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 (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.
Piccinini
M
M
Dipartimento di Morfofisiologia Veterinaria, Universit di Torino, Italy.
Merighi
A
A
Bruno
R
R
Cascio
P
P
Curto
M
M
Mioletti
S
S
Ceruti
C
C
Rinaudo
M T
MT
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D012160:Retina / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013869:Thiolester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014357:Trypsin; D043222:Ubiquitin Thiolesterase; D014452:Ubiquitins
D000906:Antibodies; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D014452:Ubiquitins; D013869:Thiolester Hydrolases; D043222:Ubiquitin Thiolesterase; D014357:Trypsin