Glutamine has been found by several workers to be present
as the free amino acid in all animal tissues but little is known
of its functions and the way it accumulates in the cells. The
existence of the enzyme, glutamine synthetase which catalyses the
synthesis of glutamine from glutamate and ammonia in the presence
of ATP and a divalent cation, has been well established in brain,
liver and kidney, but it is much less certain whether it occurs
in skeletal and cardiac muscle.
Hormones play an important role in the regulation of the
activity of several enzymes in various tissues. Although the
growth hormone is known to stimulate protein synthesis, very few
studies have been made on its effect in the regulation of
glutamine synthesis, although glutamine may be thought of as a
reservoir and transport form of amino -N, in tissues, and is
essential for nucleic acid synthesis.
The present studies were carried out as part of an
investigation into the origin and function of glutamine in
skeletal and cardiac muscles and to explore the effect of growth
hormone, if any, on its synthesis in these tissues. Since
glutamine synthetase activity is already known to exist in kidney,
this tissue was also studied in parallel with skeletal and cardiac
muscle, so as to be able both to check the efficiency of the
methods employed and to see whether the enzyme if it existed in
the skeletal and cardiac muscles would be similar to the kidney
Glutamine synthetase can be assayed by making use of
the fact that hydroxylamine will serve as second substrate in
place of ammonia. The product of the reaction is y-glutamylhydroxamic
acid, which can be quantitatively determined through
the colour of the complex which it forms with ferric ions.
In order to make sure that the colour produced was due to
the enzyme activity and to obtain a preparation giving maximum
activity, it was necessary to study extraction methods before
determining optical density. This was achieved by employing a
dialysed high -speed supernatant, the activity of which was found
to be about 3 - 4 times greater than that found in the homogenate.
On average 183.2 ± 65.1 units glutamine synthetase
activity/g. wet tissue was found in kidney by the hydroxamate
method using dialysed high-speed supernatants; 19.1 ± 10.3
units/g. were found in skeletal muscle and 2'5 ± 0'8 in heart.
In the case of cardiac muscle, no activity could be detected in
some of the extracts by the hydroxamate method, and the activity
could also not be measured by the NADH oxidation method (see
below). These results have been compared with those of others
and the possible reasons for the differences in results have been
Inorganic phosphate was found to be an inhibitor
of the enzyme, both in kidney and muscle extracts. In order to
study the kinetics of the phosphate inhibition, a method for
freeing the extracts from most of the interfering ATP -ase, and a
more sensitive assay method, in which ADP does not accumulate,
iwere developed. The enzyme was separated from ATP -ase by
precipitation with l.5 M and 1.8 M ammonium sulphate for muscle
and kidney respectively. The assay method, measures glutamine
synthetase activity by measuring the rate of ADP production.
This is coupled to oxidation of NADH by pyruvate and lactic
dehydrogenase. Adenosine diphosphate is re- phosphorylated
with phosphoenol -pyruvate and pyruvate kinase; adenylate kinase
was also added to remove traces of adenosine monophosphate which
interfered with the assay.
Inorganic phosphate was found to be a competitive
inhibitor of ATP for the muscle enzyme and a noncompetitive
inhibitor for the kidney enzyme. Possible reaction mechanisms
which would account for these findings have been discussed.
Several differences in the properties of the enzymes in
muscle and kidney extracts were found (including this difference
in inhibition by phosphate) and it is suggested that the enzyme
in muscle is an isoenzyme of that found in kidney.
Bovine growth hormone both in vivo as well as in vitro
was found to have no significant effect on the synthesis of
glutamine in any of the tissues studied. The type of inhibition
in the two tissue enzymes was also not affected.