Glutamine synthetase from rat liver. Purification, properties, and preparation of specific antisera.

As the initial aspect of investigations on the regulation of glutamine synthetase in rat hepatomas and in hepatoma cell culture systems, glutamine synthetase from rat liver was purified to apparent homogeneity. A single protein band is found in gels after electrophoresis in three separate systems, and a constant enzyme specific activity is found in fractions containing glutamine synthetase activity eluting from a hydroxylapatite column. The properties of crude liver enzyme and the purified protein were compared with those previously described (Tate, S. S., and Meister, A. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, 781-785). No differences were found, suggesting that the protein retained essential catalytic and regulatory properties through the different purification procedures used. The apparent K,,, of the enzyme for L-glutamate varied over lo-fold depending upon the concentration of divalent cation used for assay. At 8 mM &In’+, the apparent K,,, for glutamate is 0.3 mM, whereas at an Mn”+ concentration of 2 mM, the apparent Km for glutamate is 5 mM. Similar changes in the apparent K,, for glutamate are also noted with different concentrations of Mg’+. The inhibition of the enzyme by histidine, glutamine, and alanine differs as the concentration of divalent cation is changed. Free sulfhydryl groups appear necessary for the catalytic activity. Iodoacetamide quantitatively inactivates rat liver glutamine synthetase. The inactivation is blocked completely by the addition of substrates to the reaction mixture. No evidence for bound adenylyl groups, which modify the catalytic and regulatory properties of Escherichia coli glutamine synthetase, or other covalent modification of rat liver glutamine synthetase, was found by ultraviolet absorption spectroscopy or after incubation of enzyme, liver extracts, and effecters which result in adenylylation of E. coli glutamine synthetase. Antisera were obtained from rabbits immunized with purified rat liver glutamine synthetase. By immunodiffusion analysis, the antisera appear