The Use of lc Labeled Tyrosine to Explore the Synthesis of Thyroid Hormone
The 131r studies dealing with the synthesis of iodinated
led to additional work attempting to use a second isotope.
Same
unds in
Fabbits
J4c labeleli tyrosine
with the label in the carboxyl position was incubated with thyroid slices ih a nutrient
solution to study the sequence of the synthesis of thyroid hormone. As expected, the
l'tc labeled tyrosine was recovered in its original form in the thyroid tisaghe slices
but none of the l4c was found in the mono- or diiodotyrosine fractions. It[was
surprising then that some of the 1c appeared in the thyroxine and triiodothyronine
fractions recovered from these slices.
Because of the longstanding and
unipersally
attractive theory that the synthesis of thyroxine is accomplished by the combination
of two icdotyrosine molecules, this apparent by-passing of mono- and diicdotyrosine
to form thyroxine was unexpected.
See attached Figure 3.
It should be poifhted out that
the yields of l4c in the thyroxine and triiodothyronine fractions were very
smll but
Significant. This was reported in the discussion of the paper by Gross andj Pitt-Rivers
at the Laurentian Hormone Conference in September, 1953.
Dobyns, B.M. In discussion of a paper by J. Gross and R. Pitt-Rivers.
bid Hormone
Physiology and Biochemistry. Triiodothyronine in Relation to Thyroid Physiblogy.
Recent Progress in Hormone Research 10: 119-121 Academic Press, 1954.
Although the experiment demonstrating the phenomenon of tyrosine by-pabsing of the
mono-— and diicdotyrosine to form thyroxine had been successfully repeated before the
observation was reported, it was looked upon by others with same skepticism}
The
experiment has since been performed a total of 7 times confirming the synthbsis of a
small amount of thyroxine from tyrosine without forming mono- or diiocdctyropine fram
the labeled tyrosine.
These incubation experiments have been performed usilhg i3ly
to label iodide and l4c +6 label the tyrosine in the same incubation experiments.
‘The
two isotopes were counted in the various fractionated samples and their distribution
determined by first counting the 15ly and then permitting
and then counting the l4¢,
‘see attached Fi
4.
The
all of this isotope to decay
131I iodine was_abupdant in the
mono- and diiodotyrosine but there was no 1%¢ in these compournis. The l4c
sine
again appeared in the thyroxine and triiodothyronine fractions in small but] significant
amounts .
Paper Chromatography of Iodinated Campounds
Although it was possible to completely separate the iodinated compoundg by the
starch column method developed and described, it was so laborious that it rpquired
almost a week to run a single separation and count all of the samples from ft. It
was therefore_impossible to use this method of separation for a series of simples.
containing 131; and collected over a period of hours or a few days because
2 small.
amount of isotope in iodinated campounds decayed before it could be measurefi accurately.
During and following the development of the starch colum chromatography, paper
chromatography gained increasing popularity. Our method of paper chromatogrep
was developed as a quantitative method using a butanol-dioxane—ammonia solvpnt
descending fashion.
in a2
This we developed for our own use from one of the two Bteps of the
two dimensional paper chromatography originally described by LeBlond and Grpss.
general pattern became very popular and was widely used.
The paper method using butanol as a principal ingredient of the solvenk
This
in an
acid medium was extensively studied in our laboratory because it was rapid Bnd seemed
reasonably quantitative. Much time was spent studying this method as it wak being
used by others.
In the light of our experience with the acid medium used originally