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Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 2: Isolation and characterization of the transducin-activated form

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Abstract

Rod photoreceptor cGMP phosphodiesterase (PDE6) consists of a catalytic subunit complex (Pαβ) and two inhibitory subunits (Pγ). In the accompanying article, using bovine photoreceptor outer segment homogenates, we show that Pγ as a complex with the GTP-bound transducin α subunit (GTP-Tα) dissociates from Pαβγγ on membranes, and the Pαβγγ becomes Pγ-depleted. Here, we identify and characterize the Pγ-depleted PDE. After incubation with or without guanosine 5′-O-(3-thiotriphosphate) (GTPγS), Pαβ complexes are extracted. When a hypotonic buffer is used, Pαβγγ, Pαβγ, and a negligible amount of a Pαβ complex containing Pγ are isolated with GTPγS, and only Pαβγγ is obtained without GTPγS. When an isotonic buffer containing Pδ, a prenyl-binding protein, is used, Pαβγγδ, Pαβγδδ, and a negligible amount of a Pαβ complex containing Pγ and Pδ are isolated with GTPγS, and Pαβγγδ is obtained without GTPγS. Neither Pαβ nor Pαβγγ complexed with GTPγS-Tα is found under any condition we examined. Pαβγ has ~12 times higher PDE activity and ~30 times higher Pγ sensitivity than those of Pαβγγ. These results indicate that the Pγ-depleted PDE is Pαβγ. Isolation of Pαβγγδ and Pαβγδδ suggests that one C-terminus of Pαβ is involved in the Pαβγγ interaction with membranes, and that Pγ dissociation opens another C-terminus for Pδ binding, which may lead to the expression of high PDE activity. Cone PDE behaves similarly to rod PDE in the anion exchange column chromatography. We conclude that the mechanisms for PDE activation are similar in mammalian and amphibian photoreceptors as well as in rods and cones.

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Acknowledgments

We thank Dr. Richard Needleman, Mr. Masaomi Matsumoto and Ms. Marta Matsumoto for critical reading of the manuscript. We also express our gratitude to Dr. Peter Stein for his suggestion to use the method [40] to wash Coomassie blue-stained gels. This work was supported in part by National Institute of Health Grants EY07546, EY09631, and EY13877, Jules and Doris Stein Professorship and an unrestricted grant from Research to Prevent Blindness, Grants-in Aids for Scientific Research from Japan Society for the promotion of Science 18310088 and 18370062, and Iketani Foundation for Science and Engineering 0191093-A.

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Authors and Affiliations

  1. Department of Ophthalmology, Kresge Eye Institute, Wayne State University, 4717 St. Antoine St., Detroit, MI, 48201, USA

    Akio Yamazaki

  2. Department of Pharmacology, Wayne State University, Detroit, USA

    Akio Yamazaki & Russell K. Yamazaki

  3. Department of Intellectual Properties, Niigata University, Niigata, 950-2181, Japan

    Masahiro Tatsumi

  4. College of Osteopathic Medicine, Touro University, Henderson, NV, 89014, USA

    Vladimir A. Bondarenko

  5. Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73190, USA

    Sadamu Kurono, Naoka Komori & Hiroyuki Matsumoto

  6. Division of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan Town, Taiwan, ROC

    Isao Matsuura

  7. Department of Biology, Graduate School of Science, Kobe University, Kobe, 657-8501, Japan

    Fumio Hayashi

  8. Division of Integrated Project, EcoTopia Science Institute, Nagoya University, Nagoya, 464-8603, Japan

    Jiro Usukura

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  1. Akio Yamazaki
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  2. Masahiro Tatsumi
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Correspondence to Akio Yamazaki.

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Yamazaki, A., Tatsumi, M., Bondarenko, V.A. et al. Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 2: Isolation and characterization of the transducin-activated form. Mol Cell Biochem 339, 235–251 (2010). https://doi.org/10.1007/s11010-010-0404-y

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