Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver.
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.
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*
D007074:Immunoglobulin G; D046911:Macromolecular Substances; D014166:Transferases; D012696:Glycine Hydroxymethyltransferase