UBC Theses and Dissertations
Glucose metabolism in the outer segments of photoreceptor cells : its involvement in the phototransduction process Hsu, Shu-Chan
The existence and function of glucose metabolism in the outer segments of photoreceptor cells were investigated using biochemical and immunocytochemical approaches. The presence of glycolytic enzymes in photoreceptor outer segments was detected by enzyme activity assays, Western blotting, and immunofluorescence microscopy. Activities of six glycolytic enzymes including hexokinase, phosphofructokinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and lactate dehydrogenase, were found to be present in purified rod outer segment preparations in quantities similar to that found in human red blood cells. Immunofluorescence microscopy of bovine and chicken retina sections labeled with monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and lactate dehydrogenasehave confirmed that these enzymes are present in rod and cone outer segments and are not simply contaminants from the inner segments or other retinal cells. One of these glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase, was found to make up approximately 2 % of the total rod outer segment protein and over 11 % of the plasma membrane protein. It has been purified by affinity chromatography on a NAD +-agarose column and shown to associate reversibly and electrostatically with a specific protein in the rod outer segment plasma membrane. The rod outer segment plasma membrane was also found to contain aGLUT-1 type glucose transporter of Mr 45K as detected by 3-O-methylglucoseuptake and exchange studies and Western blot analyses using type-specific glucose transporter antibodies. Solid-phase radio immune competitive inhibition studies indicated that the rod outer segment plasma membrane contained 15 % the number of glucose transporters found in human red blood cell membranes and had an estimated density of 400 glucose transporter per um2 of rod outer segment plasma membrane. Immunofluorescence microscopy indicated that both rod and cone outer segments have a GLUT-1 type glucose transporter. The involvement of glucose metabolism in supporting the phototransduction process was studied by measuring the activities of glycolytic and hexosemonophosphate pathways in isolated rod outer segments by spectrophotometric and radiometric enzyme assays. Glucose metabolism in rod outer segments was found to produce both ATP and NADPH required for the maintenance of phototransduction. ATP produced by glycolysis at a rate of 35-44 nmol/min/mg rod outer segment protein can potentially support cGMP regeneration, one of the most energy-consuming processes in photo transduction, under dark but not light conditions. NADPH produced by the hexose monophosphate pathway at a maximalrate of 40 nmol/min/mg ROS protein is sufficient to support the reduction of all-trans-retinal to all-trans-retinol in rod outer segments occurring at a rate of 1.2nmol/min/mg ROS protein. A high hexose monophosphate pathway capacity suggests that the pathway may also be involved in supporting the glutathione redox cycle to protect rod outer segments from oxidative stress. In summary, photoreceptor outer segments contain enzymes involved in glucose metabolism and a GLUT-1 type glucose transporter for glucose supply. Glucose metabolism in this organelle can potentially function to maintain a constant tROS cGMP concentration in dark, to buffer against sudden changes in cytoplasmic ATP concentration upon visual excitation and to supply NADPH required for visual recovery.
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