April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Characterization of Arginine-Specific Mono-ADP Ribosyltransferase Isolated from Frog Retina and its Function in Signal Transduction
Author Affiliations & Notes
  • V. A. Bondarenko
    Basic Sciences, Touro University Nevada, Henderson, Nevada
  • A. Yamazaki
    Department of Ophthalmology and Pharmacology, Kresge Eye Institute, Wayne State University, Detroit, Michigan
  • Footnotes
    Commercial Relationships  V.A. Bondarenko, None; A. Yamazaki, None.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 5440. doi:
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      V. A. Bondarenko, A. Yamazaki; Characterization of Arginine-Specific Mono-ADP Ribosyltransferase Isolated from Frog Retina and its Function in Signal Transduction. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5440.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : The purpose of this study is to elucidate the properties and functions of retinal ADP-ribosyltransferase.

Methods: : We isolated GPI-anchored ADP-ribosyltransferase (ADP-RT) by treatment frog outer segments (OS) of retinal photoreceptors with phospholipase-C (PL-C). We analyzed the effect of different nucleotides on ADP-ribosylation of inhibitory subunit of retinal cGMP phosphodiesterase (Pγ) by membrane bound and solubilized enzymes.

Results: : ADP-RT was released from OS homogenates into soluble fraction by treatment the membranes with PL-C. The proteins in soluble fraction were further separated on Mono-Q, Mono-S, TSK-250, or Pγ-affinity columns. Previously we used a combination of Mono-S and Mono-Q column chromatographies to identify two different izozymes of ADP-RT present in frog retina. Here we identified the molecular weight of both ADP-RT izozymes using two approaches: size exclusion chromatography on TSK-250 and zymographic assay.Both techniques showed that apparent molecular weight of ADP-RT is 35kDa. To identify the mode of action of ADP-RT in signal transduction we examine the effect of various nucleotides on the activity of membrane-bound enzyme. We found that addition of 1mM cGMP to the prewashed OS membranes resulted in increased ADP-ribosylation of Pγ. Pretreatment of membranes with ATP, on the other hand, leads to inhibition of Pγ ADP-ribosylation. Addition of GDP or GTP had no effect on Pγ ADP-ribosylation.

Conclusions: : We propose that ADP-RT functions under dark conditions to reduce the dark current in photoreceptors. There is possible interplay between Pγ phosphorylation and ADP-ribosylation suggesting that regulation of PDE activity is more complex than currently accepted.

Keywords: phosphorylation • protein modifications-post translational • signal transduction 

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