Copurification of selected glycolytic enzymes with retinal S-antigen (arrestin) by hydroxyapatite agarose chromatography of bovine retina.
OBJECTIVE
A variety of methods have been developed for the purification of S-antigen but a simple and rapid procedure based on hydroxyapatite-agarose (HA) adsorbtion is most widely used. In the present study, we investigated the nature of proteins purified with the aid of HA chromatography.


METHODS
After elimination of retinal S-antigen by HA, the soluble extract of retinal tissue was readsorbed on HA. The proteins were thereafter desorbed by 10 to 500 mM phosphate buffer gradient. Two peaks obtained by SDS-PAGE were used for the generation of specific antisera for subsequent analysis by ELISA and Western blotting.


RESULTS
Four proteins (two 48 kDa , one 50 kDa and one 46 kDa) were obtained in this manner. Partial amino acid sequencing permitted the identification of these proteins as alpha-enolase (48 kDa), gamma-enolase (48 kDa), Glucose-6-phosphate-Isomerase (50 kDa) and aspartate-amino-transferase (46 kDa).


CONCLUSIONS
The selected glycolytic enzymes co-purified with retinal S-antigen by hydroxyapatite agarose chromatography of bovine retina.
Mirshahi
M
M
INSERM U86, Paris, France.
Camoin
L
L
Nicolas
C
C
Ghedira
I
I
Cozette
J
J
Faure
J P
JP
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D019393:Arrestin / Q000302:isolation & purification*; D001219:Aspartate Aminotransferases / Q000235:genetics / Q000302:isolation & purification; D002417:Cattle; D002847:Chromatography, Agarose / Q000379:methods*; D017886:Durapatite; D004798:Enzymes / Q000235:genetics / Q000302:isolation & purification*; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics / Q000302:isolation & purification; D006019:Glycolysis / Q000502:physiology; D008969:Molecular Sequence Data; D010751:Phosphopyruvate Hydratase / Q000235:genetics / Q000302:isolation & purification; D012160:Retina / Q000737:chemistry*
D019393:Arrestin; D004798:Enzymes; D017886:Durapatite; D001219:Aspartate Aminotransferases; D010751:Phosphopyruvate Hydratase; D005956:Glucose-6-Phosphate Isomerase