PubMed · 8386853
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
R H Kessin, M M Van Lookeren Campagne. 1993-04-30. Database contamination.. https://doi.org/10.1126/science.8386853
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
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 alpha and beta subunits ( approximately90 kDa each) and two copies of the inhibitory subunit gamma ( approximately 10 kDa). However, the possibility that rod PDE could exist as distinct isoforms, such as alphaalphagamma2 and betabetagamma2 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 approximately 180-190 kDa, and a doublet at approximately 210-220 kDa. Cross-linked products were analyzed using polyclonal-specific anti-PDEalphabeta, anti-PDEalpha, anti-PDEbeta, or anti-PDEgamma antibodies. The anti-PDEalpha and anti-PDEalphabeta antibodies recognized all the cross-linked products, whereas anti-PDEbeta and anti-PDEgamma antibodies did not interact with the 150-kDa band, indicating that the composition of this band is most likely alphaalpha. Similar analysis of cross-linked products of trypsin-treated PDE preparations revealed bands that are likely formed by PDEbeta 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 approximately220 and approximately150 kDa, respectively. These results indicate that PDE is likely a mixture of the major species alphabetagamma2, minor species alphaalphagamma2, and possibly betabetagamma2. Our data are consistent with the detection of low PDE activity in the rd mouse, which lacks any functional PDEbeta subunit.
Retinal rod and cone phosphodiesterases are oligomeric enzymes that consist of a dimeric catalytic core (alpha'2 in cones and alphabeta in rods) with inhibitory subunits (gamma) that regulate their activity. In addition, a 17-kDa protein referred to as the delta subunit co-purifies with the rod soluble phosphodiesterase and the cone phosphodiesterase. We report here partial protein sequencing of the rod delta subunit and isolation of a cDNA clone encoding it. The predicted amino acid sequence is unrelated to any other known protein. Of eight bovine tissue mRNA preparations examined by Northern analysis, the strongest delta subunit-specific signal was present in the retina. A less intense signal was seen in the brain and adrenal mRNA. In bovine retinal sections, rod delta subunit anti-peptide antibodies label rod but not cone outer segments. delta subunit, added back to washed outer segment membranes, solubilizes a large fraction of the membrane-bound phosphodiesterase, indicating that this subunit binds to the classical membrane associated phosphodiesterase. The subunit forms a tight complex with native, but not trypsin-released phosphodiesterase, suggesting that the isoprenylated carboxyl termini of the catalytic subunits may be involved in binding of the delta subunit to the phosphodiesterase holoenzyme.
cGMP-binding phosphodiesterases contain two kinetically distinct cGMP-binding sites (a and b), and each site contains a conserved N(K/R)XnFX3DE sequence. N276A, K277A, K277R, D289A, and E290A mutants in the N276KX7FX3DE290 sequence of site a (higher affinity site) of bovine cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE or PDE5A) were expressed in High Five cells and purified. The cGMP-binding affinities of three mutants [K277A (Kd approximately 12 microM), D289A (Kd approximately 24 microM), and N276A (Kd approximately 60 microM)] were decreased in comparison with wild-type enzyme (Kd = 1.3 microM), which suggested an important role for Asn276, Lys277, and Asp289 in cGMP binding. These residues could be presented as a putative NKXnD motif, and their functions were predicted based on analogy with the canonical NKXD motif in GTP-binding proteins. No marked differences in catalytic functions such as specific activity, Km for cGMP, and IC50 for zaprinast or 3-isobutyl-1-methylxanthine were found among wild-type and mutant cGB-PDEs. This suggested that cGMP binding to site a does not influence the catalytic properties of cGB-PDE.