Abstract
Background The literature of the past four decades contains several scattered reports that demonstrate the existence of two different 3’,5’-cCMP degrading enzymes with distinct substrate affinity and specificity. The so-called “cCMPspecific” PDE was isolated from disrupted L-1210 leukemia cells [1] and from rat liver [2,3], shows an extremely high 3’,5’-cCMP KM value of up to 9 mM and seems to be activated by iron [1]. This enzyme prefers 3’,5’-cCMP over other cyclic nucleotides. The other enzyme is called “multifunctional cCMP PDE” and was isolated from pig liver [3-5], rat liver [6,7] and human liver [8]. This enzyme shows a 3’,5’-cCMP KM value in the range of 100-300 μM and is additionally degrading 2’,3’-cCMP as well as the 2’,3’and 3’,5’-isomers of cUMP, cAMP, cGMP and cIMP. The multifunctional cCMP PDE is inhibited by inorganic phosphate and AMP. Both enzymes are calmodulin-insensitive, IBMX-resistant and show a low molecular weight of <35 kDa, which is uncommon for the “classic” phosphodiesterases. However, the exact amino acid sequence and identity of these proteins remain elusive. Our project aims at a clear and unequivocal determination of amino acid sequence and identity of these elusive cCMP-degrading enzymes by analyzing and purifying the cCMP degrading activity of organ extracts and biological fluids (e.g. serum). Moreover, we re-investigate wellknown “classic” PDEs in enzymatic assays to identify a potential 3’,5’-cCMP degrading activity.
Highlights
The literature of the past four decades contains several scattered reports that demonstrate the existence of two different 3’,5’-cCMP degrading enzymes with distinct substrate affinity and specificity
A screen of 13 commercially available recombinant PDEs revealed that these enzymes show unexpectedly broad substrate specificity and degrade “exotic” cyclic nucleotides like cUMP, cIMP, cXMP and cTMP [9]
We have identified a heat-sensitive cCMPdegrading activity in certain commercially available batches of fetal bovine serum. This enzymatic activity can be considerably enhanced by filtration of the serum through centrifugation filtration devices (30 kDa cutoff)
Summary
The literature of the past four decades contains several scattered reports that demonstrate the existence of two different 3’,5’-cCMP degrading enzymes with distinct substrate affinity and specificity. The so-called “cCMPspecific” PDE was isolated from disrupted L-1210 leukemia cells [1] and from rat liver [2,3], shows an extremely high 3’,5’-cCMP KM value of up to 9 mM and seems to be activated by iron [1]. This enzyme prefers 3’,5’-cCMP over other cyclic nucleotides. The exact amino acid sequence and identity of these proteins remain elusive
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