The intracellular distribution of glutamine synthetase in rat liver and the effect of metals on its activity

Abstract

A N D A. L. TAPPEL Differential centrifugation in 0.25 M sucrose of rat brain (Waelsch, '59 and Sellinger and de Balbian Verster, '62) and rat liver (Wu, '63b) indicate that in both tissues almost half of the total glutamine synthetase is associated with the microsomal fraction, with the remaining being distributed among the soluble, nuclear and mitochondrial fractions. In the microsomal fraction, the enzyme is loosely bonded onto the outer surface of the vesicular membranes from which place it can be solubilized with NaCl in concentration lower than that of physiological saline (Wu, '63b) and with EDTA which also has an activating effect (Sellinger and de Balbian Verster, '62 and Wu, '63b). This effect appears to be an indirect one as a two-fold increase of total glutamine synthetase activity occurred only when EDTA was incorporated into the homogenizing medium but not when added to an isolated fraction (Sellinger and de Balbian Verster, '62).In the present study, rat livers were fractionated in 0.25 M sucrose and the intracellular distribution of glutamine synthetase was determined and compared with that of enzymes selected as markers for the various subcellular organelles including lysosomes and uricase particles. The possibility that the activating effect of EDTA was due to removal of inhibitory metals was tested. tase, eight livers were fractionated, yielding six major fractions: a nuclear and cell debris fraction sedimented at 480 X g for 10 min, a mitochondrial fraction at 5000 X g for 10 min, a 'light mitochondrial" fraction at 16,300 X g for 20 min, a "heavy microsomal fraction" at 30,000 X g for 30 min, and a "light microsomal fraction" at 100,000 X g for one hour. The supernatant obtained after the last centrifugation was termed the soluble fraction. To obtain data on the effects of metals on synthetase activity, the cation under study was added to the 0.25 M sucrose suspending medium in which the livers were homogenized an3 fractionated.The activity of glutamine synthetase was determined according to the colorimetric assay outlined by Wu ('63a). The oxidation of alcohol by alcohol dehydrogenase at pH 8.5 was measured by the increase in DPNH at 340 mH. Glutamic dehydrogenase was assayed in buffer of pH 6.8 and 0.01 M in KCN and the amount of DPNH oxidized when glutamic acid is formed from a-keto glutaric acid and ammonia was taken as a measure of enzyme activity. The activity of glucose-6-phosphatase was measured at pH 6.5 with glucose-6-phosphate as substrate. Uricase was assayed at pH 9.0 by measuring the decrease in absorption at 290 mv as uric acid is converted to allantoin. Acid phosphatase, @-glucuronidase and aryl sulfatase were assayed simultaneously at pH 5.0 with an autoanalyzer program using the substrates, phenolphthalein phosphate, phenolphthalein glucuronide and nitrocatechol sulfate, respectively. Protein values were obtained by Miller's method ('59).