Glycolytic enzymes and a GLUT-1 glucose transporter in the outer segments of rod and cone photoreceptor cells.
The presence of glycolytic enzymes and a GLUT-1-type glucose transporter in rod and cone outer segments was determined by enzyme activity assays, glucose uptake measurements, Western blotting, and immunofluorescence microscopy. Enzyme 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 (ROS) preparations. Immunofluorescence microscopy of bovine and chicken retina sections labeled with monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and lactate dehydrogenase have confirmed that these enzymes are present in rod and cone outer segments and not simply contaminants from the inner segments or other cells. Rod outer segments were also found to contain glucose transport activity as detected by 3-O-[14C]methylglucose uptake and exchange. The glucose transporter had a Km of 6.3 mM and a Vmax of 0.15 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for net uptake and a Km of 29 mM and a Vmax of 1.06 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for equilibrium exchange. These Km values for net uptake and equilibrium exchange are similar to values obtained for human red blood cells and are characteristic of GLUT-1-type glucose transporter. The transport was inhibited by both cytochalasin B and phloretin. Western blot analysis and immunofluorescence microscopy using type-specific glucose transporter antibodies indicated that both rod and cone outer segment plasma membranes have a GLUT-1 glucose transporter of Mr 45K as found in red blood cells and brain microsomal membranes. Solid-phase radioimmune competitive inhibition studies indicated that rod outer segment plasma membranes contained 15% the number of glucose transporters found in human red blood cell membranes and had an estimated density of 400 glucose transporter per micron2 of plasma membrane. These studies support the view that outer segments can generate energy in the form of ATP and GTP by anaerobic glycolysis to supply at least some of the energy requirements for phototransduction and other metabolic processes.
Hsu
S C
SC
Department of Biochemistry, Faculty of Medicine, University of British Columbia, Vancouver, Canada.
Molday
R S
RS
D019325:3-O-Methylglucose; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D003571:Cytochalasin B / Q000494:pharmacology; D004798:Enzymes / Q000302:isolation & purification / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006019:Glycolysis; D006801:Humans; D007700:Kinetics; D008757:Methylglucosides / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000032:analysis / Q000378:metabolism*; D012160:Retina / Q000166:cytology / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure
D004798:Enzymes; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D019325:3-O-Methylglucose; D003571:Cytochalasin B