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Seeing the Importance of Palmitoylation

Rhodopsin is a G-protein coupled receptor in the retina that enables us to see dim light. The post-translational addition of palmitate to rhodopsin anchors the C terminus of the molecule to the membrane, creating an additional intracellular domain. Many residues in this domain are involved in signal transduction activation. Phosphorylation of several serines in the C-terminal domain by rhodopsin kinase terminates rhodopsin activity and deactivates light-induced signaling.

Mice with unpalmitoylated rhodopsin experience mild retinal degradation.

To gain a better understanding of the role of palmitoylation in visual transduction, Zhongyan Wang and colleagues generated a transgenic knock-in mouse expressing nonpalmitoylated rhodopsin. The mutant rhodopsin was expressed to levels equivalent to wild-type and correctly localized to the rod outer segment membrane. However, the mutant mice exhibited reduced light sensitivity and faster photoresponse recovery times. In addition, phosphorylation of the mutant rhodopsin by rhodopsin kinase in response to light was faster and occurred to a greater extent, compared to wild-type rhodopsin. These findings allow the authors to conclude that palmitoylation slows the shutoff of photoexcited rhodopsin, thereby extending the activity of this G-protein coupled receptor in vivo.

FOOTNOTES

See referenced article, J. Biol. Chem. 2005, 280, 24293-24300

Global Analysis Reveals Concentrations of Free and Enzyme-bound NADH

Nicotinamide adenine dinucleotide (NADH) is the principal electron donor in metabolism and thus one of the most important coenzymes in the cell. The fact that the molecule is a natural fluorophore makes it an ideal non-invasive fluorescent probe of metabolic state. However, quantitating free and enzyme-bound NADH within cells has not been easy. Current analytical chemistry techniques generally entail destroying the tissue and limiting their use to single-shot measurements, and fluorescent techniques cannot distinguish between free and enzyme-bound NADH.

Hippocampal tissue fluorescence during base line (A) is enhanced after a brief episode of hypoxia (B).

In this Paper of the Week, Harshad D. Vishwasrao and colleagues report that a combined analysis of time-resolved fluorescence and anisotropy decays allowed them to follow changes in amounts of free and enzyme-bound NADH in living tissue. In particular, binding of NADH to an enzyme greatly increases the rotation time of the bound fluorophore, so anisotropy measurements give a sensitive approach to quantitation of free and enzyme-bound NADH in living cells. Importantly, the authors demonstrated that a substantial fraction of NADH in the tissue analyzed is free, in contrast to earlier reports, which maintained that all NADH is enzyme-bound. Also, a metabolic transition to hypoxia changes the population of bound NADH molecules, as reflected in the distribution of species with different lifetimes.

FOOTNOTES

See referenced article, J. Biol. Chem. 2005, 280, 25119-25126

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