Purification and characterisation of phosphates responsible for the dephosphorylation of phospho-opsin in bovine rod outer segments
Purification and characterisation of phosphates responsible for the dephosphorylation of phospho-opsin in bovine rod outer segments
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 2A family. The work described in this thesis provides new evidence for a more definite characterisation of the phospho-opsin phosphatase and shows that the enzyme is almost certainly the Type 2A1 protein phosphatase. This conclusion was based on the use of classical approaches involving stimulatory, inhibitory and Western blot experiments.
The dephosphorylation of phospho-opsin (1 nmol) was found to be stimulated (4-10 fold) by the presence of protamine (250 μg/ml), which is diagnostic of phosphatase 2A activity. 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 the Type 1 catalytic subunit had little activity against these phospho-peptides. Furthermore, if phospho-opsin was pretreated with protamine, 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-pretreated substrate with 1 M NaCl, whence the protamine-dependent stimulation returned to normal levels.
University of Southampton
1993
King, Alastair James
(1993)
Purification and characterisation of phosphates responsible for the dephosphorylation of phospho-opsin in bovine rod outer segments.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
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 2A family. The work described in this thesis provides new evidence for a more definite characterisation of the phospho-opsin phosphatase and shows that the enzyme is almost certainly the Type 2A1 protein phosphatase. This conclusion was based on the use of classical approaches involving stimulatory, inhibitory and Western blot experiments.
The dephosphorylation of phospho-opsin (1 nmol) was found to be stimulated (4-10 fold) by the presence of protamine (250 μg/ml), which is diagnostic of phosphatase 2A activity. 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 the Type 1 catalytic subunit had little activity against these phospho-peptides. Furthermore, if phospho-opsin was pretreated with protamine, 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-pretreated substrate with 1 M NaCl, whence the protamine-dependent stimulation returned to normal levels.
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Published date: 1993
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Local EPrints ID: 462435
URI: http://eprints.soton.ac.uk/id/eprint/462435
PURE UUID: b0b0d0be-7cd2-4d12-9f10-be306363c944
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Date deposited: 04 Jul 2022 19:08
Last modified: 04 Jul 2022 19:08
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Author:
Alastair James King
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