title: Large-scale production and purification of the human green cone pigment: characterization of late photo-intermediates.
abstract: We present the first characterization of the late photo-intermediates (Meta I, Meta II and Meta III) of a vertebrate cone pigment in a lipid environment. Marked differences from the same pathway in the rod pigment were observed. The histidine-tagged human green cone pigment was functionally expressed in large-scale suspension cultures in Sf9 insect cells using recombinant baculovirus. The recombinant pigment was extensively purified in a single step by immobilized metal affinity chromatography and displays the expected spectral characteristics. The purified pigment was able to activate the rod G-protein transducin at about half the rate of the rod pigment. Following reconstitution into bovine retina lipid proteoliposomes, identification and analysis of the photo-intermediates Meta I, Meta II and Meta III was accomplished. Similar to the rod pigment, our results indicate the existence of a Meta I-Meta II equilibrium, but we find no evidence for pH dependence. Replacement of native Cl- by NO3- in the anion-binding site of the cone pigment affected the spectral position of the pigment itself and of the Meta I intermediate, but not that of Meta II and Meta III. The decay rate of the 'active' intermediate Meta II did not differ for the Cl- and NO3- state. However, in qualitative agreement with results reported before for chicken cone pigments, the rate of Meta II decay was significantly higher in the human cone pigment than in the rod pigment.
authors.affiliation: Institute of Cellular Signalling, Department of Biochemistry, FMW-160, University of Nijmegen, P.O. Box 9101, NL-6500 HB, The Netherlands.
authors.forename: P M
authors.initials: PM
authors.lastname: Vissers
authors.forename: P H
authors.initials: PH
authors.lastname: Bovee-Geurts
authors.forename: M D
authors.initials: MD
authors.lastname: Portier
authors.forename: C H
authors.initials: CH
authors.lastname: Klaassen
authors.forename: W J
authors.initials: WJ
authors.lastname: Degrip
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D005136:Eye Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008027:Light; D008081:Liposomes; D010777:Photochemistry; D011510:Proteolipids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012160:Retina / Q000502:physiology; D017949:Retinal Cone Photoreceptor Cells / Q000737:chemistry / Q000502:physiology*; D017948:Retinal Rod Photoreceptor Cells / Q000737:chemistry / Q000502:physiology; D017299:Rod Opsins; D016324:Sequence Tagged Sites; D013053:Spectrophotometry; D018411:Spodoptera; D015293:Transducin / Q000378:metabolism; D014162:Transfection
chemical_list: D005136:Eye Proteins; D008081:Liposomes; D011510:Proteolipids; D011994:Recombinant Proteins; D017299:Rod Opsins; C095854:middle-wavelength opsin; C020485:proteoliposomes; D015293:Transducin