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Expression and characterization of deletion recombinants of two cGMP-inhibited cyclic nucleotide phosphodiesterases (PDE-3)

He, R; Komas, N; Ekholm, Dag; Murata, T; Taira, M; Hockman, S; Degerman, Eva LU and Manganiello, V C (1998) In Cell Biochemistry and Biophysics 29(1-2). p.89-111
Abstract
cDNAs encoding two PDE-3 or cyclic GMP-inhibited (cGI) cyclic nucleotide phosphodiesterase (PDE) isoforms, RPDE-3B (RcGIP1) and HPDE-3A (HcGIP2), were cloned from rat (R) adipose tissue and human (H) heart cDNA libraries. Deletion and N- and C-terminal truncation mutants were expressed in Escherichia coli in order to define their catalytic core. Active mutants of both RPDE-3B and HPDE-3A included the domain conserved among all PDEs plus additional upstream and downstream sequences. An RPDE-3B mutant consisting of the conserved domain alone and one from which the RPDE-3B 44-amino acid insertion was deleted exhibited little or no activity. All active recombinants exhibited a high affinity (< 1 microM) for cyclic AMP (cAMP) and cyclic GMP... (More)
cDNAs encoding two PDE-3 or cyclic GMP-inhibited (cGI) cyclic nucleotide phosphodiesterase (PDE) isoforms, RPDE-3B (RcGIP1) and HPDE-3A (HcGIP2), were cloned from rat (R) adipose tissue and human (H) heart cDNA libraries. Deletion and N- and C-terminal truncation mutants were expressed in Escherichia coli in order to define their catalytic core. Active mutants of both RPDE-3B and HPDE-3A included the domain conserved among all PDEs plus additional upstream and downstream sequences. An RPDE-3B mutant consisting of the conserved domain alone and one from which the RPDE-3B 44-amino acid insertion was deleted exhibited little or no activity. All active recombinants exhibited a high affinity (< 1 microM) for cyclic AMP (cAMP) and cyclic GMP (cGMP), were inhibited by cAMP, cGMP, and cilostamide, but not by rolipram, and were photolabeled with [32P]-cGMP. The IC50 values for cGMP inhibition of cAMP hydrolysis were lower for HPDE-3A than for RPDE-3B recombinants. The deduced amino acid sequences of HPDE-3A and RPDE-3B catalytic domains are very similar except for the 44-amino acid insertion not found in other PDEs. It is possible that this insertion may not only distinguish PDE-3 catalytic domains from other PDEs and identify catalytic domains of PDE-3 subfamilies or conserved members of the PDE-3 gene family, but may also be involved in the regulation of sensitivity of PDE-3s to cGMP. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Cell Biochemistry and Biophysics
volume
29
issue
1-2
pages
89 - 111
publisher
Humana Press
external identifiers
  • pmid:9631240
  • scopus:0031634909
ISSN
1085-9195
DOI
10.1007/BF02737830
language
English
LU publication?
yes
id
b6bdee3e-f905-449e-8b2b-010b111bbbee (old id 1113551)
date added to LUP
2008-07-15 12:27:08
date last changed
2017-04-30 14:58:50
@article{b6bdee3e-f905-449e-8b2b-010b111bbbee,
  abstract     = {cDNAs encoding two PDE-3 or cyclic GMP-inhibited (cGI) cyclic nucleotide phosphodiesterase (PDE) isoforms, RPDE-3B (RcGIP1) and HPDE-3A (HcGIP2), were cloned from rat (R) adipose tissue and human (H) heart cDNA libraries. Deletion and N- and C-terminal truncation mutants were expressed in Escherichia coli in order to define their catalytic core. Active mutants of both RPDE-3B and HPDE-3A included the domain conserved among all PDEs plus additional upstream and downstream sequences. An RPDE-3B mutant consisting of the conserved domain alone and one from which the RPDE-3B 44-amino acid insertion was deleted exhibited little or no activity. All active recombinants exhibited a high affinity (&lt; 1 microM) for cyclic AMP (cAMP) and cyclic GMP (cGMP), were inhibited by cAMP, cGMP, and cilostamide, but not by rolipram, and were photolabeled with [32P]-cGMP. The IC50 values for cGMP inhibition of cAMP hydrolysis were lower for HPDE-3A than for RPDE-3B recombinants. The deduced amino acid sequences of HPDE-3A and RPDE-3B catalytic domains are very similar except for the 44-amino acid insertion not found in other PDEs. It is possible that this insertion may not only distinguish PDE-3 catalytic domains from other PDEs and identify catalytic domains of PDE-3 subfamilies or conserved members of the PDE-3 gene family, but may also be involved in the regulation of sensitivity of PDE-3s to cGMP.},
  author       = {He, R and Komas, N and Ekholm, Dag and Murata, T and Taira, M and Hockman, S and Degerman, Eva and Manganiello, V C},
  issn         = {1085-9195},
  language     = {eng},
  number       = {1-2},
  pages        = {89--111},
  publisher    = {Humana Press},
  series       = {Cell Biochemistry and Biophysics},
  title        = {Expression and characterization of deletion recombinants of two cGMP-inhibited cyclic nucleotide phosphodiesterases (PDE-3)},
  url          = {http://dx.doi.org/10.1007/BF02737830},
  volume       = {29},
  year         = {1998},
}