The Enzymes Causing Thyroid Hormone Activation 
p. Reed Larsen, M.D. — Investigator 
Dr. Larsen is also Professor of Medicine at Harvard Medical School and both Director of the Thyroid 
Diagnostic Center and Senior Physician at Brtgham and Women's Hospital. He obtained his bachelor's 
degree in English literature from Princeton University and his M.D. degree from Columbia University 
College of Physicians and Surgeons. He received postdoctoral training at Presbyterian Hospital, New York 
City, and the NIH and served for five years on the faculty at the University of Pittsburgh School of Medi- 
cine. He has been a member of the Harvard Medical School faculty for the past 16 years. Honors for his 
contributions to research on thyroid physiology and disease include the Van Meter- Armour Award and 
the Parke-Davis Distinguished Lectureship of the American Thyroid Association. 
THYROID hormone is critically important in 
the development and regulation of metabolic 
processes. In humans the hormone is especially 
necessary for normal development of the central 
nervous system. Its importance is exemplified by 
the irreversible mental retardation that ensues if 
congenital hypothyroidism is not recognized and 
treated within the first few months of life. In in- 
fancy and childhood, thyroid hormone is abso- 
lutely required for normal growth. 
Our laboratory is interested in learning how 
this pluripotent hormone produces its effects. 
The synthesis of thyroid hormone, thyroxine, is a 
tightly regulated process requiring oxidation of 
iodine and its incorporation into thyroglobulin, a 
thyroxine precursor. This protein is present 
in large quantities in the thyroid gland, and 
under regulation by pituitary thyroid-stimulat- 
ing hormone, thyroxine is released into the 
bloodstream. 
The thyroxine molecule has four iodine atoms 
distributed over its two-ring structure. In this 
form, the hormone is relatively inactive. To be- 
come fully potent, thyroid hormone must be 
deiodinated to form 3,5,3 -triiodothyronine. Dur- 
ing this process, a specific atom of iodine is lost 
from the thyroxine molecule, and it is therefore 
termed T3. 
The process of thyroid hormone activation is 
thus one of great general interest. The enzymes 
that carry out this reaction are termed deiodin- 
ases, since they cause the removal of an iodine 
atom. There are two major deiodinases in the 
body. One is present in the liver and kidney and 
acts on thyroxine to form T3, which then reenters 
the bloodstream. By this route T, is carried to 
other organs in the body, such as the skeletal 
muscle and heart, which do not have the capacity 
to produce it. The enzyme requires an intracellu- 
lar cofactor for its function, which in turn re- 
quires adequate nutrition and the absence of 
stress for its production. In persons who have 
fasted or become ill, conversion of thyroxine to 
T3 by this enzyme is markedly slowed, allowing 
thyroid hormone to fall to conserve energy. 
A major focus of our laboratory has been to iso- 
late and clone this deiodinase. The purification 
of this membrane-bound protein is made difficult 
by its insolubility in water and its inactivation by 
oxygen. To circumvent these difficulties, we 
have used a sensitive measurement for the en- 
zyme's activity. We quantitate the release of radio- 
active iodine from an artificially synthesized ana- 
logue of thyroxine. This analogue is an even 
better substrate for the enzyme's deiodination 
process than is thyroxine itself. 
Having a sensitive assay has allowed us to de- 
vise a strategy for cloning the enzyme without 
purifying it. For this purpose we have adopted 
the technique called expression cloning. In this 
process, one tries to identify sequences of DNA 
coding for a protein by measuring the capacity of 
the DNA to program a cell to make the protein. 
The strategy is labor intensive, but it assures an 
accurate sequence if successful. One common 
approach to expression cloning is to employ the 
egg (oocyte) of the African clawed toad {Xeno- 
pus) as a highly efficient factory for making pro- 
teins from the messenger RNA (mRNA) encoded 
by the DNA of interest. If this system is to be suc- 
cessful, an active form of the protein must be pro- 
duced in the egg after it is injected with the 
mRNA from the tissue that expresses the enzyme. 
In the case of the deiodinase, injection of mRNA 
into Xenopus oocytes led to the appearance of 
the deiodinase in the egg proteins, indicating the 
feasibility of the approach. 
To obtain the optimal starting material for pre- 
paring artificial DNA sequences to code for the 
deiodinase, we analyzed the effect of altering an 
animal's thyroid hormone status on the activity of 
this enzyme. If an animal is made hyperthyroid — 
i.e., given large quantities of thyroid hormone — 
the activity of the enzyme is increased and the 
amount of mRNA coding for the enzyme in liver 
and kidney is increased. Conversely, when an ani- 
mal's capacity to synthesize thyroid hormone is 
blocked, the mRNA levels for the enzyme fall. 
Thus we have chosen hyperthyroid animals to use 
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