Abstract
A major 38-kDa protein associated with bovine rod outer segment plasma membranes, but not disk membranes, has been identified as glyceraldehyde-3-phosphate dehydrogenase on the basis of its N-terminal sequence and specific enzyme activity. This enzyme was extracted from lysed rod outer segments or isolated rod outer segment plasma membrane with 0.15 M NaCl and purified to homogeneity by affinity chromatography on a NAD(+)-agarose column. A specific activity of 90-100 units/mg of protein is within the range of activity obtained for glyceraldehyde-3-phosphate dehydrogenase isolated from other mammalian cells. Enzyme activity measurements indicate that this enzyme makes up approximately 2% of the total rod outer segment protein and over 11% of the plasma membrane protein. Protease digestion and binding studies on purified rod outer segment plasma and disk membranes suggest that glyceraldehyde-3-phosphate dehydrogenase reversibly interacts with a protease-sensitive plasma membrane-specific protein of rod outer segments. The finding that glyceraldehyde-3-phosphate dehydrogenase is present in large quantities in rod outer segments suggests that at least some of the energy required for the synthesis of ATP and GTP for phototransduction and other processes of the outer segment is derived from glycolysis which takes place within this organelle.
Highlights
A major 38-kDa protein associated with bovine rod outer segment plasma membranes, but not disk membranes, has been identified as glyceraldehyde-3-phosphate dehydrogenase on the basis of its N-terminal sequence and specific enzyme activity
Phosphate dehydrogenase is present in large quantities in rod outer segments suggests that at least some of the energy required for the synthesis of ATP and GTP for phototransduction and other processes of the outer segment is derived from glycolysis which takes place within this organelle
Partial N-terminal sequence analysis and specific enzyme activity measurements have confirmed that the major 38kDa protein associated with purified rod outer segment (ROS) plasma membrane is GSPD
Summary
Materials-Fresh bovine eyes and blood were obtained from Intercontinental Packers (Vancouver, British Columbia). EDTA, 5 mM Na2HP0.+ and 10 mM Tris acetate at pH 7.4, G3PD was eluted with the NaCl extraction buffer containing 10 mM NAD+. The ROS and purified ROS plasma membranes after extraction were further washed with the NaCl extraction buffer by centrifugation until no significant GBPD activity (less than 1% of the total GBPD activity associated with the membranes before NaCl extraction) was det.ected The disk and plasma membranes were separated on a 25-50% (w/v) sucrose gradient in 20 mM Tris acetate, LH 7.4, by centrifugation at 45,000 mm for 20 min in a TLS-55 rotor (Beckman Instruments). The digested ROS membranes were washed three times in 0.2 ml of buffer A before incubation for 30 min with 40 ~1 of 0.1 mg/ml NAD’ affinity-purified. The extent of GBPD binding to the membranes was monitored by measuring G3PD activity in both supernatants and membrane pellets following centrifugation
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