| Summary: | Thesis (M.Sc.)--Memorial University of Newfoundland, 2009. Biochemistry Includes bibliographical references (leaves 114-133) The goal of this work was to localize one of the enzymes involved in proline oxidation, Δ1 -pyrroline-5-carboxylate dehydrogenase (P5CDh) and to gain an understanding of the factors affecting proline catabolism in rat liver. In this regard we performed a systematic subcellular localization for P5CDh and studied proline catabolism in response to dietary protein and exogenous glucagon. Our results indicate that P5CDh is located solely in mitochondria in rat liver. With respect to factors affecting proline catabolism we observed that rats fed a diet containing excess protein (45% casein) display a 1.5 fold increase in activity of P5CDh and proline oxidase (PO), and a 40% increase in flux through the pathway resulting in complete oxidation of proline in isolated mitochondria. We also observed that rats administered exogenous glucagon exhibit a 2 fold increase in PO activity and a 1.5 fold increase in P5CDh activity, and a 2 fold increase in flux through the pathway resulting in complete oxidation of proline in isolated mitochondria. 14 CO2 production from 14 C-proline in the isolated nonrecirculating perfused rat liver was also elevated 2 fold in the glucagon treated rat. We also studied the transport of proline into isolated hepatocytes and observed a 1.5 fold increase in the transport of proline in rats given exogenous glucagon. -- Conclusions. Subcellular localization: (a) The spectrophotometric assay is valid and provides a quick, easy method for assays of P5CDh (b) P5CDh is located strictly in the mitochondrial matrix. High protein diet increases: (a) flux through the proline catabolic pathway in mitochondria resulting in the production of CO 2 (b) activity of PO, P5CDh, and ornithine aminotransferase (OAT) in rat liver mitochondria Glucagon increases: (a) proline transfer from plasma to hepatocytes (b) oxidation of proline by perfused liver (c) flux through the proline catabolic pathway resulting in the production of CO 2 (d) activity of PO, P5CDH and glutamate dehydrogenase (GDH) in rat liver mitochondria
|
|---|