Solubilization and partial purification of S-adenosyl-L-homocysteine binding proteins from the rat brain.
Rat brain proteins presenting high-affinity binding of S-adenosyl-L-homocysteine were solubilized and purified. Extraction of binding protein was carried out in the presence of Triton X-100 and 1 M NaCl; this solubilized fraction exhibits similar kinetic properties than the membrane proteins. Purification was performed using affinity chromatography on S-adenosyl-L-homocysteine carboxyhexyl Sepharose 48 conjugate. The analysis of the affinity gel eluate by SDS-PAGE showed high purification ratios for two proteins exhibiting 54 and 68 kDa. Three activity peaks were separated when solubilized membrane proteins were submitted to isoelectric focusing; the activity peaks corresponded to proteins of pH1 6.0, 6.5, and 7.2. SDS-PAGE separation of proteins contained in each peak showed protein aggregation; a 54-kDa subunit was present in each aggregate. Solubilized membrane proteins were labeled by photoaffinity labeling with tritiated S-adenosyl-L-homocysteine; the 54- and 68-kDa proteins were found among the specifically labeled proteins. Finally, according to the previous data from the literature, the purified S-adenosyl-L-homocysteine binding proteins do not seem to be the same as adenosine receptors or phosphatidylethanolamine-N-methyltransferase.
Fonlupt
P
P
Unité INSERM, Institute National des Sciences Appliquées, Villeurbanne, France.
Comte
N
N
Rey
C
C
Pacheco
H
H
D000345:Affinity Labels; D000818:Animals; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006710:Homocysteine; D007525:Isoelectric Focusing; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D010777:Photochemistry; D051381:Rats; D012435:S-Adenosylhomocysteine / Q000378:metabolism*; D012995:Solubility
D000345:Affinity Labels; D002352:Carrier Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D006710:Homocysteine; D012435:S-Adenosylhomocysteine