The characterization of a new enzyme from rat liver mitochondria, oligophosphoglyceroyl-ATP 3′-phosphodiesterase

Abstract

By using an assay based on the precipitation of intact 14C-labelled substrate, an activity has been located in the mitochondrial fraction of rat liver which selectively hydrolyses the 3′ ester link in the fairly recently discovered oligomeric tetraphosphate derivative of ATP and glyceric acid for which the structure 3-phospho[glyceroyl-gamma-triphospho-5′-adenosine-3′-3-phospho]n-glyceroyl- gamma-triphospho-5′-adenosine has been proposed [Hutchinson, Morris & Mowbray (1986) Biochem. J. 234, 623-627]. This enzyme activity (Mr 85,000) has been purified approx. 30-fold from washed mitochondria by (NH4)2SO4 precipitation and f.p.l.c. The apparent Km for substrate (adenosine equivalents) is around 35 microM. The recovery of total activity is about 20%, and this, allied to the relatively low Vmax. found in contrast with the rapid turnover of oligomer seen in post-ischaemic tissues, suggests that some activating factors have been lost in purification. Percoll-gradient studies confirm that the activity is mitochondrial and not lysosomal or endoplasmic-reticular. The activity is latent in intact mitochondria; it is not, however, associated with intact inner-membrane vesicles but released during their preparation, implying an intermembrane-space location. The product of the enzyme is proposed to be the monomeric unit 3-phosphoglyceroyl-gamma-triphospho-5′-adenosine, from which digestion with snake-venom phosphodiesterase releases ADP.

Evidence that the recently discovered high‐energy nucleotide derivative, oligo(phosphoglyceroyl‐ATP) forms the end chains of purinogen, a complex polymeric major stroage form of adenine nucleotide and phosphate in heart

“…D(-)Ribose was from Sigma Chemical Co. The (PG-ATP), and its monomer, 3-phosphoglyceroyl-y-triphospho(5')adenosine (PG-ATP) were prepared as previously de-scribed [8,91. The triphospho(5')adenosine(3')-3-phosphoglycerate (PPPAdoPGri) was synthesised [lo] in the laboratory of Professor C. B. Reese at Kings College London.…”

“…Selective digestion and chromatography, along with "P-NMR and mass spectrometry studies, led us to propose a structure and the name of oligo-(phosphoglyceroyl-ATP) (PG-ATP), for this compound [7]. In subsequent work we have purified from liver a specific 3'-phosphodiesterase which releases 3-phospho-glyceroyl-ytriphospho(5')adenosine (PG-ATP) units [8] : furthermore in liver and heart, this enzyme is found only in mitochondrial intermembrane space [8] tightly complexed with its substrate [9], suggesting that (PG-ATP), may be exclusively located there too.In perfused rat hearts the acid-insoluble species was observed to reach specific activity equilibrium with [8-I4C]ATP Correspondence to J. Mowbray, …”

Purification of a Crude Mitochondrial Fraction by Density‐Gradient Centrifugation

“…Thus, the centrifugation conditions that are required are not unlike those used for purification through preformed gradients of other gradient media. One of the problems of using Percoll, however, is the significant overlap of mitochondria and endoplasmic reticulum (ER; Patel et al, 1991). The method of Hovius et al (1990) uses a relatively low RCF (10,000 × g for 10 min) to produce the crude mitochondrial fraction in order to reduce the ER content (see Basic Protocol 3).…”