Biomedical subjects

T A Shuster

Publications and source records attributed to T A Shuster.

14 recordsLinked to original sources

PubMed1996

Zinc causes an apparent increase in rhodopsin phosphorylation.

PURPOSE: Rhodopsin is a zinc-binding protein. We investigated the effect of low concentrations of zinc on the initial phosphorylation of rhodopsin. METHODS: Dark-adapted bovine rod outer segments (ROS) were incubated with (gamma 32P) ATP and 5 mM magnesium in the presence and absence of micromolar amounts of zinc. The ROS were exposed to light to initiate phosphorylation under conditions which allow only limited initial phosphorylation. RESULTS: We found that zinc enhanced the rhodopsin phosphorylation apparent on autoradiographies by several fold. Phosphorylation reactions conducted in the presence of potent phospho-opsin phosphatase inhibitors show a comparable zinc-enhanced phosphorylation of rhodopsin. Under our reaction conditions, ROS membranes also appear more red upon initial exposure to light when zinc is present. CONCLUSIONS: Zinc can increase initial rhodopsin phosphorylation, apparently acting at the substrate rhodopsin and not at relevant phosphatases or rhodopsin kinase. How zinc binding to rhodopsin might increase its ability to serve as a substrate for phosphorylation is under investigation.

Animals↗

PubMed1995

ATP-binding proteins on the external surface of synaptic plasma membranes: identification by photoaffinity labeling.

8-Azidoadenosine triphosphate labeled in the alpha or gamma position with 32P was used as a photoaffinity reagent for identifying ATP binding sites on the external surface of intact rat brain synaptosomes. As revealed by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns. UV irradiation of intact synaptosomes in the presence of the above radioactive compounds at 5-10 microM resulted in the formation of several major radioactive conjugates with approximate molecular masses of 29, 45/46, 58, and 93 kDa. Minor bands of 20, 39, 52/54, 82/84, 120, and 140 kDa were also consistently labeled in these experiments. The possibility that labeling of these proteins was due to the presence of contaminating subcellular particles or intrasynaptosomal proteins was excluded. The major 8-azidoadenosine [alpha-32P]triphosphate-labeled protein complex of approximately 45/46 kDa was resolved into several subbands that are labeled differently depending on the type of divalent cations added to the photoaffinity reaction. In the presence of magnesium only, the major labeled band appeared at 45 kDa. With calcium, two additional subbands (43 and 46 kDa) could be distinguished. In the presence of 1 mM EDTA, a band at 44 kDa was labeled within this ATP-binding complex. The labeling pattern of the subbands of this 45/46-kDa complex is consistent with these bands being extracellular ATP-binding proteins on the surface of the synaptosome.

Adenosine Triphosphate↗

PubMed1992

Direct zinc binding to purified rhodopsin and disc membranes.

Using the radionuclide 65Zn, we have demonstrated the direct binding of zinc to purified rhodopsin. 65Zn is eluted with detergent-solubilized rhodopsin from concanavalin A columns and remains bound to the visual pigment through a subsequent gel-filtration step. Zinc binding to purified disc membranes is highly specific and, of the ions tested, copper is the best competitor. Equilibrium-dialysis experiments indicate that zinc binding to detergent-solubilized forms of rhodopsin may increase on bleaching the photopigment. These results may have important implications for studies that indicate that zinc plays a role in retinal degeneration and normal photoreceptor physiology.

Animals↗

PubMed1989

Isolation of rod outer segments on Percoll gradients: effect of specific protease inhibition.

Rat rod outer segments (ROSs) were isolated by vortexing retinas and separating the detached components on performed Percoll gradients. A lighter band of 20 x 10(6) unsealed ROSs per ten retinas, and a heavier band of 60 x 10(6) sealed ROSs per ten retinas were obtained from each 12 ml gradient. The yield of sealed ROSs (but not unsealed ROSs) was increased up to twofold in the presence of the specific cysteine protease inhibitor, Ep-475. Aprotinin, pepstatin, PMSF, TPCK and EGTA plus EDTA had no effect. These results indicate that during isolation, ROSs are vulnerable to damage by cysteine protease activity either from damaged retinal cells or from within.

Animals↗

PubMed1989

Rat brain synaptosomal ATP:AMP-phosphotransferase activity.

Adenylate kinase activity (ATP:AMP-phosphotransferase; EC 2.7.4.3) was studied in various subcellular fractions of rat brain tissues. Because of the presence of other adenosine nucleotide-utilizing enzymes, adenylate kinase activity was assayed in both the forward and reverse directions by using coupled enzyme systems and by using a specific adenylate kinase inhibitor, P1,P5-di(adenosine-5') pentaphosphate. As expected, the highest specific adenylate kinase activity (2.89 mumol/min/mg of protein) was detected in the cytosolic brain fraction. However, substantial enzyme activity (0.68 mumol/min/mg) was also found in the intact synaptosomal fraction isolated on Percoll/sucrose gradients. The increased specific enzyme activity of purified synaptosomes and the differences found between the kinetic parameters of the membrane-bound and cytosolic enzyme forms suggest that the synaptosomal adenylate kinase activity cannot be attributed to the small amount of contaminating cytosol present in our preparations. The adenylate kinase enzyme adhered to purified synaptic plasma membranes and was not released by washings with isoosmotic sucrose medium. The facts that the adenylate kinase enzyme activity could be measured in intact synaptosomal preparations and that both its substrates and its inhibitors do not cross intact plasma membranes support the possibility that the synaptosomal adenylate kinase is an ecto-enzyme.

Adenylate Kinase↗

PubMed1988

8-Azido-ATP (alpha 32P) binding to rod outer segment proteins.

ATP has important roles in the vertebrate rod outer segment (ROS) physiological response to light. One of them is the quench of light-activated cGMP-phosphodiesterase activity. How ATP quenches PDE is not established; however, leading hypotheses favor the intervention of a 48-kDa ATP-binding protein and/or an ATP-utilizing rhodopsin kinase in this reaction. We have investigated the binding of [alpha 32P]8-azido-ATP to rat ROS proteins in the presence and absence of various divalent cations and competitive nucleotides. An event we have detected which might further clarify the role of ATP in PDE inactivation is a zinc-induced binding of azido-ATP to rhodopsin. Manganese is also effective in inducing this binding, while magnesium and calcium are not. The azido-ATP binding is eliminated by the addition of ATP, but not GTP, UTP, cGMP, or cAMP. A nucleotide-binding site on the rim protein is also suggested from these studies.

Adenosine Triphosphate↗

PubMed1988

Nucleotide binding to the rod outer segment rim protein.

The rod photoreceptor outer segment maintains a remarkable morphology. Two of the proteins which have been implicated in the maintenance of this structure are the 240 kDa spectrin-like protein, and the 220 kDa glycoprotein often referred to as the rim protein. We have probed rat rod outer segment proteins with light-activated (azido-labeled) radioactive nucleotides and found a nucleotide binding site(s) on the rim protein which has a preference for guanine nucleotides. Binding to this site is stimulated by the divalent cations zinc, manganese and magnesium, but not calcium. This site is under investigation and may play a role in stabilizing protein structure.

ATP-Binding Cassette Transporters↗

PubMed1987

Identification of a peanut agglutinin-binding protein from human retina.

With the use of an antiserum raised to peanut agglutinin (PNA)-binding protein(s) from human retinas, we have identified a glycoprotein that has the same subunit molecular weight (135,000) as human interphotoreceptor retinol-binding protein (IRBP) on human retinal protein transblots, and, like IRBP, is localized in the interphotoreceptor matrix surrounding both rods and cones. We show that this PNA-binding protein is not IRBP by comparing the binding patterns of antiserum to the PNA-binding protein to binding patterns obtained with antisera to bovine and monkey IRBP.

Arachis↗

PubMed1986

Ecto-ATPase of mammalian synaptosomes: identification and enzymic characterization.

Intact synaptosomes isolated from mammalian brain tissues (rat, mouse, gerbil, and human) have an ATP hydrolyzing enzyme activity on their external surface. The synaptosomal ecto-ATPase(s) possesses characteristics consistent with those that have been described for ecto-ATPases of various other cell types. The enzyme has a high affinity for ATP (the apparent Km values are in the range of 2-5 X 10(-5) M), and is apparently stimulated equally well by either Mg2+ or Ca2+ in the absence of any other cations. The apparent activation constant for both divalent cations is approximately 4 X 10(-4) M in all mammalian brain tissues studied. The involvement of a non-specific phosphatase in the hydrolysis of externally added ATP is excluded. ATP hydrolysis is maximal in the pH range 7.4-7.8 for both divalent cation-dependent ATPase activities. Dicyclohexylcarbodiimide, 2,4-dinitrophenol, trifluoperazine, chlorpromazine, and p-chloromercuribenzoate (50 microM) inhibit the ecto-ATPase, whereas ouabain (1 mM) and oligomycin (3.5 micrograms X mg-1 protein) show little or no inhibition of this enzyme activity. Inhibitor data suggest that the Mg2+- and Ca2+-dependent ecto-ATPase may represent two different enzymes on the surface of synaptosomes.

Adenosine Triphosphatases↗

PubMed1986

Rhodopsin phosphorylation in developing normal and degenerative mouse retinas.

The developmental pattern of rhodopsin phosphorylation in degenerative (rdle homozygote) and normal (rd/+ heterozygote) mouse retina has been investigated. The results indicate that rhodopsin levels are comparable in the 2 retinas up to about 10 days of age but that rhodopsin phosphorylation is not. The phosphorylation of rhodopsin is substantially reduced in the degenerative retina during development. This abnormality may be an expression of the rd lesion. The rhodopsin kinase/phosphatase system, the G protein, and the visual pigment are all involved in the modulation of cGMP-phosphodiesterase activity in normal retinas. A defect in any of these components could account for the reduced level of cGMP-phosphodiesterase activity in rd retinas, resulting in cGMP accumulation and subsequent photoreceptor degeneration.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1986

Light-induced phosphorylation of proteins from the all-cone retina of the lizard, Anolis carolinensis.

Proteins from the all-cone retina of the lizard Anolis carolinensis were phosphorylated using [gamma 32P] ATP, separated by SDS-PAGE and detected by autoradiography. Several proteins incorporated 32P. Exposure of the retinal homogenates to light brought about a dramatic increase in phosphorylation of the protein(s) with a molecular weight nearly identical to that of rat rhodopsin. It is likely that these proteins are the cone visual pigments, and that they incorporate phosphate when bleached by light. Increasing the time of the phosphorylation reaction from 1 to 30 min led to an increase in the amount of incorporation of labeled phosphate by the putative cone visual pigments, but changing the temperature from 4 degrees C to 20 degrees C decreased it. The amount of phosphate incorporation was substantially increased by NaF, a phosphatase inhibitor. This latter finding, along with the changes in incorporation of 32P with increased temperature, suggest that a phosphoprotein phosphatase is active in the lizard retina. The cation requirements, as well as the effects of cyclic nucleotides on light-induced phosphorylation of retinal lizard proteins, were also investigated.

Animals↗

PubMed1984

Phosphorylation in sealed rod outer segments: effects of cyclic nucleotides.

Rod outer segments (ROS) from rat were purified on Percoll gradients. These ROS had intact plasma membranes since they were impermeable to small molecules. Protein phosphorylation in the purified ROS was studied after the plasma membrane was disrupted by freeze/thawing. [gamma-32P]ATP was used as phosphate donor. ATP concentration, time, temperature, and light or dark adaptation were varied in the assays. The 32P-labeled proteins were separated by polyacrylamide gel electrophoresis and autoradiographed. Rhodopsin was the dominant phosphorylated protein, and the addition of adenosine cyclic 3',5'-phosphate (cAMP) or guanosine cyclic 3',5'-phosphate (cGMP) (10(-4) M) did not qualitatively alter the ROS phosphorylation pattern. The only cyclic nucleotide effect we could establish in these experiments was the inhibition of rhodopsin phosphorylation by cGMP. This inhibition did not appear to be competitive with ATP since cAMP was much less inhibitory than cGMP and the phosphorylation in the presence of cGMP reached a plateau at a much lower level than in control conditions. Hypotheses implying an involvement of protein phosphorylation/dephosphorylation in dark adaptation have been formulated [Miller, J. A., & Paulsen, R. (1975) J. Biol. Chem. 250, 4427-4432; Kuhn, H., McDowell, J. H., Leser, K. H., & Bader, S. (1977) Biophys. Struct. Mech. 3, 175-180]; we suggest that cGMP may control this process through the modulation of the extent of inhibition of phosphorylation of the visual pigment.

Adenosine Triphosphate↗

PubMed1983

Adenosine triphosphatase activity at the external surface of chicken brain synaptosomes.

An ATP-hydrolysing activity on the external surface of intact synaptosomes from chicken forebrain has been investigated. The observed ATPase activity was not due to leakage of the intracellular ATPase activities, of artefacts resulting from breakage of the nerve endings during the incubation and isolation periods, or to possible contamination by other subcellular particles. Disruption of the synaptosomes resulted in an approximately 2.5-fold increase of the basal, Mg2+-dependent ATPase activity, suggesting that the plasma membrane was acting as permeability barrier to the substrate. ATP hydrolysis was maximal (0.8 mumol Pi/min/mg protein) at pH 8.2 in a medium containing either Mg2+ or Ca2+ ions. Ouabain (0.2 mM) and oligomycin (2 micrograms/mg protein) had no appreciable effect on this ATPase activity. Kinetic studies of the enzyme revealed an apparent Km value of ATP of approximately 4 x 10(-5) M. These data are consistent with the view that the observed ATP hydrolysis was being catalysed by an ectoenzyme, i.e., an enzyme in the plasma membrane of the nerve endings with its active site facing the external medium. The rapid hydrolysis of the released ATP is a suspected function for this ecto-ATPase.

Adenosine Triphosphatases↗

PubMed1983

Characterization of a protein kinase from chicken oviductal fluids.

Protein kinase has been found extracellularly in avian oviductal secretions. The enzyme has been isolated and shown to be primarily type II cAMP enhanced. The Ka for cAMP activation, binding and elution from ion-exchange columns, molecular weight of subunits, pH optimum as a histone kinase, response to cations, kinetic properties, and preference for lysine-rich histone are similar to those of mammalian type II protein kinase. Gel filtration data suggest that the dimer form is the functional entity in the reproductive tract. The catalytic subunit has been purified to greater than 90% homogeneity and has a Km of 4 mumol/l and Vmax of 10(6) U/mg, comparable to published values for bovine catalytic subunit.

Animals↗