Patel, Brinda; (1993) The purification and metabolism of a mitochondrial high phosphate derivative, oligophosphoglyceroyl-ATP, in rat heart and liver. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Radioactive labelling of rat heart and kidney nucleotides by perfusion with [8-14C]- adenosine has shown that about 15% of tissue radioactivity was incorporated into a trichloroacetic-acid/methanol insoluble precipitable derivative of ATP and glyceric acid, for which the structure 3-phosphoglyceroyl [-γ-triphospho-5'-adenosine-3'-3-phospho-glyceroyl]n-γ-triphospho-5'-adenosine (OPG-ATP) has been proposed. Analogous techniques to those used with hearts for specifically labelling tissue purine nucleotides followed by extraction and purification from TCA-precipitable fraction show the existence of substantially higher quantities of OPG-ATP in rat livers than in heart and kidney. Cell-fractionation experiments have shown that OPG-ATP co-distributes with succinate-dehydrogenase and can be readily purified from crude liver mitochondrial fraction. The very labile mixed anhydride bond in OPG-ATP was found to be remarkably stabilised in neutral solution by Mg2+ ions above 2mM. The monomeric unit (PG-ATP) also appears to be so stabilised and exhibits unusual properties on anion-exchange h.p.l.c. By using an assay based on the precipitation of 14C-OPG-ATP in 50% cold ethanol, an activity has been located in the mitochondrial fraction of rat heart and liver which selectively hydrolyses the 3'-phosphoester link in OPG-ATP. Percoll-gradient studies confirm that the activity is mitochondrial. Sub-fractionation of liver mitochondria shows that the activity is confined to the inter-membrane space. This enzyme activity has been purified approximately 40-fold from washed liver mitochondria by (NH4)2SO4 precipitation and f.p.l.c. This enzyme, OPG-ATP 3'-phosphodiesterase has 4 sub-units and a Mr of 165,000. The enzyme contains tightly bound OPG-ATP which can be released by alkali treatment without loss of activity. The apparent Km for substrate is about 35µM and the Vmax 6nmol/min./mg of protein. Evidence is presented that the product of the enzyme is the monomer 3-phosphoglyceroyl-γ-triphospho-5'-adenosine (PG-ATP). The likely role of OPG-ATP in control of cellular adenine nucleotides is discussed.

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