title: Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver.
abstract: The homogeneous serine hydroxymethyltransferase purified from monkey liver, by the use of Blue Sepharose affinity chromatography, exhibited positive homotropic co-operative interactions (h = 2.5) with tetrahydrofolate and heterotropic interactions with L-serine and nicotinamide nucleotides. The enzyme had an unusually high temperature optimum of 60 degrees C and was protected against thermal inactivation by L-serine. The allosteric effects were abolished when the monkey liver enzyme was purified by using a heat-denaturation step in the presence of L-serine, a procedure adopted by earlier workers for the purification of this enzyme from mammalian and bacterial sources. The enzyme activity was inhibited completely by N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, dichloromethotrexate, aminopterin and D-cycloserine, whereas methotrexate and dihydrofolate were partial inhibitors. The insoluble monkey liver enzyme-antibody complex was catalytically active and failed to show positive homotropic co-operative interactions with tetrahydrofolate (h = 1) and heterotropic interactions with NAD+. The enzyme showed a higher heat-stability in a complex with its antibody than as the free enzyme. These results highlight the pitfalls in using a heat-denaturation step in the purification of allosteric enzymes.
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D012696:Glycine Hydroxymethyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007074:Immunoglobulin G; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008255:Macaca radiata; D046911:Macromolecular Substances; D008970:Molecular Weight; D011817:Rabbits; D012756:Sheep; D014018:Tissue Distribution; D014166:Transferases / Q000302:isolation & purification*
chemical_list: D007074:Immunoglobulin G; D046911:Macromolecular Substances; D014166:Transferases; D012696:Glycine Hydroxymethyltransferase