Mark W. Bitensky

The effects of additive solution pH and metabolic rejuvenation on anaerobic storage of red cells

BACKGROUND: Red cell (RBC) storage can be extended to 9 weeks under anaerobic or alkaline conditions. Simultaneous use of these approaches has not provided additive benefit. Our objective was to determine whether anaerobic storage with acidified additive solution (AS) coupled with metabolic rejuvenation might further improve the benefits of anaerobic storage.

Modulation of the G Protein Regulator Phosducin by Ca2+/Calmodulin-dependent Protein Kinase II Phosphorylation and 14-3-3 Protein Binding

Phototransduction is a canonical G protein-mediated cascade of retinal photoreceptor cells that transforms photons into neural responses. Phosducin (Pd) is a Gβγ-binding protein that is highly expressed in photoreceptors. Pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. Dephosphorylated Pd binds Gβγ with high affinity and inhibits the interaction of Gβγ with Gα or other effectors, whereas phosphorylated Pd does not. These results have led to the hypothesis that Pd down-regulates the light response. Consequently, it is important to understand the mechanisms of regulation of Pd phosphorylation. We have previously shown that phosphorylation of Pd by cAMP-dependent protein kinase moderately inhibits its association with Gβγ. In this study, we report that Pd was rapidly phosphorylated by Ca2+/calmodulin-dependent kinase II, resulting in 100-fold greater inhibition of Gβγ binding than cAMP-dependent protein kinase phosphorylation. Furthermore, Pd phosphorylation by Ca2+/calmodulin-dependent kinase II at Ser-54 and Ser-73 led to binding of the phosphoserine-binding protein 14-3-3. Importantly, in vivodecreases in Ca2+ concentration blocked the interaction of Pd with 14-3-3, indicating that Ca2+ controls the phosphorylation state of Ser-54 and Ser-73 in vivo. These results are consistent with a role for Pd in Ca2+-dependent light adaptation processes in photoreceptor cells and also suggest other possible physiological functions.

[63] Purification of rod outer segment GTP-Binding protein subunits and cgmp phosphodiesterase by single-step column chromatography

Publisher Summary This chapter describes a novel and simple single-step chromatographic method for the extensive purification of the subunits of the GTP-binding protein and the phosphodiesterase (PDE). There is a well-established homology between the hormone-sensitive adenylate cyclase and the light-activated cGMP phosphodiesterase in rod outer segments (ROS). The light-activated PDE of ROS has three functional segments. The photopigment rhodopsin, the GTP-binding protein, and the PDE catalytic complex. Signal transduction from rhodopsin to PDE is dependent on the binding of GTP to the α subunit of the GTP-binding protein. Photon capture is necessary for the binding of GTP to the Gα subunit. The GTP/Gα complex is rapidly released from the disk membrane surface. The activation of PDE in ROS prepared from the amphibian retina occurs as a consequence of the presentation of photons and the addition of GTP. Moreover, this activation depends upon the physical release of an inhibitory moiety from the PDE that remains bound to the disk membrane throughout the activation cycle. The major components of the ROS light-sensitive PDE cascade have been isolated and purified. The purification of rhodopsin has been well characterized in a number of laboratories.

[66] Photoaffinity labeling of high-affinity cGMP-specific noncatalytic binding sites on cGMP phosphodiesterase of Rod Outer segments

Publisher Summary The use of photoaffinity-labeling techniques has gained prominence in many areas of biochemistry. Photoaffinity-labeling reagents have been especially productive for the specific labeling of proteins that contain cyclic nucleotide binding sites. Rod outer segments (ROS) is used for membrane preparations and a crude ROS PDE preparation for the purpose of detecting the noncatalytic cGMP-binding sites with photoaffinity probes. This chapter emphasizes that the binding of cGMP to these noncatalytic high-affinity cGMP-binding sites on PDE is markedly stimulated by the presence of the heat-stable PDE inhibitor. Thus any preparation of PDE from ROS membranes should, for the purpose of demonstrating this class of cGMP-binding sites, contain or be supplemented with a preparation of the heat-stable inhibitory moiety. Moreover, because the PDE preparation often contains the GTP-binding protein subunits, it is essential to exclude either GTP or its nonhydrolyzable analogs from such a preparation inasmuch as the complex formed between GTP and the a subunit of the GTP-binding protein markedly interferes with the binding of cGMP to the noncatalytic sites, presumably as a result of the release of the inhibitory moiety.

[71] Purification and characterization of a light-activated rod outer segment phosphodiesterase

Publisher Summary This chapter discusses the purification and characterization of a light-activated rod outer segment phosphodiesterase. There are abundant data to demonstrate that levels of cyclic guanosine monophosphate (cGMP) in the vertebrate rod outer segment (ROS) are rapidly reduced by light. The explosive hydrolysis of cGMP in vertebrate rods is mediated by a light and guanosine triphosphate (GTP)-dependent cGMP phosphodiesterase (PDE). This PDE is a peripheral membrane protein that has been found in association with amphibian and mammalian rods. Action spectra and reconstitution experiments show that rhodopsin (Rho) is the photopigment that mediates light activation of rod PDE. The purification and characterization of rod PDE has provided significant advantages in studying the mechanism of its activation and its physiological significance. When the PDE is in its native position, activation is supported by light and GTP. The isoelectric point for the purified PDE has been determined. Using an isoelectric focusing technique adapted for nondenaturing polyacryl-amide gels, the purified PDE behaves as a single protein with an isoelectric point of pH.

[20] Preparation of vertebrate photoreceptor membranes for study of adenylate cyclase, guanylate cyclase, and cyclic nucleotide phosphodiesterase

Publisher Summary Photoreceptor membranes (vertebrate rod outer segments) are purified by flotation on heavy sucrose. All procedures except deliberate light adaptation are carried out in the complete absence of visible light with the aid of infrared light sources (made from ordinary microscope or high intensity tungsten lamps fitted with a Corning CS No. 7–56 infrared filter) and infrared image converters or “sniperseopes.” Adenylate cyclase and guanylate eyclase are assayed using radioactive substrate ([ 3 H] ATP or [ 3 H]GTP) and isolating the cAMP or GMP by thin-layer chromatography. The assay mixtures for the cyclases contain a regenerating system (creatine phosphokinase and phosphoereatine) and an inhibitor of phosphodiesterase (isobutyl methylxanthine). Phosphodiesterase is assayed using [ 3 H] cAMP or [ 3 H] cGMP and isolating the remaining substrate by thin-layer chromatography. The standard reaction mixture for phosphodiesterase contains only substrate, buffer, and magnesium chloride.