Subunit structure of rod cGMP-phosphodiesterase

Nikolai O. Artemyev, Rajendran Surendran, James C. Lee, Heidi E. Hamm

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

The rod cGMP phosphodiesterase (PDE) is the G-protein-activated effector enzyme that regulates the level of cGMP in vertebrate photoreceptor cells. Rod cGMP PDE is generally viewed as a heterotrimeric protein composed of catalytic α and β subunits (~90 kDa each) and two copies of the inhibitory subunit γ (~10 kDa). However, the possibility that rod PDE could exist as distinct isoforms, such as ααγ2 and ββγ2 has not been ruled out. We have studied this question using cross-linking of PDE subunits with maleimidobenzoyl-N-hydroxysuccinimide ester and para-phenyldimaleimide. The cross-linking resulted in major products with molecular mass of 100 and 150 kDa, a doublet at ~180-190 kDa, and a doublet at ~210-220 kDa. Cross- linked products were analyzed using polyclonal-specific anti-PDEαβ, anti- PDEα, anti-PDEβ, or anti-PDEγ antibodies. The anti-PDEα and anti-PDEαβ antibodies recognized all the cross-linked products, whereas anti-PDEβ and anti-PDEγ antibodies did not interact with the 150-kDa band, indicating that the composition of this band is most likely αα. Similar analysis of cross- linked products of trypsin-treated PDE preparations revealed bands that are likely formed by PDEβ subunit. The molecular size of holo-PDE and trypsin- activated PDE were studied using analytical ultracentrifugation in order to determine if oligomerization of PDE could account for the cross-linking of identical PDE subunits. The sedimentation analysis of both holo-PDE and ta- PDE revealed homogeneous samples with molecular masses of ~220 and ~150 kDa, respectively. These results indicate that PDE is likely a mixture of the major species αβγ2, minor species ααγ2, and possibly ββγ2. Our data are consistent with the detection of low PDE activity in the rd mouse, which lacks any functional PDEβ subunit.

Original languageEnglish (US)
Pages (from-to)25382-25388
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number41
DOIs
StatePublished - 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Artemyev, N. O., Surendran, R., Lee, J. C., & Hamm, H. E. (1996). Subunit structure of rod cGMP-phosphodiesterase. Journal of Biological Chemistry, 271(41), 25382-25388. https://doi.org/10.1074/jbc.271.41.25382