Affinity purification and characterization of protein gene product 9.5 (PGP9.5) from retina

Protein gene product 9.5 (PGP9.5) is a cytosolic protein that is highly expressed in vertebrate neurons, which is now included in the ubiquitin C-terminal hydrolase subclass (UCH) on the basis of primary-structure homology and hydrolytic activity on the synthetic substrate ubiquitin ethyl ester (UbO...

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Bibliographic Details
Published in:Biochemical journal 1996-09, Vol.318 ( Pt 2) (2), p.711-716
Main Authors: Piccinini, M, Merighi, A, Bruno, R, Cascio, P, Curto, M, Mioletti, S, Ceruti, C, Rinaudo, M T
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Antibodies
Blotting, Western
Cattle
Chromatography, Affinity
Chromatography, High Pressure Liquid
Chromatography, Ion Exchange
Humans
Hydrogen-Ion Concentration
Kinetics
Molecular Sequence Data
Molecular Weight
Nerve Tissue Proteins - isolation & purification
Nerve Tissue Proteins - metabolism
Peptide Fragments - chemistry
Peptide Fragments - isolation & purification
Retina - metabolism
Sequence Homology, Amino Acid
Thiolester Hydrolases - chemistry
Thiolester Hydrolases - isolation & purification
Thiolester Hydrolases - metabolism
Trypsin
Ubiquitin Thiolesterase
Ubiquitins
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Description
Summary:Protein gene product 9.5 (PGP9.5) is a cytosolic protein that is highly expressed in vertebrate neurons, which is now included in the ubiquitin C-terminal hydrolase subclass (UCH) on the basis of primary-structure homology and hydrolytic activity on the synthetic substrate ubiquitin ethyl ester (UbOEt). Some UCHs show affinity for immobilized ubiquitin, a property exploited to purify them. In this study we show that this property can also be applied to PGP9.5, since a protein has been purified to homogeneity from bovine retina by affinity chromatography on a ubiquitin-Sepharose column that can be identified with: (a) PGP9.5 with respect to molecular mass, primary structure and immunological reactivity; (b) the known UCHs with respect to some catalytic properties, such as hydrolytic activity on UbOEt, (which also characterizes PGP9.5), Km value and reactivity with cysteine and histidine-specific reagents. However, it differs with respect to other properties, e.g. inhibition by UbOEt and a wider pH range of activity.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj3180711