Solubilization of membrane-bound rod phosphodiesterase by the rod phosphodiesterase recombinant delta subunit.

Retinal rod and cone phosphodiesterases are oligomeric enzymes that consist of a dimeric catalytic core (alpha'2 in cones and alphabeta in rods) with inhibitory subunits (gamma) that regulate their activity. In addition, a 17-kDa protein referred to as the delta subunit co-purifies with the rod soluble phosphodiesterase and the cone phosphodiesterase. We report here partial protein sequencing of the rod delta subunit and isolation of a cDNA clone encoding it. The predicted amino acid sequence is unrelated to any other known protein. Of eight bovine tissue mRNA preparations examined by Northern analysis, the strongest delta subunit-specific signal was present in the retina. A less intense signal was seen in the brain and adrenal mRNA. In bovine retinal sections, rod delta subunit anti-peptide antibodies label rod but not cone outer segments. delta subunit, added back to washed outer segment membranes, solubilizes a large fraction of the membrane-bound phosphodiesterase, indicating that this subunit binds to the classical membrane associated phosphodiesterase. The subunit forms a tight complex with native, but not trypsin-released phosphodiesterase, suggesting that the isoprenylated carboxyl termini of the catalytic subunits may be involved in binding of the delta subunit to the phosphodiesterase holoenzyme.

Affinities of bovine photoreceptor cGMP phosphodiesterases for rod and cone inhibitory subunits.

Rods and cones have analogous phototransduction components and cycles, but differ from each other in their physiological response to light. Differences between the affinities of rod and cone phosphodiesterase (PDE) catalytic subunits for their respective inhibitory subunits could potentially contribute to these physiological differences. To test this idea, we expressed both the 13 kDa PDE subunit, unique to a subset of bovine retinal cones [(1990) J. Biol. Chem. 265, 11259-11264], and the rod PDE 11 kDa inhibitory subunit in E. coli, purified them, and compared their abilities to inhibit rod and cone PDE catalytic subunits. Rod PDE has similar Ki values (approximately 80 pM) for both the rod and cone recombinant inhibitory subunits. Activated cone PDE has Ki values of 200 pM for the cone 13 kDa subunit and 600 pM for rod PDE gamma.

Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG.

Agrobacterium tumefaciens virulence genes are induced by plant signals through the VirA-VirG two-component regulatory system. The VirA protein is a membrane-spanning sensor molecule that possesses an autophosphorylating activity, and the VirG protein is a sequence-specific DNA-binding protein. In this report, we demonstrate that the VirG protein is phosphorylated by the VirA protein and that the phosphate is directly transferred from the phosphorylated VirA molecule (phosphohistidine) to the VirG protein. The chemical stability of the phospho-VirG bond suggested that the VirG protein was phosphorylated at the aspartate and/or glutamate residue. The phosphorylated VirG protein was reduced with tritiated sodium borohydride and subjected to proteolytic digestion with the Achromobacter protease I enzyme. The resulting peptide fragments were separated by C8 reversed-phase high-pressure liquid chromatography, and the tritium-labeled peptide was sequenced. Amino acid sequence data showed that the aspartate residue at position 52 was the only site phosphorylated. Changing this aspartate into asparagine resulted in a nonphosphorylatable and biologically nonfunctional gene product. As a control, a randomly chosen aspartate was changed into an asparagine (position 72), and no effect on its phosphorylation or biological activity was observed. Unlike its homologs, including CheA-CheY, EnvZ-OmpR, and NtrB-NtrC, the phospho-VirG molecule was very stable in vitro. The possible implications of these observations and the function of VirG phosphorylation in vir gene activation are discussed.

Identification of a noncatalytic cGMP-binding domain conserved in both the cGMP-stimulated and photoreceptor cyclic nucleotide phosphodiesterases.

Partial amino acid sequence has been determined for the cone, alpha' subunit of the bovine photoreceptor cyclic nucleotide phosphodiesterase (PDE) and deduced from nucleotide sequences of a partial cDNA clone. These sequences identify the alpha' subunit as the product of a gene that is distinct from those encoding the alpha or beta subunits of the membrane-associated rod photoreceptor PDE. Comparisons between the recently determined cGMP-stimulated-PDE sequence and those of the alpha and alpha' photoreceptor PDE subunits reveal an unexpected sequence similarity. In addition to the catalytic domain conserved in eukaryotic PDEs, all three PDEs possess a second conserved segment of approximately 340 residues that contains two internally homologous repeats. Limited proteolysis and direct photolabeling studies indicate that the noncatalytic, cGMP-binding site(s) in the cGMP-stimulated PDE is located within this conserved domain, suggesting that it also may serve this function in the photoreceptor PDEs. Moreover, other PDEs that do not bind cGMP at noncatalytic sites do not contain this conserved domain. The function of the conserved segment in the photoreceptor PDEs is not known, but the homology to allosteric sites of the cGMP-stimulated PDE suggests a role in cGMP binding and modulation of enzyme activity.

The Agrobacterium tumefaciens virC1 gene product binds to overdrive, a T-DNA transfer enhancer.

In Agrobacterium tumefaciens, a cis-active 24-base-pair sequence adjacent to the right border of the T-DNA, called overdrive, stimulates tumor formation by increasing the level of T-DNA processing. Recent results from our laboratory have suggested that the virC operon which enhances T-DNA processing probably does so because the VirC1 protein interacts with overdrive (N. Toro, A. Datta, M. Yanofsky, and E. W. Nester, Proc. Natl. Acad. Sci. USA 85:8558-8562, 1988). We report here the purification of the VirC1 protein from cells of Escherichia coli harboring a plasmid containing the coding sequences of the virC locus of the octopine Ti plasmid. By gel mobility shift and DNase I footprinting assays, we showed that this purified virC1 gene product binds to overdrive but not to the right border of T-DNA.

A soluble form of bovine rod photoreceptor phosphodiesterase has a novel 15-kDa subunit.

A substantial fraction (20-30%) of the bovine rod outer segment phosphodiesterase (PDE) activity is not associated with outer segment membranes prepared with buffers of moderate ionic strength; this PDE activity appears to represent a distinct, soluble isozyme. Although this PDE isozyme can be demonstrated to be present in sealed rod outer segments, it is discarded from most standard rod outer segment preparations. A method was developed that allowed the rapid purification of the soluble rod PDE by 2600-fold, to apparent homogeneity, using a monoclonal antibody column (ROS-1a). The soluble rod PDE isozyme has a novel Mr = 15,000 subunit (delta) in addition to subunits of Mr = 88,000 (alpha sol), 84,000 (beta sol), and 11,000 (gamma sol). The delta subunit comigrates with and may be identical to the cone PDE 15-kDa subunit. The small subunits of the soluble rod PDE and the membrane-associated rod PDE were isolated by reverse-phase chromatography. The gamma sol subunit was a potent inhibitor of trypsin-activated rod PDE, inhibiting 50% of 1 pM PDE activity at a concentration of 11 pM. This concentration was similar to that observed for the gamma subunit of the membrane-associated rod PDE. The purified delta subunit did not appear to affect PDE activity; this subunit was, however, unusually difficult to keep in solution. All of the kinetic and physical properties of the soluble rod PDE tested thus far are similar to those of the membrane-associated form, except for the presence of the delta subunit, suggesting that this unique subunit could mediate the solubility of the soluble rod PDE and the cone PDE in the intact photoreceptor.

Catechol stimulation of ferricyanide Hill reaction by spheroplasts of cyanobacterium, Synechococcus cedrorum: effect of temperature on catechol-stimulated oxygen evolution.

Catechol(o-dihydroxybenzene) at low concentrations (20-100 microM) stimulates FeCN-dependent O2 evolution of spheroplasts isolated from the cyanobacterium Synechococcus both in the presence and absence of DBMIB, an inhibitor of electron flow from PSII to PSI, the stimulation being two-fold with saturating concentration of (60 microM) catechol. Catechol thus appears to mediate the acceptance of electrons at the reducing side of PSII. Similarly it may act on the component of electron donor to PSII and caused the photoreduction of FeCN when O2 evolution capacity of spheroplasts is damaged by heat treatment. Analysis of the temperature effect on FeCN-supported O2 evolution by spheroplasts suggests that catechol shifts the temperature maxima to a lower temperature and thereby hastens the decay of O2 evolution capacity by heat as compared to the normal spheroplasts. Catechol also induces a change in the magnitude of activation energy for ferricyanide Hill activity of spheroplasts and lowers the transition temperature. These results suggest that lipophilic catechol brings about an alteration in membrane fluidity in cyanobacterial spheroplasts. Catechol is involved in a thermotropic destabilization of the membrane of the cyanobacterium. However, Al3+ was found to stabilize the membrane and raise the phase transition temperature. Further increase in temperature caused a gradual decline in the rate of O2 evolution.

Light-adaptation in the photophobic response by Stentor coeruleus.

Effects of preillumination on photophobic response (light-adaptation) and recovery of the photophobic sensitivity in the dark (dark-adaptation) in Stentor coeruleus were examined. When the cells were preilluminated with white light of 7.80 W/m2 for 2 min, the fluence-rate response curve of photophobic response was shifted toward higher light intensities by half an order of magnitude compared to the one without preillumination. Preillumination with a higher light intensity resulted in a further shift of the fluence-rate response curve. An action spectrum for light-adaptation showed a primary peak at 610 nm and secondary peaks at 540 and 480 nm which are almost identical to the peaks observed in the photophobic action spectrum. The light-adapted cells showed a recovery of their photophobic sensing ability following dark treatment. Dark-adaptation resulted in total recovery of photophobic sensing ability in 8 minutes for the most cases examined.

Phototaxis and photophobic responses in Stentor coeruleus. Action spectrum and role of Ca2+ fluxes.

Negative phototactic orientation, step-up photophobic responses and light-induced action potentials have been studied in the ciliate Stentor coeruleus. A resolved action spectrum, based on fluence rate-response curves, is consistent with stentorin as the photoreceptor. Calcium flux blockers prolong the response time for ciliary stop and reversal and inhibit step-up photophobic responses. Drugs believed to affect the membrane-bound calcium pump likewise inhibit phobic responses. On the other hand, alpha-phosphatidic acid promotes Ca2+-influx and enhances the photophobic sensitivity of the organism, thus providing an unambiguous evidence for the role of Ca2+ influx. A change in the response time decreases the degree of phototactic orientation, indicating that negative phototaxis in this organism is brought about by subsequent phobic responses of individual rows of cilia as the associated photoreceptor granules experience an increase in light intensity when the organism rotates during forward locomotion in lateral light.