Development, distribution and properties of purine phosphoribosyltransferases in mammals
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The object of the work described in this thesis has been to determine the tissue distribution, development, and properties of the purine phosphoribosyltransferases (PRTases), especially in man but also in some other animals.A major part of the work was directed to the study of hypoxanthine guanine phosphoribosyl- transferase EC 220.127.116.11 (HGPRTase) in man in view of its importance in human metabolism as shown by pathological abnormalities resulting from its deficiency.As existing assay methods would tend to give falsely low results, an assay method was developed for the purine PRTases from tissues, which incorporated thymidine triphosphate as a selective inhibitor of 5'- nucleotidase EC 18.104.22.168, to prevent the hydrolysis of the PRTase reaction products.The specific activity of HGPRTase and adenine phosphoribosyltransferase EC 22.214.171.124 (APRTase) was determined in tissues from foetuses, children and adults. In the central nervous system and, unexpectedly, in the testis, HGPRTase activity increased during childhood to reach adult levels which were several times higher than those found in the foetus and in other tissues in the adult. The activity of HGPRTase in the other tissues studied remained constant or increased only slightly during development. In central nervous system, APRTase activity decreased during the period between foetal and neonatal life, while liver APRTase activity increased during childhood. In the adult, liver activity was about three times that found in foetal liver.Since it was difficult to obtain human tissues between the ages of 2 years and 60 years, a study was carried out on the developmental changes in rat tissue HGPRTase activity. Complex changes in activity were detected, being most marked in testis, liver, and cerebral cortex. However, some evidence was obtained for a rise in testicular HGPRTase occurring during sexual maturation. Low activities of HGPRTase were detected in the testis, liver, and cerebral cortex of rats at 16 months of age which, for the P.V.G. strain of rats used, was very old age.The electrophoretic properties of the purine PRTases were investigated using a newly developed detection technique and four isoenzyme bands were detected for both HGPRTase and APRTase.HGPRTase from several human tissues was studied by determining the thermal denaturation rate constants and the results suggested the possible presence of a tissue specific testicular HGPRTase.Autoradiographic localisation of testicular HGPRTase showed that the majority of the activity was in the basal layers of the epithelium of the seminiferous tubule.As hypoxanthine is the major purine released by erythrocytes, and the uptake of hypoxanthine into erythrocytes is dependent on HGPRTase, the possible effect of differing tissue enzyme activities on the uptake of purines by tissues from erythrocytes, was investigated in the rabbit using prelabelled rabbit erythrocytes. The results demonstrated that erythrocyte purines were transported to tissues and incorporated into cellular nucleic acids. The uptake of purines by tissues from the erythrocytes did not bear a simple direct relationship to the tissue HGPRTase activity.In conclusion, the developmental changes detected in HGPRTase activity in the central nervous system may provide an explanation for the delayed onset of the neurological symptoms found in children with the Lesch -Nyhan syndrome and would appear to be broadly related to the development of function of some parts of the central nervous system. The unexpectedly high HGPRTase activity located in the seminiferous tubule of the testis, and the developmental changes in activity found to occur in this tissue, suggest a role for HGPRTase in spermatogenesis which may be important.