Compartmentalization of S-adenosylhomocysteine in rat liver. Determination and characterization of the in vivo protein binding.
A method for the determination of S-adenosylhomocysteine (AdoHcy), which is associated with proteins in vivo, was developed. This method involves homogenization of tissue or cells in saturated, ice-cold solution of ammonium sulfate containing adenosine and S-adenosylmethionine to suppress unspecific binding of AdoHcy during sample processing. The homogenate was then extensively diluted, filtered, the precipitated protein washed, and AdoHcy extracted with perchloric acid. With this method it could be demonstrated that 30-50% of AdoHcy in rat liver and isolated rat hepatocytes is associated with proteins. Under physiological conditions, the major fraction of protein-bound AdoHcy resides in the microsomal fraction, whereas most free AdoHcy was recovered in the cytosol. The total AdoHcy content in hepatocytes could be markedly elevated by addition of adenosine or methionine. Under both conditions, protein-bound AdoHcy increased 2-fold and then leveled off, whereas free AdoHcy accounts for a further, massive, rise in intracellular AdoHcy. This suggests that AdoHcy is binding to saturable sites in vivo. AdoHcy in hepatocytes was labeled by incubating the cells with L-[35S]methionine, and within the first 60 min, free AdoHcy attained a significantly higher specific activity than protein-bound AdoHcy. Furthermore, chase with excess unlabeled methionine or with cycloleucine, revealed a shorter half-life of radioactive sulfur in free AdoHcy than in protein-bound AdoHcy. This shows that protein-bound and free AdoHcy represent kinetically distinct AdoHcy pools.
Svardal
A M
AM
Department of Pharmacology, University of Bergen, Norway.
Ueland
P M
PM
D000818:Animals; D003600:Cytosol / Q000378:metabolism; D006710:Homocysteine / Q000031:analogs & derivatives*; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D012435:S-Adenosylhomocysteine / Q000378:metabolism*
D006710:Homocysteine; D012435:S-Adenosylhomocysteine