. (1981) Nature, 292, 70-72. mer, W.J., Rajkumar, T.. Penhoet, E., Kochman, M. and Valentine, R. (1968) Ann. New York Acad. Sci., 151, 102-117. __ Hers, Hx and Joassig. (1961) Enzymol. Biol. Clin., 1, 4-14. Schapira, F., Nordmann, Y. and Dreyfus, J.CT(T96~lE.~ranc. -Et. Clin. Biol., 2, 267-269. SchapicF., Gregori, C. and Hatzfeld, A. (1977) --Clin. Chim. @, 78, l-7. Ebherz, H.G. and Doyle, D. (1976) J. Biol. Chem., 251, 4355-4358. Sakiyama, S., Akiba, N. and Fujimu%, -1979) Cancer Res., 39, ___502-506. Schapira, F., Hatzfeld, A. and Weber, A. (1975) in : Isozymes, C.L. Markert ed., Acad. Press, III, 987-1003. Oda, T. and Omura, T.980) J. Biochem.(Tokyo), 88, 437-442. Tsutsumi, K.I.4;:d41;shikawa, K. (I9IBiochen: B=phys. Kes. Conmun, 100, Penhoet, E. and Rutter, W.J. (1975) Methods Enzymol., M. Press, 2, part C, 240-249. Cox, R.A. (1968) ___. Methods ___ Enzymol., Press, -12, 120-129. --Acad. -_.374

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BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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