title: The phospho-opsin phosphatase from bovine rod outer segments. An insight into the mechanism of stimulation of type-2A protein phosphatase activity by protamine.
abstract: The vertebrate visual transduction system involves a cycle of phosphorylation and dephosphorylation of a transmembranous photoreceptor (rhodopsin). Upon illumination, the activated photoreceptor (metarhodopsin-II) is phosphorylated by a specific kinase on up to seven serine and threonine residues. A dephosphorylation process must then be undertaken to return the photoreceptor to its ground state. Initial work, along with studies using the rabbit skeletal muscle catalytic subunit of protein phosphatase 2A, indicated that the phosphatase responsible was a member of the type-2 family. The work has been further extended and using 1000 bovine retinae, the catalytic subunit and a holoenzyme form of phospho-opsin phosphatase were purified 2100-fold and 550-fold respectively. The stimulation of the activities of both these fractions with protamine sulphate and the inhibition by okadaic acid are consistent with the fact that these phosphatases belong to the type-2A family. Western blotting using a variety of specific antibodies established that the catalytic subunit (36 kDa, C subunit) was indeed of type 2A, while the holoenzyme was a heterotrimer comprising the preceding catalytic subunit complexed to two other polypeptides of 55 kDa (B subunit) and 65 kDa (A subunit), both of which were of alpha subtype; phospho-opsin phosphatase may thus be described as a trimeric enzyme containing the ABC subunits of type-2A protein phosphatase, i.e. PP2A1. The dephosphorylation of phospho-opsin by both fractions was found to be stimulated (4-8-fold) by the presence of protamine sulphate (250 micrograms/ml; 50 microM). However, when phospho-peptides corresponding to the C-terminal region of opsin were used, these were maximally dephosphorylated without requiring the presence of protamine; at equivalent concentrations of substrates the phospho-peptides were dephosphorylated (in the absence of protamine) at rates which were approximately equal to those obtained with phospho-opsin (in the presence of protamine). It was shown that type-1 phosphatases had little activity against these phospho-peptides. Furthermore, if phospho-opsin was treated with protamine, the activity of the phosphatase assumed an elevated level and was not significantly stimulated by the addition of exogenous protamine. This effect could be reversed by washing the protamine-treated substrate with 1 M NaCl, whence the protamine-dependent stimulation returned to normal levels. To this end, studies revealed that protamine was binding to the particulate substrate in a ratio of protamine/opsin of 0.7:1. The cumulative finding may be rationalised by suggesting that the effect of protamine is a substrate-directed phenomenon and a hypothetical mechanism for this effect is considered.
authors.affiliation: Department of Biochemistry, University of Southampton, England.
authors.forename: A J
authors.initials: AJ
authors.lastname: King
authors.forename: N
authors.initials: N
authors.lastname: Andjelkovic
authors.forename: B A
authors.initials: BA
authors.lastname: Hemmings
authors.forename: M
authors.initials: M
authors.lastname: Akhtar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011479:Protamines / Q000494:pharmacology*; D054648:Protein Phosphatase 2; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D046911:Macromolecular Substances; D010455:Peptides; D011479:Protamines; D012243:Rhodopsin; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2