Abstract
Catabolism of AMP during ATP breakdown produces adenosine, which restores energy balance. Catabolism of IMP may be a key step regulating purine nucleotide pools. Two, cloned cytosolic 5'-nucleotidases (cN-I and cN-II) have been implicated in AMP and IMP breakdown. To evaluate their roles directly, we expressed recombinant pigeon cN-I or human cN-II at similar activities in COS-7 or H9c2 cells. During rapid (more than 90% in 10 min) or slower (30-40% in 10 min) ATP catabolism, cN-I-transfected COS-7 and H9c2 cells produced significantly more adenosine than cN-II-transfected cells, which were similar to control-transfected cells. Inosine and hypoxanthine concentrations increased only during slower ATP catabolism. In COS-7 cells, 5'-nucleotidase activity was not rate-limiting for inosine and hypoxanthine production, which was therefore unaffected by cN-II- and actually reduced by cN-I- overexpression. In H9c2 cells, in which 5'-nucleotidase activity was rate-limiting, only cN-II overexpression accelerated inosine and hypoxanthine formation. Guanosine formation from GMP was also increased by cN-II. Our results imply distinct roles for cN-I and cN-II. Under the conditions tested in these cells, only cN-I plays a significant role in AMP breakdown to adenosine, whereas only cN-II breaks down IMP to inosine and GMP to guanosine.
Highlights
ATP levels are a key determinant of cellular function
We proposed that adenosine acts as a “retaliatory metabolite,” being formed when there is an excess of ATP breakdown over its formation and mediating the spectrum of actions designed to restore energy balance [7, 8]
The cN-I displays a preference for AMP over IMP and is stimulated by ADP but not ATP [15, 16], implying that it is active against AMP during ATP catabolism
Summary
Isolation of Pigeon cN-I and Human cN-II cDNA—The full-length cDNA clone coding for pigeon cN-I has already been described [9]. Metabolite and protein concentrations were determined as described below. Nucleosides and nucleotides were measured by reverse phase high pressure liquid chromatography as described previously [27], but performed at pH 6.7 and using a modified gradient of B (15% acetonitrile in Buffer A containing 150 mM KCl and 150 mM KH2PO4) changed linearly as follows: 0 min, 0% B; 0.1 min, 3% B; 3 min, 9% B; 7.5 min, 100% B; 8.5 min, 0% solution B for 3.5 min. Characterization of Recombinant cN-I and -II in Cell Extracts—To measure cN-I activity, control- and cN-I-transfected cells were extracted with a buffer containing 40 mM N-dimethylglutarate, 40 mM -glycerophosphate, 200 mM KCl, 0.2 mM dithiothreitol, 0.2% Triton X-100, pH 6.9. Cells transfected 48 hours previously were solubilized and total extracts were assayed as described under “Experimental Procedures.” cN-I and cN-II activity are expressed (mean Ϯ SE) as mol 1⁄7 minϪ1 1⁄7 mg proteinϪ1.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
