Differential effects of N-acetylglutamate on citrulline synthesis by coupled and uncoupled mitochondria.
When rats were placed on a low-protein (5%) diet for 24 h or less, liver mitochondrial acetylglutamate decreased rapidly, carbamyl phosphate synthetase (ammonia) and ornithine transcarbamylase decreased little, and carbamyl phosphate synthesis (measured as citrulline) by isolated mitochondria occurred at very low rates. The matrix acetylglutamate content of these mitochondria, whether coupled or uncoupled, was increased similarly by preincubating them with added acetylglutamate, but citrulline synthesis increased from less than 1 to 2.3 nmol min-1 mg-1 in the coupled state, and from less than 1 to 35 nmol min-1 mg-1 in the uncoupled state. However, when coupled mitochondria were incubated with the substrates required for the synthesis of acetylglutamate in the matrix, citrulline synthesis increased to 48 nmol min-1 mg-1; this rate was similar to that of mitochondria from control rats (fed a normal diet). When mitochondria from controls were incubated with up to 5mM acetylglutamate, citrulline synthesis by coupled mitochondria was increased by 10 to 40%, while synthesis by uncoupled mitochondria was 1.5 to 4 times higher than that observed with the coupled mitochondria; matrix acetylglutamate in both conditions rose to levels similar to those in the medium. The reason for the different behavior of carbamyl phosphate synthetase (ammonia) in coupled and uncoupled mitochondria was not apparent; neither oxidative phosphorylation nor ornithine transport were limiting in the coupled system. These observations are an example of the restrictions imposed upon enzymatic systems by the conditions existing in the mitochondrial matrix, and of the different behavior of carbamyl phosphate synthetase in situ and in solution. In addition, they show that conclusions about the characteristics of the enzyme in coupled mitochondria based on observations made in uncoupled mitochondria are not necessarily justified.
D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D002956:Citrulline / Q000096:biosynthesis*; D004789:Enzyme Activation / Q000187:drug effects; D005971:Glutamates / Q000378:metabolism / Q000494:pharmacology*; D066298:In Vitro Techniques; D008025:Ligases / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism*; D009952:Ornithine / Q000378:metabolism; D010085:Oxidative Phosphorylation / Q000187:drug effects; D011488:Protein Deficiency / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D005971:Glutamates; D002956:Citrulline; D009952:Ornithine; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C016195:N-acetylglutamic acid