cGMP is tightly bound to bovine retinal rod phosphodiesterase

Research output: Contribution to journalArticle

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Abstract

Although the total concentration of cGMP in rod outer segments is thought to be substantially greater than the free concentration, no quantitatively relevant site for the bound cGMP has been described in mammalian photoreceptors. We have found that preparations of purified bovine rod photoreceptor cyclic nucleotide phosphodiesterase (PDE) contain 1.8 ± 0.3 mol of tightly bound cGMP per mol of PDE. When subunits of the purified PDE were separated by reverse-phase HPLC in 0.1% trifluoroacetic acid and acetonitrile, a peak of material having spectral properties characteristic of a guanine ring was seen. This material was identified as cGMP by comigration with authentic cGMP on HPLC, conversion to 5-GMP by trypsin-activated rod PDE, and conversion to guanosine by a combination of trypsin-activated PDE and 5'-nucleotidase-containing snake venom. When incubated with 1 μM [3H]cGMP, only 0.1 mol of [3H]cGMP bound per mol of purified PDE, presumably because nearly all binding sites were occupied by tightly bound endogenous cGMP carried through the purification. Scatchard plots of [3H]cGMP binding have indicated that two classes of binding sites are present on the rod PDE. The off-rate of cGMP from the slowly dissociating site is extremely slow; it has a t( 1/2 ) of ~ 4 hr at 37°C. At lower temperatures, very little cGMP dissociates; the amount of [3H]cGMP bound to rod PDE after 2 hr at 4°C was essentially the same as at the beginning of the incubation. The observation that stoichiometric amounts of cGMP are tightly bound to PDE accounts for the inability to purify the bovine rod PDE on cGMP affinity columns or to demonstrate stoichiometric high-affinity binding sites with [3H]cGMP. More significantly, the tightly bound cGMP may resolve the apparent discrepancy between the free and total cGMP concentrations of photoreceptor outer segments.

Original languageEnglish (US)
Pages (from-to)4311-4315
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume86
Issue number11
StatePublished - 1989
Externally publishedYes

Fingerprint

Retinal Rod Photoreceptor Cells
Phosphoric Diester Hydrolases
Binding Sites
Trypsin
High Pressure Liquid Chromatography
Type 5 Cyclic Nucleotide Phosphodiesterases
Guanosine Monophosphate
Rod Cell Outer Segment
Trifluoroacetic Acid
5'-Nucleotidase
Snake Venoms
Guanosine
Cyclic Nucleotides
Guanine
Temperature

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

cGMP is tightly bound to bovine retinal rod phosphodiesterase. / Barr-Gillespie, Peter; Beavo, J. A.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, No. 11, 1989, p. 4311-4315.

Research output: Contribution to journalArticle

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AU - Beavo, J. A.

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AB - Although the total concentration of cGMP in rod outer segments is thought to be substantially greater than the free concentration, no quantitatively relevant site for the bound cGMP has been described in mammalian photoreceptors. We have found that preparations of purified bovine rod photoreceptor cyclic nucleotide phosphodiesterase (PDE) contain 1.8 ± 0.3 mol of tightly bound cGMP per mol of PDE. When subunits of the purified PDE were separated by reverse-phase HPLC in 0.1% trifluoroacetic acid and acetonitrile, a peak of material having spectral properties characteristic of a guanine ring was seen. This material was identified as cGMP by comigration with authentic cGMP on HPLC, conversion to 5-GMP by trypsin-activated rod PDE, and conversion to guanosine by a combination of trypsin-activated PDE and 5'-nucleotidase-containing snake venom. When incubated with 1 μM [3H]cGMP, only 0.1 mol of [3H]cGMP bound per mol of purified PDE, presumably because nearly all binding sites were occupied by tightly bound endogenous cGMP carried through the purification. Scatchard plots of [3H]cGMP binding have indicated that two classes of binding sites are present on the rod PDE. The off-rate of cGMP from the slowly dissociating site is extremely slow; it has a t( 1/2 ) of ~ 4 hr at 37°C. At lower temperatures, very little cGMP dissociates; the amount of [3H]cGMP bound to rod PDE after 2 hr at 4°C was essentially the same as at the beginning of the incubation. The observation that stoichiometric amounts of cGMP are tightly bound to PDE accounts for the inability to purify the bovine rod PDE on cGMP affinity columns or to demonstrate stoichiometric high-affinity binding sites with [3H]cGMP. More significantly, the tightly bound cGMP may resolve the apparent discrepancy between the free and total cGMP concentrations of photoreceptor outer segments.

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