Arthur S. Polans

The effect of recoverin-like calcium-binding proteins on the photoresponse of retinal rods.

The rod photoresponse is triggered by an enzyme cascade that stimulates cGMP hydrolysis. The resulting fall in cGMP leads to a decrease in Ca2+, which promotes photoresponse recovery by activating guanylate cyclase, causing cGMP resynthesis. In vitro biochemical studies suggest that Ca2+ activation of guanylate cyclase is medicated by recoverin, a 26 kd Ca(2+)-binding protein. To evaluate this, exogenous bovine recoverin and two other homologous Ca(2+)-binding proteins from chicken and Gecko retina were dialyzed into functionally intact Gecko rods using whole-cell recording. All three proteins prolonged the rising phase of the photoresponse without affecting the kinetics of response recovery. These results suggest that recoverin-like proteins affect termination of the transduction cascade, rather than mediate Ca(2+)-sensitive activation of guanylate cyclase.

Calbindin D-28K immunoreactivity of human cone cells varies with retinal position

Calbindin D-28K is a calcium-binding protein found in the cone but not rod photoreceptor cells in the retinas of a variety of species. Recent studies of the monkey retina indicated that calbindin D-28K may be expressed preferentially in non-foveal regions of the retina. In the current studies of human retinas, immunohistochemical experiments demonstrated that calbindin D-28K is reduced or absent in the fovea and parafovea, but prevalent in the perifovea and periphery. These findings were supported by the quantification of calbindin D-28K in 1-mm trephine punches obtained from different regions of the human retina. The specificity of the anti-calbindin D-28K antibodies used in these studies was confirmed by Western blot analysis using purified calbindin D-28K. The protein was purified from retinal tissue and its identity confirmed by partial amino-acid sequence analysis. The expression of calbindin D-28K did not correlate with the spectral properties of the cones, rather to their position in the retina. The study of spatially expressed genes, like the one encoding calbindin D-28K, may help explain the patterns of retinal degeneration seen in some human cone-rod dystrophies.

Calcium-Binding Proteins in the Retina

Publisher Summary This chapter discusses the purification and characterization of some of the major retinal calcium-binding proteins, such as recoverin and visinin. It is a fact that calcium has been implicated in a wide variety of physiological processes. On binding calcium, calmodulin becomes able to activate a diverse set of enzymes, including adenylate cyclase, protein kinases, and cyclic nucleotide phosphodiesterases. Other binding proteins regulate the diffusion and buffering of intracellular calcium. However, the functions of the majority of calcium-binding proteins are unknown. Since these proteins are expressed in a cell-specific manner, they are thought to be involved in specialized functions of the cell. The portions of their sequences not involved in calcium binding remain highly conserved, thus suggesting that these regions can be important for interacting with effector molecules. In the retina, calcium is thought to act as a modulator of light adaptation. Alterations in calcium concentration affect the recovery phase of the photoresponse as well as the sensitivity of the cell to a flash of light in the presence of background illumination.

Distribution of 5-HT1 binding sites in cat spinal cord.

Quantitative analysis of high affinity [3H]5-HT binding to 5-HT1 receptors in the cervical, thoracic, lumbar, and sacral spinal cord of the cat revealed specific binding throughout the grey matter, with the highest levels of binding in laminae II and III, and the lowest levels in laminae I and VII. Relatively high levels were also observed in the thoracic intermediolateral cell column. There were no significant differences in the degree of binding between various segmental levels. Comparison of these data with published maps of 5-HT immunoreactivity reveals--with the exception of lamina I--a close correspondence between the degree of immunoreactivity and the degree of 5-HT binding. These results suggest that 5-HT plays an important role in a variety of spinal cord sensory, motor and autonomic functions.

Methods for the purification and characterization of calcium-binding proteins from retina

Publisher Summary More than 200 EF hand calcium-binding proteins have been identified; however, the function of only a few is known. This chapter discusses methods that have been used for purifying and characterizing calcium-binding proteins from ocular tissues and for determining their calcium binding parameters. Several strategies have been provided for the purification and characterization of calcium-binding proteins of the EF hand family. Members of this family often can be enriched through calcium-dependent binding to a hydrophobic matrix. Although, Calbindin D-28K, for example, does not bind to phenyl-Sepharose, it can be purified through binding to DEAE-Sepharose in low calcium. In addition to these relatively soluble calcium-binding proteins, other less soluble EF hand proteins (eg. GCAP) can be purified by relatively simple procedures. Such isolation procedures will be of increasing importance for studying signal transduction pathways that involve calcium. The chapter explains that the procedures are adaptable to a variety of tissues and hopefully will facilitate further investigations of calcium-binding proteins.

Recoverin is the tumor antigen in cancerassociated retinopathy

Considerable progress has been made toward understanding the involvement of recoverin in a cancer-associated retinopathy (CAR) that results in blindness. We describe the expression of recoverin in tumors of individuals afflicted with CAR, characterize the immunological response towards recoverin in these patients, and demonstrate how the disease can be induced in rodents using recoverin as an immunogen.

The use of gold reagents to quantitate antibodies eluted from nitrocellulose blots application to electron microscopic immunocytochemistry

An assay is described in which gold reagents were used to quantitate nanogram amounts of antibody that had been eluted from antigens immobilized on nitrocellulose paper. Standard curves were generated by the application of rabbit immunoglobulin G (IgG) to nitrocellulose sheets assembled in a dot blot matrix apparatus. Blots were stained using either colloidal gold or immunogold, enabling quantitation of IgG concentration by scanning densitometry. Linear and reproducible standard curves were obtained. As little as 1 ng IgG/dot could be quantified using either gold reagent. In contrast to colloidal gold, immunogold could be used specifically to quantitate rabbit IgG regardless of the presence of bovine serum albumin or antigen coeluted from the nitrocellulose blot. The applicability of the immunogold assay was demonstrated by fractionating a complex rabbit antiserum raised against the RIM protein of frog retinal rod outer segments. Anti-RIM antibody was affinity-purified, quantitated by the immunogold assay, and subsequently employed in immunocytochemical studies using thin sections of retina embedded in a hydrophilic plastic, LR-Gold.

Introducing Krzysztof Palczewski, the 2014 Recipient of the Friedenwald Award

Kris was raised and educated in Wroclaw, a city in southwestern Poland. He attended the University of Wroclaw, obtaining his Master’s degree in Organic Chemistry. Kris then attended the Technical University, receiving his PhD in Biochemistry under the tutelage of Professor Marion Kochman. Kris studied the structure of the active site and the mononucleotide binding site of an aldolase. Using photo-affinity labeling, Kris identified the amino acid residues responsible for coordinating the mononucleotide. I mention this early study, because it represents the beginning of what will be a theme throughout Kris’ research—his interest in molecular structure, and the relationship between structure and function. Of further significance, Paul Hargrave was a collaborator on this project, and in 1986 Kris came to the United States to join Paul’s laboratory as a postdoctoral fellow at the University of Florida (Gainesville, FL, USA). This was his introduction to vision research. While a postdoctoral fellow, Kris studied the inactivation of photolyzed rhodopsin. Specifically, he isolated and characterized rhodopsin kinase and the protein phosphatase responsible for determining the phosphorylated state of rhodopsin, and then isolated and further characterized arrestin, a modulator of rhodopsin dephosphorylation. In 1989, while in Portland, I had the pleasure to serve as the chair of a search committee looking for a young vision scientist to join the faculty. Of the many talented applicants, Kris was chosen owing to the quality of his work and an unbridled enthusiasm expressed in his cover letter and then in person. Kris postulated that we were nearing the point when further biochemical and structural studies of phototransduction would provide a formalism for understanding disparate retinal pathologies. While in Portland, Kris continued his studies of rhodopsin kinase and arrestin, in what is a second theme characterizing his research, that Kris has never really concluded a project, he simply adds new ones, like rings to a tree. He continued with his interests in molecular structure, crystallizing and then determining the structure of sangivamycin, a small kinase inhibitor. He then added an interest in calcium and calcium-binding proteins; initially describing a protein to become known as recoverin or s-modulin. It’s an EF hand calcium-binding protein found in photoreceptors, now thought to interact with rhodopsin kinase. It’s also involved in a human disease that causes retinal degeneration. The project initiated a long-term collaboration with John Crabb and later a collaborative reunion with Grazyna Adamus. In 1992, Kris was recruited to the University of Washington (Seattle, WA, USA), where he would stay for approximately 13 years. In addition to his studies of rhodopsin kinase, arrestin, and recoverin, Kris expanded his interests in calcium-binding proteins, discovering the family of guanylate cyclase activating proteins (GCAPs). Following light absorption and a decline in intracellular calcium, GCAPs stimulate the cyclase to synthesize cyclic guanosine monophosphate (cGMP), thereby returning its levels toward the dark-adapted state. The project involved electrophysiological collaborations with Peter Detweiler, and genetic and molecular studies that would initiate a long-term collaboration with Wolfgang Baehr, this year’s recipient of the Proctor Award. Continuing with his interests in molecular structure, Kris and his colleagues determined in a seminal study the structure of rhodopsin. We finally learned how rhodopsin is folded in the membrane, as well as critical information about the binding site of the chromophore and interaction sites for other members of the phototransduction cascade. The structural determination also provided a basis for understanding