Mutations and deletions in mitochondrial DNA (mtDNA) lead to a number of human diseases characterized by neuromuscular degeneration. Accumulation of truncated mtDNA molecules (∆-mtDNA) lacking a specific 4977-bp fragment, the common deletion, leads to three related mtDNA diseases : Pearson's syndrome; Kearns-Sayre syndrome; and chronic progressive external ophthalmoplegia (CPEO). In addition, the proportion of ∆-mtDNA present increases with age in a range of tissues. Consequently, there is considerable interest in the effects of the accumulation of ∆-mtDNA on cell function. The 4977-bp deletion affects genes encoding 7 polypeptide components of the mitochondrial respiratory chain, and 5 of the 22 tRNAs necessary for mitochondrial protein synthesis. To determine how the accumulation of ∆-mtDNA affects oxidative phosphorylation we constructed a series of cybrids by fusing a human osteosarcoma cell line depleted of mtDNA (ρ 0 ) with enucleated skin fibroblasts from a CPEO patient. The ensuing cybrids contained 0Ϫ86 % ∆-mtDNA and all had volumes, protein contents, plasma-membrane potentials and mitochondrial contents similar to those of the parental cell line. The bioenergetic consequences of accumulating ∆-mtDNA were assessed by measuring the mitochondrial membrane potential, rate of ATP synthesis and ATP/ADP ratio. In cybrids containing less than 50Ϫ55% ∆-mtDNA, these bioenergetic functions were equivalent to those of cybrids with intact mtDNA. However, once the proportion of ∆-mtDNA exceeded this threshold, the mitochondrial membrane potential, rate of ATP synthesis, and cellular ATP/ADP ratio decreased. These bioenergetic deficits will contribute to the cellular pathology associated with the accumulation of ∆-mtDNA in the target tissues of patients with mtDNA diseases.
Spin-echo NMR spectroscopy was used to record the cleavage of a gamma-glutamyl--amino-acid by (5-L-glutamyl)-L-amino-acid 5-glutamyltransferase (cyclizing) (gamma-glutamylcyclotransferase) in human erythrocyte hemolysates. The Michaelis-Menten steady-state kinetic parameters were obtained by fitting the integrated Michaelis-Menten equation to the reaction time curves. The product, L-5-oxoproline, was shown to be an inhibitor of the reaction. The active site of the enzyme was probed by studies of the inhibition by D- and L-beta-aminoglutaryl-L-alanine which are the beta-amino-acid isomers of D- and L-gamma-glutamyl-L-alanine (the latter being a natural substrate of the enzyme); the D-isomer was the more potent inhibitor (Ki = 0.30 +/- 0.02 mmol/l water). When the alanyl alpha-carboxyl of the inhibitor was reduced to a hydroxyl (i.e. to give D-beta-aminoglutaryl-L-alaninol) the potency of inhibition was reduced. The previously reported kinetic isotope effect of solvent 2H2O on the enzyme-catalyzed reaction has been further studied using a proton inventory. We propose that the solvent kinetic isotope effect is due to an intramolecular proton transfer between the glutamyl amino group and the peptide bond nitrogen.
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