A soluble form of bovine rod photoreceptor phosphodiesterase has a novel 15-kDa subunit.
A substantial fraction (20-30%) of the bovine rod outer segment phosphodiesterase (PDE) activity is not associated with outer segment membranes prepared with buffers of moderate ionic strength; this PDE activity appears to represent a distinct, soluble isozyme. Although this PDE isozyme can be demonstrated to be present in sealed rod outer segments, it is discarded from most standard rod outer segment preparations. A method was developed that allowed the rapid purification of the soluble rod PDE by 2600-fold, to apparent homogeneity, using a monoclonal antibody column (ROS-1a). The soluble rod PDE isozyme has a novel Mr = 15,000 subunit (delta) in addition to subunits of Mr = 88,000 (alpha sol), 84,000 (beta sol), and 11,000 (gamma sol). The delta subunit comigrates with and may be identical to the cone PDE 15-kDa subunit. The small subunits of the soluble rod PDE and the membrane-associated rod PDE were isolated by reverse-phase chromatography. The gamma sol subunit was a potent inhibitor of trypsin-activated rod PDE, inhibiting 50% of 1 pM PDE activity at a concentration of 11 pM. This concentration was similar to that observed for the gamma subunit of the membrane-associated rod PDE. The purified delta subunit did not appear to affect PDE activity; this subunit was, however, unusually difficult to keep in solution. All of the kinetic and physical properties of the soluble rod PDE tested thus far are similar to those of the membrane-associated form, except for the presence of the delta subunit, suggesting that this unique subunit could mediate the solubility of the soluble rod PDE and the cone PDE in the intact photoreceptor.
Gillespie
P G
PG
Department of Pharmacology, University of Washington, Seattle 98195.
Prusti
R K
RK
Apel
E D
ED
Beavo
J A
JA
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D010786:Photoreceptor Cells / Q000201:enzymology*; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D015293:Transducin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D010727:Phosphoric Diester Hydrolases; D015293:Transducin