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

Abstract

Previous work in our laboratory has shown that free adenine nucleotides in heart, liver and kidney exchange rapidly with a major acid-insoluble species which we have characterised as oligo[3-phospho-glyceroyl-y-triphospho(5')adenosine(3')], abbreviated to (PG-ATP),. This has been found in liver and in heart to be complexed to a specific 3'-phosphodiesterase which releases PG-ATP monomers and is located in mitochondrial inter-membrane space. In ['4C]adenosine-perfused hearts, (PG-ATP), has been shown to reach specific radioactivity equilibrium with free ATP within 30 min. Attempts to estimate the quantities of nucleotide in (PG-ATP), and free nucleotides in response to a variety of stimuli using previously labelled hearts showed by contrast that the free and sequestered nucleotide pools were not at equilibrium. Re-examination of the rapidly labelled acid-insoluble species led to the recognition of radioactive inhomogeneity and of three additional nucleotide components. Perfusion of hearts with phosphate-free medium increased the proportion of 14C incorporated into the sequestered form by 70% and caused the net transfer of 0.9 pmol . g-' of purine from free adenine nucleotides to the sequestered form. The findings point to the existence of a more complex polymer whose rapidly exchanging end chains we had previously isolated and characterised as (PG-ATP),; we suggest the name purinogen for this polymer and show that it can contain between 25 % and 55 % of the tissue nucleotide pool in rat heart. Large changes in the amounts of adenine nucleotides extracted at different times from steady-state perfused rat hearts could not be explained by interconversion with known derivatives or polymers and led us to propose that some unknown major source of nucleotides capable of rapid exchange with the known nucleotides must exist in heart [l -31. Labelling studies located a trichloroacetic acidmethanol-precipitable species which .rapidly exchanged units with ATP in rat heart [4], kidney [5] and liver [6]. 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,

Purinogen is not an endogenous substrate used in endothelial cells during substrate deprivation

“…This chromatogram is virtually identical with that of the acid-soluble fraction of PAEC that was prepared by the method of Mowbray et al [4,5] (see Figure 1A for comparison).…”

“…PAEC grown to confluency were extracted with trichloroacetic acid\methanol as described by Mowbray et al [4,5] and subsequently analysed by HPLC. Analysis of the trichloroacetic acidsoluble fraction of PAEC revealed the presence of ATP, ADP, GTP and GDP, as shown in Table 1.…”

“…It has been reported that, quantitatively, this pool corresponds to approx. 25-55 % of the total acid-soluble adenine nucleotide pool [4,5]. Preparative biochemistry combined with diverse analytical methods showed that a phosphate derivative of adenosine is a main building block of this polymeric adenine nucleotide, named purinogen [3,6].…”

“…Preparative biochemistry combined with diverse analytical methods showed that a phosphate derivative of adenosine is a main building block of this polymeric adenine nucleotide, named purinogen [3,6]. It has been proposed that purinogen might play an important role as an energy reserve in the heart and also in other tissues such as liver and kidney [5][6][7][8].…”

“…Preparative biochemistry combined with diverse analytical methods showed that a phosphate derivative of adenosine is a main building block of this polymeric adenine nucleotide, named purinogen [3,6]. It has been proposed that purinogen might play an important role as an energy reserve in the heart and also in other tissues such as liver and kidney [5][6][7][8].In view of these findings we explored whether purinogen is present in endothelial cells and whether its presence could explain the remarkable ability of this cell type to survive extended Abbreviations used : PAEC, porcine aortic endothelial cells ; FID, free induction decay. 1 To whom correspondence should be addressed (e-mail ognjen!herzkreis.uni-duesseldorf.de).…”