BIOCHEMICAL

Vol. 79, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CONVERSION OF THYROXINE TO 3,3',5'-TRIIODOTHYRONINE (REVERSE-T3)

Ralph

BY A SOLUBLE ENZYME SYSTEM OF RAT LIVER

R. Cavalieri, Frances

Veterans

Received

Laurence

McMahon,

A. Gavin,

and Margaret

Franc0

Bui,

Hammond

Nuclear Medicine Service and Department of Medicine, Administration Hospital and University of California, San Francisco, California 94121

October

7,1977

The reaction by which thyroxine (T4) undergoes monodeiodination in the nonphenolic ring to form 3,3' ,5'-triiodothyronine (reverse-T3) has been studied in rat liver. In whole homogenate the conversion of T4 to rT3 is obscured by rapid degradation of the product, whereas in cytosol accumulation of rT3 is linear for 60 min of incubation. In the cytosol system, the rate of rT3 formation is maximal at pH 8.2, is thiol-dependent, is inactivated by heat, but is not inhibited by anaerobiosis or absence of light. Propylthiouracil is a potent inhibitor of the reaction. The higher pH-optimum of the rT3-forming system compared to that of T4-to-T3 conversion indicates that the former reaction is mediated by an enzyme which is distinct from that controlling 3,5,3'-triiodothyronine (T3) formation.

INTRODUCTION The formation roxine

(T4)

a major

has not

by monodeiodination

pathway

in perfused

rTjdegrading scribes

(4)

been convincingly that

due to rapid

the

the nonphenolic (l-3).

This

and in cultured

failure

to

of rT3

rT3-forming

properties

(reverse-T3,

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

has been

preparations.

system which

liver

thy-

(7).

converts

is

demonstrated cells

(5) but

A recent

T4 5-deiodination

in rat

from

(5-deiodination)

We have succeeded

activities

of a soluble

rT3)

monkey hepatocarcinoma

demonstrate (6).

ring reaction

shown in broken-cell

degradation

from the

in

of T4 metabolism

dog liver

has suggested is

of 3,3',5'-triiodothyronine

report

in homogenates in separating This

report

the de-

T4 to rT3 and pre-

897 ISSN

0006-291

X

Vol. 79, No. 3, 1977

sents

evidence

dinase

which

that forms

BIOCHEMICAL

it

is

enzymic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in nature

3,5,3'-triiodothyronine

MATERIALS

and distinct

from

the

T4 5'-deio-

(T3).

AND METHODS

L-T4 and dithioerythritol (DTE) were obtained from Sigma Chemical Co., St. Louis, Reverse-T generously provided by Dr. Eugene C. ui'-$:,,3 and 125:-ys 3, each labeled Jorgensen. in the phenolic ring positions at specific radioactivities of 500-900 UCi/ug, were purchased from Abbott Laboratories, North Chicago, U.S.A. Goat anti-rabbit gamma globulin serum was obtained from Antibodies, Inc., Davis, CA, U.S.A. Preparation of liver homogenate and cytosol. Sprague-Dawley male rats, weighing 180-220 gm, fed Purina Lab Chow until time of sacrifice, were killed by decapitation and bled. The liver was chilled on ice, weighed and homogenized in 3 volumes ice-cold buffer (0.25 M sucrose - 0.05 M Tris-HCl, pH 7.4) using a Polytron homogenizer for 2-3 seconds. The crude homogenate was centrifuged at 2,000 x g for 10 minutes and the pellet discarded. Part of the supernatant from the initial low-speed spin was used for incubation studies (homogenate) and part for preparation of post-microsomal supernatant (cytosol) by a final centrifugation at 100,000 x g for 1 hour. In most experiments, homogenate and cytosol were dialyzed for 16 hours against two changes of 30 volumes each of either 0.05 M Tris-HCl, pH 7.0-8.5, or 0.1 M sodium acetatebarbital-HCl buffer over a pH range from.6.5 to 8.4. Each of the buffers contained DTE, 5 mM. All operations to this stage were performed at 4'. Incubation procedure. To 1 ml of homogenate or cytosol was added L-T4 The mixture was incubated at 37' in (1.5 up) in 10 ul of the same buffer. glass tubes open to the air. At intervals up to 2 hours, 100 ul samples were removed and added to 0.9 ml ice-cold normal human serum which had been treated with activated charcoal (8) to remove iodothyronine and which contained 1 mM propylthiouracil (PTU). These mixtures of serum and incubation media ("serum extracts") were centrifuged when necessary to remove particulates derived from homogenate and then analyzed for products of T4 deiodination. Control experiments consisted of (a) incubation of T4 without tissue fractions, and (b) addition of T4 to the tissue fraction without incubation (time-zero tubes). Degradation of rT3 was studied by adding rT3 (20 ng) to 1 ml homogenate or cytosol (in Tris-HCl, pH 7.2 and 8.2) and incubating as described above. Measurements of rT3 and T3 in "serum extracts" Analytical methods. were made by specific radioinsnunoassays previously described (3). In each assay the sensitivity was 30 pg per ml serum (300 pg per ml of incubation mixture). The presence of liver fractions in the serum, in the proportions used, had no significant effect either on the recovery of rT3 and of T3 or on the sensitivity of these assays. There was negligible cross-reactivity in the rT3 assay by T4, T3, the acetic acid derivatives of T4 and T3, and by glucuronide and sulfa-conjugates of rT3. In control incubations (tubes containing T4 in tissue-free buffer and unincubated,time-zero tubes) the rT3 content of the incubation mixture was less than 1 ng per ml and was subtracted from the measured rT3 values in the experimental tubes. method

The protein concentration of Lowry et al (9).

of incubation

898

mixtures

was determined

by the

BIOCHEMICAL

Vol. 79, No. 3, 1977

0

30

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

90

60

30

INCUBATION

TIME ImInI

FIGURE 1. THE GENERATION (A) AND DEGRADATION (B) OF REVERSE-T3 IN LIVER A. Incubation HOMOGENATE (OPEN CIRCLES) AND CYTOSOL (CLOSED CIRCLES). mixtures contained 2 pM T4, 5 mM dithioerythritol (DTE), in 0.05 M Tris-HCl, The rT3 content of unincubated tubes (time-zero) was less than pH 8.2. B. Reverse-T3 (20 rig/ml) 0.8 rig/ml and has been subtracted from the results. was added at time zero to homogenate or cytosol in 0.05 M Tris-HCl, pH 8.2, containing 5 mM DTE. In both A and B, each point is the average of duplicate tubes, which varied by less than 10 per cent.

RESULTS AND DISCUSSION During only

incubation

during

cytosol

the initial

showed

difference

of liver

a linear

in the

was explained

lo-20 rate

than

mined

rT3 degradation

at pH 8.2. liver

in cytosol

Preliminary

The properties ed. protein a uM.

The rate

of a 20-fold (Fig.

The activity

remained for

60 minutes

rate

In separate was only

have localized

slightly

rT3 accumulated

constant.

between

faster

1B).

in cytosol

the

In contrast, (Fig.

1A).

two preparations

of rT3 degradation experiments faster

This

it

in

was deter-

at pH 7.2 than

the rTj-degrading

activity

of

fraction. of the cytosol

of conversion

concentration

and then

T4 at pH 8.2,

of rT3 accumulation

studies

to the microsomal

with

of rT 3 generation

finding

homogenate that

minutes

time-course

by the

homogenate

of T4 5-deiodinase

of T4 to rT3 was directly

in the range of cytosol

system

did

0.2

to 10 mg/ml,

not vary

899

significantly

were

proportional

investigatto cytosol

at a Tk concentration when homogenization

of

Vol. 79, No. 3, 1977

Table

1. Effects on conversion

Condition A.

B.

C.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of possible cofactors of T4 to rT3 by liver

or Agent

and inhibitors cytosol.

Reverse-T

Basal System* plus 8-mercaptoethanol, ,t dithioerythritol, I, Hg C12' Complete System** Nitrogen Atmosphere Dark Heat (60°/30 min) EDTA Complete System** Propylthiouracil

5 50 500 100

Methimazole

5mM 5mM 5mM

1.10 3.15 5.01 0.07

5mM

6.56 8.33 6.61 0.22 5.90

t

(ng/ml/hr)

5.51 4.85 2.80 0.05 5.46

I-IM uM uM uM

3,5,3'-triiodothyronine

3 Formed

5.60 5.96

1llM 5W

* Basal System: Cytosol in 0.05 M Tris pH 8.2, 2 @I T4. ** Complete System: Basal System plus dithioerthyritol, 5 mM. t Results shown are averages of duplicate experiments which varied by less than 10 per cent. Cytosol protein concentration in these incubation mixtures ranged from 6 to 8 mglml.

of liver

was performed

action

rate

at different

revealed

rT3 formation

rates

Michaelis-Menten

against

or by different

kinetics;

T4 concentration

a double

was linear,

with

methods. reciprocal an apparent

The replot

of

Km of

5 x 1O-6 M. Dialysis effect -20°,

of cytosol

on ratesof caused

for

gen or light,

thiol

loss

Other

that

less

More

is

than

prolonged

of activity.

properties

compounds

indicating

periods

formation.

progressive

one week at -2OO. quirement

rT3

for

Fifty

of the system evident.

a non-enzymic,

900

Activity

24 hours

had no significant

storage,

however,

per cent

loss

occurred

are given

in Table

was not

dependent

light-dependent

at 4' or

deiodination

after 1.

A re-

on oxy(10)

BIOCHEMICAL

Vol. 79, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMWNICATIONS

5

FIGURE 2. EFFECT ON pH ON GENERATION OF T3 FROM T4 BY LIVER HOMOGENATE (BROKEN LINE) AND OF RT3 FROM T4 BY CYTOSOL (CONTINUOUS LINE). Incubation mixtures contained 2 nM T4 and 5 mM DTE in 0.1 M sodium Each point is the average of duplicates, which acetate-barbital-HCl. varied by less than 10 per cent.

is not led

involved.

The system

to irreversible

inhibited

inactivation.

The finding

centration

tested

petitively

inhibits

that

is of interest

The pH-dependency 2 and compared

Almost

also

present

in

than

in homogenate.

ases by subcellular most of the also

activity

cytosol

of Chopra's

approximately

fractionation.

Isolated

but

equal

at its

T4-to-T3

we have been unable

T4 to rT3,

liver

of homogenate at a low

901

rate

(11).

is

shown in

in homogenate.

buffer.

To date,

activity

rT3 com-

homogenates

reaction

is nearly

concentration.

in this in the con-

that

in cytosol

of the T4-to-T3 in Tris

not methimazole

conversion

T4 in liver

to 60'

degradation

report

activity

of cytosol

(pH optimum

rT3

T4-to-rT3

of T3 from

of heating

(PTU) but

when each was assayed

5'-deiodinase

to convert

in view

Were obtained

of protein

periods

increase

inhibit

the p&curve

of homogenate

in terms

not

of the 5-deiodinase

with

The 5-deiodinase

pressed

not

formation

identicalresults

ase activity

(PTU did

T3 did

the

short

Propylthiouracil

T4 to rT3 formation.

system.)

Fig.

is heat-labile;

(13).

to the 5'-deiodin-

optimum

converting 7.0)

but

activity

at a lower

to separate microsomes, (12 ),

pH and ex-

the

level

two deiodin-

which

have been

was

contain

observed

Vol. 79, No. 3, 1977

The major is present and (b)

findings

the pH optimum In several

T4 5'-deiodinase and inhibited The difference that

fraction of the

respects, activity

by PTU but

alternative

study

of liver

however,

not

is the

is

(a)

T4 5-deiodinase

higher

thiol-sensitive

demonstrated

(11,14

of T4 deiodination

that

are

in nature,

of the

similar.

(12 ),

heat

study,

5)-deioThus,

labile

) or anaerobiosis

in the present

activity

to be enzymic

than

two enzymes

by methimazole

pathways

are

and appears

5-deiodinase

in liver

in pH optima, the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the present

in the soluble

dinase.

dence

BIOCHEMICAL

(15 ).

provides

are mediated

(11 ),

evi-

by differ-

ent enzymes. Acknowledgements This work was supported by the Medical Research Service of the Veterans Administration and by the Edward Rice and the Springer Funds of the University of California, San Francisco. Dr. Bui was supported by a Fellowship from the Fondazione Anna Villa Rusconi. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15.

Chopra, I. J., Sack, J., and Fisher, D.A. (1975) Endocrinology, 97, 10801088. Chopra, I. J. (1976) J. Clin. Invest. 58, 32-40. Gavin, L., Castle, J., McMahon, F., Martin, P., Hammond, M., and Cavalieri, R. R. (1977) J. Clin. Endocrinol. Metab. 44, 733-742. Flock, E. V., Bollman, J. L., Grindlay, J. H., and Stobie, G. H. (1961) Endocrinology 69, 626-637. Sorimachi, K., and Robbins, J. (1977) J. Biol. Chem. 252, 4458-4463. Hufner, M., Grussendorf, M., and Ntokalou, M. (1977) Clin. Chim. Acta 78, 251-259. Cavalieri, R. R., Gavin, L. A., McMahan, F., and Hammond, M. (1977) Clin. Res. 25, 462A. Mitsuma, T., Colucci, J., Shenkman, L., and Hollander, C. S. (1972) Biochim. Biophys. Res. Commun. 46, 2107-2113. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1955) J. Biol. Chem. 193, 265-275. J. E. (1966) J. Biol. Chem. 241, 1636-1643. Reinwein, D., and Rall, Chopra, I. J. (1977) Endocrinology, 101, 453-463. Visser, T. J., van der Does-Tobe, I., Doctor, R., and Hennemann, G. (1976) Biochem. J. 157, 479-482. Hoffken, B., Kodding, R., and Hesch, R-D. (1977) Clin. Chim. Acta 78, 261-266. I., Doctor, R., and Hennemann, G. (1975) Visser, T. J., van der Does-Tobe, Biochem. J. 150, 489-493. Green, W. L., In Robbins, J., and Braverman, L. E., eds. (1976) Thyroid Research, pp 239-243, American Elsevier, New York.

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