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Title: Studies on the metabolism of glutamine
Author: Iqbal, Khalid
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1969
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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 enzyme. 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 discussed. 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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available