Send to:

Choose Destination
See comment in PubMed Commons below
Biochem Pharmacol. 1989 Nov 15;38(22):4123-36.

Resolution of soluble cyclic nucleotide phosphodiesterase isoenzymes, from liver and hepatocytes, identifies a novel IBMX-insensitive form.

Author information

  • 1Department of Biochemistry, University of Glasgow, U.K.


DEAE chromatography of a high speed supernatant fraction from a homogenate of rat liver, prepared under isotonic conditions in the presence of protease inhibitors, yielded three peaks of cyclic nucleotide phosphodiesterase activity (PDE activity). The first peak could be resolved on Affi-gel Blue chromatography to yield a Ca2+/calmodulin stimulated cyclic GMP specific PDE and a cyclic AMP and cyclic GMP hydrolysing PDE whose activity was insensitive to Ca2+/calmodulin. These two activities could also be clearly resolved by Mono-Q chromatography of soluble extracts from both liver and hepatocytes. These had different molecular weights, kinetics of substrate utilization, thermostabilities, dependence on Mg2+ and inhibitor sensitivities. The cyclic AMP and cyclic GMP utilizing PDE resolved in these procedures appears to be a novel enzyme form (PDE-MQ-I) which is insensitive to inhibition by the so-called non-selective PDE inhibitor IBMX and displays catalytic activity in the absence of Mg2+. None of the inhibitors tested were capable of inhibiting this form showing that the catalytic activity of this species could be distinguished from all the other soluble activities. This novel enzyme hydrolysed both cyclic AMP and cyclic GMP with Km values of 25 microM and 237 microM, respectively. The Vmax ratio of hydrolysis of cyclic GMP/cyclic AMP was above unity (1.4). It accounted for 30% of the soluble cyclic AMP PDE activity and 10% of the cyclic GMP PDE activity assessed at 1 microM substrate. Gel filtration of PDE-MQ-I indicated a size of 33,150 Da, in contrast to the size of 237,500 Da observed for the Ca2+/calmodulin PDE-MQ-II. Thermal inactivation of PDE-MQ-I and PDE-MQ-II yielded single exponential decays with t1/2 values of 6.33 min and 0.7 min at 60 degrees respectively. In the presence of saturating Ca2+, PDE-MQ-II was activated by calmodulin with an EC50 of ca. 30 ng/ml. In the presence of calmodulin, PDE-MQ-II was activated by Ca2+ with an EC50 of ca. 20 microM. Chromatography of homogenates on Mono-Q also identified a cyclic GMP-activated cyclic nucleotide PDE (PDE-MQ-III) and two cyclic AMP specific activities (PDE-MQ-IV and PDE-MQ-V). These exhibited very different inhibitor sensitivities and could be readily distinguished using the compound Ro-20-1724 which yielded IC50 values for inhibition of greater than 500 microM, 13 microM and 1.5 microM, respectively, for the hepatocyte enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

How to join PubMed Commons

    Supplemental Content

    Loading ...
    Write to the Help Desk