Regulation of Arrestin Binding by
Rhodopsin Phosphorylation Level: Critical Analysis and Review
What is the novelty of the research (what was known before and how this work is different?)
Prior to the experiment the level of understanding regarding arresting binding in relation to rhodopsin phosphorylation was rather limited. Specifically, although a link was established between arresting binding and rhodopsin phosphorylation, the implications of the level phosphorylation were still not elucidated (Vishnivetskiy et. al., 2007). Hence, the research conducted by Vihnivetskiy and colleagues were basically aimed at providing a better level of understanding regarding the potential effects of different amounts of phosphates bound to rhodopsin in relation to arresting binding as well as other related parameters such as stability in the presence of certain substances and salt sensitivity.
How were the experiments carried out (principles behind the techniques and why this model system)
The phosphorylated rhodopsin was created and the varying levels of phosphorylation were determined by the researchers with strict consideration of external factors and the environment. To expound, in terms of lighting, strict timing was used in order to successfully produce varied phosphorylation levels (Vihnivetskiy et. al., 2007). Aside from this, appropriate selection of procedures such as in vitro translation and transcription were also used. The use of an in vitro process allows for a better control of the parameters and to assure the proper observation of processes such as the specific activity of the radiolabeled arresting (Vihnivetskiy et al., 2007). Of course, arresting purification and binding along with statistical data processing were also conducted in order to determine the effects of rhodopsin phosphorylation level and to ensure the reliability of the results.
What is the purpose of the experiment on Fig 1. What can be concluded from the experiment?
The purpose of the experiment was to determine the effects of various rhodopsin phosphorylation levels to arresting binding due to the fact that the specific effects of different levels of phosphorylation were formerly unidentified (Vishnivetskiy et. al., 2007). The experiment yielded a number of different results. For one, through the experiment, it has been determined that there was a discrepancy in terms of stability and salt sensitivity between rhodopsin with less than five phosphates and rhodopsin having phosphates of five and above (Vishnivetskiy et. al., 2007). Another finding derived from the research pertains to the function of photoreceptors. In this sense, having excessive rhodopsin phosphorylation levels increases the chances of having errors and defects in the function of photoreceptors, which possibly leads to problems at a cellular level which might cause defects or disease for an organism (Vishnivetskiy et. al., 2007).
Acting as a referee for the paper, would you accept this paper for publication (what would you have done differently or changed to make the research better?)
Although the research is highly acceptable for publication, having been revised several times to address certain points more efficiently, there are several points wherein the research might still be improved upon albeit not considering material availability and budget. In this sense, one limitation as noted in the research was in terms of using the direct binding assay which presents limitations in relation to concentration measurement (Vishnivetskiy et. al., 2007). Thus in this sense, although the researchers were able to circumvent such a limitation, it would have been better if a more suitable method was utilized. Another limitation of the study was that purified monophosphorylated rhodopsin was not acquired for use. Since monophosphorylated rhodopsin was not tested, the implications of having a single phosphate effective arresting binding was not determined (Vishnivetskiy et. al., 2007). Therefore, it would have been better if the researchers were able to test monophosphorylated rhodopsin as well.
Vishnivetskiy, S.A., Raman, D., Wei, J., Kennedy, M.J., Hurley, J.B., and Gurevich, V.V.
(2007). Regulation of Arrestin Binding by Rhodopsin Phosphorylation Level. The Journal of Biological Chemistry, 282 (44): 32075 – 32083.