DEPENDENCE OF IN-VIVO GLUTAMINE-SYNTHETASE ACTIVITY ON AMMONIA CONCENTRATION IN RAT-BRAIN STUDIED BY H-1-N-15 HETERONUCLEAR MULTIPLE-QUANTUM COHERENCE-TRANSFER NMR

The dependence of the in vivo rate of glutamine synthesis on the subst rate ammonia concentration was studied in rat brain by H-1-N-15 hetero nuclear multiple-quantum coherence-transfer NMR in combination with bi ochemical techniques. In vivo rates were measured at various steady-st ate blood and brain ammonia concentrations within the ranges 0.4-0.55 mu mol/g and 0.86-0.98 mu mol/g respectively, after low-rate intraveno us (NH4+)-N-15 infusion to N-15-enrich brain glutamine, followed by (N H4+)-N-14 infusion (isotope chase). The rate of glutamine synthesis at steady state was determined from the change in brain [5-N-15]glutamin e levels during isotope chase, observed selectively through the amide proton by NMR, and N-15 enrichments of brain glutamine and of blood an d brain ammonia measured by gas chromatography-MS. The in vivo rate (v ) was 3.3-4.5 mu mol/h per g of brain at blood ammonia concentrations (s) of 0.40-0.55 mu mol/g. A linear increase of 1/v with 1/s permitted estimation of the in vivo glutamine synthetase (GS) activity at a phy siological blood ammonia concentration to be 0.4-2.1 mu mol/h per g. T he observed ammonia-dependence strongly suggests that, under physiolog ical conditions, in vivo GS activity is kinetically limited by sub-opt imal in situ concentrations of ammonia as well as of glutamate and ATP . Comparison of the observed in vivo GS activity with the reported in vivo rates of glutaminase and of gamma-aminobutyrate (GABA) synthesis suggests that, under mildly hyperammonaemic conditions, glutamine is s ynthesized at a sufficiently high rate to serve as a precursor of GABA , but glutaminase-catalysed hydrolysis of glutamine is too slow to be the sole provider of glutamate used for GABA synthesis.