AbstractAbstract
[en] Using isolated rod outer segment discs from bovine retina the authors have studied the effect of pH on the release of 45Ca. 45Ca was loaded into the discs using the ATP-dependent calcium uptake activity of the disc membrane. Measurements were made at 1 or 5 min. 250 μM cGMP released 12.9 +/- 7% of the 45Ca over the pH range 6.9-7.9 and 3.1 +/- 2.8% at pH 6.4 (45Ca is only a portion of the total disc calcium; actual release is greater). 25 μM 8-BrcGMP (more potent than cGMP at releasing disc calcium) released 10.6 +/- 2.0% of the 45Ca over the 6.9-7.9 pH range and 1.4 +/- 1.5% at pH 6.4. 250 μM cAMP released 1.0 +/- 1.3% of the 45Ca at pH 6.9. The external calcium level was submicromolar in the above experiments. When the external calcium was micromolar or above there was a release of 45Ca from discs incubated at pH 7.9 as compared to those at pH 6.9 of 11.2 +/- 1.9%. The release was apparently due to pH alone as no cyclic nucleotide was present in the incubation buffers. This release at pH 7.9 was not seen when the external calcium was submicromolar. This data indicates that the 45Ca release being studied is specific to cGMP (as cAMP at the same conc. caused minimal release) and that the ability of cGMP to release calcium from ROS discs is attenuated at pH 6.4 as compared to the release at pH 6.9-7.9 (submicromolar external free calcium). It also indicates that high pH alone can cause calcium release from discs when the external free calcium level is micromolar or above
Primary Subject
Source
76. annual meeting of the Federation of American Society for Experimental Biology; Washington, DC (USA); 8-12 Jun 1986; CONF-8606151--
Record Type
Journal Article
Literature Type
Conference
Journal
Federation Proceedings. Federation of American Societies for Experimental Biology; ISSN 0014-9446; ; CODEN FEPRA; v. 45(6); p. 1831
Country of publication
ALKALINE EARTH METALS, ANIMALS, BETA DECAY RADIOISOTOPES, BETA-MINUS DECAY RADIOISOTOPES, BODY, CALCIUM ISOTOPES, DAYS LIVING RADIOISOTOPES, DOMESTIC ANIMALS, ELEMENTS, EVEN-ODD NUCLEI, EYES, INTERMEDIATE MASS NUCLEI, ISOTOPE APPLICATIONS, ISOTOPES, MAMMALS, METALS, NUCLEI, ORGANS, RADIOISOTOPES, RUMINANTS, SENSE ORGANS, VERTEBRATES
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AbstractAbstract
[en] Glucosylation (NEG) (nonenzymatic) of proteins is a posttranslational protein modification that occurs readily in the diabetic environment. As a consequence of NEG some proteins are known to undergo a change in function. Their studies of red blood cell (RBC) cytoskeletal proteins indicate that calmodulin is glucosylated in the diabetic RBC and this is followed by a change in function. Here they present new data in support of their earlier findings. Purified bovine brain calmodulin was glucosylated in vitro in the presence of 28 mM glucose. After six days of incubation at room temperature 2.75 moles of glucose were incorporated per mole of calmodulin. Glucosylated calmodulin exhibited a marked reduction in calcium dependent functions. Its ability to stimulate neuronal phosphodiesterase (PDE) and adenylate cyclase was reduced by 65 and 80% respectively. Its ability to stimulate rat brain protein kinase was reduced by 40%. Glucosylated calmodulin exhibited a 56% drop in its 45Ca binding as compared with unmodified calmodulin. These data provide an additional example in which NEG markedly alters protein function
Primary Subject
Source
76. annual meeting of the Federation of American Society for Experimental Biology; Washington, DC (USA); 8-12 Jun 1986; CONF-8606151--
Record Type
Journal Article
Literature Type
Conference
Journal
Federation Proceedings. Federation of American Societies for Experimental Biology; ISSN 0014-9446; ; CODEN FEPRA; v. 45(6); p. 1693
Country of publication
ALDEHYDES, ALKALINE EARTH METAL COMPOUNDS, ANIMALS, BETA DECAY RADIOISOTOPES, BETA-MINUS DECAY RADIOISOTOPES, BIOLOGICAL MATERIALS, BLOOD, BLOOD CELLS, BODY, BODY FLUIDS, CALCIUM ISOTOPES, CARBOHYDRATES, CENTRAL NERVOUS SYSTEM, DAYS LIVING RADIOISOTOPES, DISEASES, DOMESTIC ANIMALS, ENDOCRINE DISEASES, ENZYMES, EVEN-ODD NUCLEI, HEXOSES, HYDROLASES, INTERMEDIATE MASS NUCLEI, ISOTOPES, KINETICS, LYASES, MAMMALS, MATERIALS, MONOSACCHARIDES, NERVOUS SYSTEM, NUCLEI, ORGANIC COMPOUNDS, ORGANS, PHOSPHORUS-GROUP TRANSFERASES, RADIOISOTOPES, REACTION KINETICS, RUMINANTS, SACCHARIDES, TRANSFERASES, VERTEBRATES
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AbstractAbstract
[en] The calmodulin (CaM) content of fully intact frog rod outer segments (ROS) has been measured using radioimmunoassay. The molar ratio between rhodopsin and total CaM in ROS is 800:1. In the absence of Ca2+, the ROS membrane fraction contains only 4% of total ROS CaM. In contrast, in the presence of Ca2+, 15% of total ROS CaM is found in the membrane fraction. For half-maximal binding of CaM to CaM-depleted ROS membranes, 3 x 10-7 M Ca2+ is required. This CaM binding is inhibited by trifluoperazine. CaM binding proteins in the ROS membrane fraction are identified by using two different methods: the overlay method and the use of 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), a bifunctional cross-linking reagent. Ca2+-dependent CaM binding proteins with apparent molecular weights of 240,000, 140,000, 53,000, and 47,000 are detected in the ROS membrane fraction by the overlay method. Anomalous, Ca2+-independent CaM binding to rhodopsin is also detected with this method, and this CaM binding is inhibited by the presence of Ca2+. With the bifunctional cross-linking reagent, DTSSP, three discrete proteins with molecular weights of 240,000, 53,000, and 47,000 are detected in the native ROS membrane fraction. CaM binding to rhodopsin is not detected with this method. These data suggest that both the Ca2+-independent binding of CaM to rhodopsin and the Ca2+-dependent binding of CaM to the M/sub r/ 140,000 protein represent binding of CaM to a site(s) which is (are) exposed only after denaturation. Ca2+-dependent CaM binding in the cytoplasmic fraction is also evaluated with the overlay method. These data suggest that CaM and its binding proteins participate in the regulation of Ca2+-sensitive processes primarily on the ROS disk membranes
Primary Subject
Record Type
Journal Article
Journal
Country of publication
ALKALI METAL COMPOUNDS, ALKALINE EARTH METAL COMPOUNDS, ANIMALS, AQUATIC ORGANISMS, BETA DECAY RADIOISOTOPES, BODY, CELL CONSTITUENTS, CHEMICAL REACTIONS, CHEMISTRY, DAYS LIVING RADIOISOTOPES, ELECTRON CAPTURE RADIOISOTOPES, HALIDES, HALOGEN COMPOUNDS, HALOGENATION, INTERMEDIATE MASS NUCLEI, IODIDES, IODINE COMPOUNDS, IODINE ISOTOPES, ISOTOPE APPLICATIONS, ISOTOPES, MEMBRANES, NUCLEI, ODD-EVEN NUCLEI, ORGANIC COMPOUNDS, ORGANS, PIGMENTS, RADIOISOTOPES, SENSE ORGANS, SODIUM COMPOUNDS, TRACER TECHNIQUES, VERTEBRATES
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Yamazaki, A.; Uchida, S.; Stein, P.J.; Wheeler, G.L.; Bitensky, M.W.
Advances in cyclic nucleotide and protein phosphorylation research. Volume 161984
Advances in cyclic nucleotide and protein phosphorylation research. Volume 161984
AbstractAbstract
[en] Light initiates a series of biochemical and biophysical changes in vertebrate rod outer segments (ROS). A variety of studies have indicated that several discrete biochemical steps occur between photon capture and the subsequent change in rod membrane voltage. Cyclic GMP is a possible candidate for a transmitter molecule in the photon transduction process. Electrophysiological studies have indicated that iontophoresis of cyclic GMP into dark adapted rods is accompanied by a reduction in plasma membrane voltage (33). Characterization of rod phosphodiesterase (PDE) indicates a large turnover number and rapid rates of activation following illumination, which are compatible with a role of cyclic GMP in the (approximately 100 msec) time span for visual excitation. An attractive hypothesis assigns a transmitter role to disc Ca2+ and is supported by many intriguing experiments. There is, however, still disagreement concerning the effect of cyclic GMP on rod Ca2+ dynamics. These and other studies have suggested that light regulation of PDE plays an important role either in support of transduction or in support of light adaptation or the regulation of photoreceptor sensitivity. Work has continued to focus on elaboration of the mechanism for the light activation of ROS PDE. In the following section, the authors briefly review the current understanding of the activation mechanism and describe some newer findings now emerging from our ongoing studies
Primary Subject
Source
Strada, S.J.; Thompson, W.J. (eds.); p. 381-392; 1984; p. 381-392; Raven Press; New York, NY (USA)
Record Type
Book
Country of publication
ALKALINE EARTH METAL COMPOUNDS, BODY, CHARGED PARTICLES, ELECTROMAGNETIC RADIATION, ENZYMES, HETEROCYCLIC COMPOUNDS, HYDROGEN COMPOUNDS, HYDROLASES, IONS, NUCLEOSIDES, NUCLEOTIDES, ORGANIC COMPOUNDS, ORGANIC NITROGEN COMPOUNDS, ORGANS, OXYGEN COMPOUNDS, PIGMENTS, PROTEINS, PURINES, RADIATIONS, RIBOSIDES, SENSE ORGANS, SENSITIVITY
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AbstractAbstract
[en] In illuminated rod outer segment membranes, GTP and guanosine 5'-[ß, ß-imido]triphosphate (p[NH]ppG) have reciprocal effects on cGMP phosphodiesterase (PDEase; 3':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) activity and cGMP binding to noncatalytic sites on that enzyme. Two micromolar p[NH]ppG increased PDEase activity more than 2-fold while inhibiting cGMP binding more than 40%. Reduction of noncatalytic cGMP binding, which followed addition of p[NH]ppG, was not a result of PDEase activation. Both effects of p[NH]ppG were completely dependent on the presence of bleached rhodopsin. A heat-stable factor has been found to inhibit PDEase activity and also to stimulate cGMP binding to noncatalytic cGMP binding sites. Addition of p[NH]ppG reversed the effects of this factor on both PDEase activity and cGMP binding. During purification of this material, the activity peaks for both PDEase inhibition and activation of noncatalytic cGMP binding comigrated on both Blue Sepharose CL-6B column chromatography and sucrose density gradients centrifugation, suggesting that the same factor could be responsible for both inhibition of PDEase activity and enhancement of noncatalytic cGMP binding. Limited tryptic proteolysis of PDEase, which markedly reduced cGMP binding to the noncatalytic sites, and experiments using highly purified cAMP (free of cGMP) as substrate for PDEase showed that the binding of cGMP to noncatalytic sites was not required for the heat-stable inhibitory factor to inhibit PDEase activity. We discuss possible relationships between the regulation of PDEase and the binding of cGMP to noncatalytic sites
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Record Type
Journal Article
Literature Type
Numerical Data
Journal
Proceedings of the National Academy of Sciences of the United States of America; ISSN 0027-8424; ; v. 79 p. 3702-3706
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