Robert L. Somers

The Oxidative DNA Lesion 8,5′-(S)-Cyclo-2′-deoxyadenosine Is Repaired by the Nucleotide Excision Repair Pathway and Blocks Gene Expression in Mammalian Cells

Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide excision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a characteristic neurodegeneration believed to result from their inability to repair neuronal DNA damaged by endogenous metabolites since the harmful UV radiation in sunlight does not reach neurons. Free radicals, which are abundant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis for 8,5′-(S)-cyclo-2′-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hamster ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is repaired by NER and not by base excision repair. We measured host cell reactivation, which reflects a cells capacity for NER, by transfecting CHO and XP cells with DNA constructs containing a single cyclo-dA or a cyclobutane thymine dimer at a specific site on the transcribed strand of a luciferase reporter gene. We found that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poorly in NER-deficient CHO cells and in cells from patients in XP complementation group A with neurodegeneration. Based on these findings, we propose that cyclo-dA is a candidate for an endogenous DNA lesion that might contribute to neurodegeneration in XP.

IGF-I receptors in the bovine neural retina: Structure, kinase activity and comparison with retinal insulin receptors

The retina contains specific high-affinity receptors for insulin-like growth factor-I (IGF-I). Although IGF-I binding was observed in photoreceptor outer segments, the level of this binding was only 10% of that found in whole retina or mixed preparations of rod outer (ROS) and inner (RIS) segments. The higher IGF-I binding activity in RIS and non-photoreceptor regions of the retina suggests these sites as candidates for putative IGF-I action. Data from crosslinking experiments with and without neuraminidase treatment indicate that the binding subunits of the retinal IGF-I receptor exist in two subpopulations (Mr = 121- and 131 kDa), and that the larger of the two subunits has either a greater number or more exposed sialic acid residues. In these characteristics, the retinal IGF-I receptor is similar to the retinal insulin receptor. Retinal IGF-I and insulin receptors possess kinase activity towards their own beta-subunits, a tyrosine containing copolymer, and various molecular forms and subunits of transducin (T alpha-GDP, T alpha-GTP, T beta). The transducin forms are phosphorylated with different efficiencies (e.g. T alpha-GDP is 10-15 times more effective than T alpha-GTP as substrate). These differences are also observed in basal conditions and may reflect differences in transducin subunit affinity for the IGF-I and insulin receptor. In all retinal areas examined, tracer IGF-I binding is 10 to 20-fold higher than insulin binding; however, autophosphorylation levels are approximately equal.

GTP binding protein: properties and lack of activation by phosphorylated rhodopsin.

Taking advantage of the capability of GTP binding protein to bind GTP, we purified the catalytic subunit (G alpha) of bovine rod GTP binding protein by nucleotide-affinity chromatography on Blue Sepharose CL6B. Purified G alpha was essentially free of bound guanine nucleotide and activated by photoactivated rod membranes. Circular dichroism spectra suggested that a significant portion of the protein would be in alpha-helical conformation. No appreciable differences were detected in the circular dichroism spectra when G alpha . GDP and G alpha . GppNp were compared. The extent of G protein activation by rod membranes was reduced moderately by phosphorylation of rhodopsin during photolysis. However, if the pigment had been phosphorylated and regenerated, the ability of rhodopsin to activate G protein was markedly suppressed.

Antibodies against a retinal guanine nucleotide-binding protein cross-react with a single plasma membrane protein in non-retinal tissues.

Antisera (AS/1‐AS/6) to purified bovine retinal transducin, a guanine nucleotide‐binding protein, were produced in 6 rabbits. Immunoblots showed that the antisera varied in their reactivity with the subunits of transducin; AS/1 reacted strongly with all 3 subunits, while the others reacted with only the β and/or γ subunits. Only AS/1 specifically immunoprecipitated the α subunit radiolabeled with non‐covalently bound guanine nucleotides. Immunostaining of plasma membrane proteins from non‐retinal tissues with AS‐1 revealed a single protein (approx. 35 kDa), most likely representing the β subunit of the guanine nucleotide‐binding proteins (Gs and gi associated with adenylate cyclase. Cerebral cortex showed the highest content of this protein. Antisera against transducin provide a highly specific and sensitive probe for quantitation of the β subunit of Gs and Gi

Development and Regulation of Rhodopsin Kinase in Rat Pineal and Retina

Abstract: Rhodopsin kinase, once thought to be a retinal enzyme, was recently found at high levels in the pineal gland. In the present study the developmental pattern and the regulation by environmental lighting of this enzyme in both tissues was studied in the rat. Enzyme activity was present in the neonatal pineal gland several days earlier than in the retina, and increased gradually up to 20 days of age and remained at that level thereafter; the retinal enzyme appeared to increase until day 60. Pineal and retinal rhodopsin kinase activities showed a 25% increase in in the middle of the dark and the beginning of the light period, respectively. Exposure to constant light caused a 50% decrease in rhodopsin kinase levels in both tissues. However, only pineal rhodopsin kinase activity declined followed bilateral superior cervical ganglionectomy. This indicates pineal rhodopsin kinase activity is similar to other pineal enzymes in that it is controlled by light acting through the sympathetic nervous system. In contrast, the light‐induced decrease in retinal rhodopsin kinase may be due to the direct destructive effect of light on the retina. The finding of neural control of pineal rhodopsin kinase in the pineal gland of adult rats is consistent with a function of the enzyme in the neural regulation of pineal function.

Cholera toxin modifies diverse GTP-modulated regulatory proteins

Abstract Cholera toxin (with NAD) has been employed to ADP-ribosylate the putative GTP regulatory proteins in a range of plasma membrane preparations which are associated with either stimulation or both stimulation and inhibition of adenylate cyclase activity. In systems subject to dual regulation, additional bands are encountered which may reflect the inhibitory GTP regulatory unit. In addition, the GTP regulatory proteins involved in the light activation of retinal phosphodiesterase and protein synthesis initiation factor, eIF-2, have been ADP-ribosylated by cholera toxin. In the latter case, the activity of the factor is grossly impaired following the covalent modification. It is suggested that considerable conservation of structure is maintained in this rather diverse group of GTP regulatory proteins.

SPATIAL ARRANGEMENT OF RHODOPSIN IN THE DISK MEMBRANE AS STUDIED BY ENZYMATIC LABELING

Abstract—Intact disks prepared from fresh bovine retinas were phosphorylated with γ‐[32P]‐ATP and highly purified rhodopsin kinase. After regeneration. the visual pigment was cxtracted and purified on ECTEOLA‐cellulose. The purified pigment contained [32P]‐radioactivity. When intact disks were frozen and thawed. the membrane was inverted and became capable of binding concanavalin A. The inversion process could be followed by measuring the entrapment by the disk of [3H]‐inulin. the impermeable polysaccharide. When the inverted disks were incubated with UDP‐[3H]‐galactose and galactosyltransferase and rhodopsin was extracted and purified. [3H]‐gahctose was incorporated into the pigment. From these results we conclude that rhodopsin is a transmembrane protein with its carbohydrate moiety on the internal (intradiscal) surface and its phosphorylation sites on the external (interdiscal) surface

[38] Rhodopsin kinase

Publisher Summary This chapter focuses on rhodopsin kinase. Rhodopsin kinase—the enzyme involved in the reaction—catalyzes the transfer of the terminal (γ) phosphate group of adenosine triphosphate (ATP) to the opsin protein. In the assay method of rhodopsin kinase, bovine rod outer segments are treated with urea to prepare the substrate. This treatment denatures membrane-associated rhodopsin kinase but has little effect on rhodopsin. The substrate is then incubated in the light with rhodopsin kinase, [y -32p ]ATP, and [adenine- 3 H]ATP. The inclusion of [ 3 H]ATP is to estimate the extent of binding of ATP itself. In an alternative method, rod membranes are incubated with [γ -32P ]ATP in dark and then in light. The kinase activity is determined by subtracting the 32 P incorporated in dark from the 32 P incorporated in light. The phosphorylated protein is identified as rhodopsin after the separation of the labeled protein by chromatographic and electrophoretic methods.

Light-stimulated GTP binding to a membrane protein in rod outer segments.

Abstract Bovine as well as frog rod outer segments contain a membrane-bound protein which binds the GTP analog GppNp in the light (Kd=0.3μM). The amount of GppNp bound is 2.5–3.5 mmole per mol rhodopsin. The binding protein (M.Wt. ⋍ 54,000) can be extracted from rod membranes with detergent and purified on an Agarose column. The chromatographic profile indicates that the binding protein is distinct from rhodopsin, GTPase or cyclic nucleotide phosphodiesterase.

Isolation of three isochromic forms of rhodopsin in digitonin

Abstract Rhodopsin can be chromatographically purified by a combination of ECTEOLA-cellulose and an aqueous digitonin solution. The purification procedure separates three fractions of rhodopsin which are spectrally identical but are different in their bleaching kinetics.