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Stentorin serves as the primary photosensor in the single cell ciliate, Stentor coeruleus, for its photophobic and phototactic response to light of visible wavelengths. We separated two subunits, stentorin-2A and -2B, from the previous stentorin complex ('stentorin-2') of greater than half a million molecular mass isolated from the photoreceptor organelle (pigment granule). Stentorin-2B bears the chromophore covalently linked to an approx. 50 kDa apoprotein, as determined by SDS-urea-PAGE. Partial amino acid sequences were obtained from this 50 kDa subunit. Its visible and CD spectra were found to be similar to those of stentorin-2. The steady-state and time-resolved fluorescence spectra of stentorin-2B, in H2O and D2O buffers, were also similar to those of stentorin-2. This suggests that the 50 kDa subunit retains the spectral integrity and primary photoreactivity of the stentorin-complex. The picosecond time-resolved fluorescence study revealed that the short picosecond emission component (tau F approximately equal to 8-10 ps) was the predominant emitting species in stentorin-2B and -2, followed by longer decaying species. No deuterium solvent effect was seen in this fast-decaying species. The possible mechanism for the primary photoreaction appears to involve electron transfer coupled with proton transfer.

Nucleoside diphosphate kinases (NDPKs) catalyze the transfer of high-energy phosphates from nucleoside triphosphates to nucleoside diphosphates and may be involved in the regulation of growth, development, and signal transduction processes. We report here the purification and characterization of NDPK from detergent-solubilized extracts of dark-grown oat (Avena) tissue. The purification was achieved primarily through adsorption to GTP-agarose, followed by elution with ATP. SDS-polyacrylamide gel electrophoresis and gel filtration chromatography indicated that the purified protein is composed of six 18 kDa subunits. Substrate specificity experiments indicated that the purified kinase is capable of using all tested nucleosides as substrates. N-terminal sequencing of the Avena protein revealed that 87% of the 23 amino acids sequenced were identical to the human Nm23 protein, a nucleoside diphosphate kinase identified as a possible tumor metastasis suppressor and transcriptional activator of the myc oncogene.

Blepharismin is the photoreceptor for the photophobic response in the ciliate Blepharisma japonicum (Scevoli, P., Bisi, F., Colombetti, G., Ghetti, F., Lenci, F., and Passarelli, V. (1987) J. Photochem. Photobiol.: B. Biol. 1, 75-84; Lenci, F., Ghetti, F., Gioffre, D., Heelis, P.F., Thomas, B., Phillips, G.O., and Song, P.-S. (1989) J. Photochem. Photobiol.: B. Biol. 3, 449-453). Blepharismin was solubilized from the red cells with 2% n-octylglucopyranoside. A crude pigment-protein preparation was then successively subjected to Bio-Gel A1.5 filtration, FPLC/hydroxyapatite and FPLC/DEAE ion-exchange chromatography. At least two spectrally distinct forms of blepharismin, with the respective absorbance maxima at 597 ± 1 and 601 ± 1 nm, were resolved. The steady state fluorescence emission maxima were at 602.5 and 617.5 nm, respectively. The fluorescence decay curves for these pigments were non-exponential. The major component possesses relatively short fluorescence lifetime (200–500 ps) for the former, according to a global analysis. This analysis suggests that the excited state of the shorter wavelength-absorbing form of blepharismin undergoes primary photoprocess faster than that of the free parental chromophore hypericin. Photolysis of blepharismin in solution yielded a irreversible product, accompanied by a 10–12 nm bathochromic shift of the absorbance maximum. However, the mechanistic nature of the time-resolved fluorescence and the photochemistry of blepharismin remains to be elucidated.

The unicellular ciliary protozoan, Stentor coeruleus, exhibits photophobic and phototactic responses to visible light stimuli. The pigment granule contains the photoreceptor chromoproteins (stentorins). Stentorin localized in the pigment granules of the cell serves as the primary photoreceptor for the photophobic and phototactic responses in this organism. An initial characterization of the pigment granules has been described in terms of size, absorbance spectra and ATPase activity. Two forms of the stentorin pigments have been isolated from the pigment granules. Stentorin I has an apparent molecular weight of 68,600 and 52,000 by SDS-PAGE (at 10 and 13% gel, respectively) or 102,000 by steric exclusion HPLC, whereas stentorin II is a larger molecular assembly probably composed of several proteins (mol. wt. greater than 500,000). Stentorin I is composed of at least two heterologous subunits corresponding to apparent mol. wts. of 46,000 (fluorescent, Coomassie blue negative) and 52,000 (fluorescent, Coomassie blue positive) on SDS-PAGE (13% gel). However, these values were found to be strongly dependent on the degree of crosslinking in the acrylamide gel. Stentorin II appears to be the primary photoreceptor whose absorption and fluorescence properties are consistent with the action spectra for the photoresponses of the ciliate to visible light.

1. 1.|The absorption spectrum of retinol when bound to β-lactoglobulin is vibrationally resolved. The circular dichroism spectrum exhibits the same structure, as does the fluorescence excitation spectrum. 2. 2.|Two molecules of retinol are bound per protein dimer, with a binding constant (Kd) of 2 · 10−8 M. Also, by fluorescence titration it was found that the monomer binds one molecule of retinol with essentially the same Kd. 3. 3.|Energy transfer occurs from tryptophan (donor) to retinol (acceptor) with a rate constant, k, of 4.4 · 108 s−1. The distance between the centers of mass of the transition is 34 A, corresponding to the energy transfer efficiency of 44%. 4. 4.|The fluoresence lifetime of retinol increases dramatically on binding to β-lactoglobulin, from approx. 2 to approx. 10 ns, as does the fluorescence quantum yield. 5. 5.|The retinol binding to β-lactoglobulin does not show a pH dependence and the binding site is hydrophobic. 6. 6.|On the Sephadex G-100 column, retinol is chemically modified to a retro derivative which binds even more strongly to β-lactoglobulin than does retinol. 7. 7.|The β-lactoglobulin-retinol complex rotates anisotropically in solution with a fast (3 ns) and a slower (12 ns) component. This may be attributed to retinol being bound at a flexible region of the protein, where only segmental flexibility is observed, weighted by its proximity to one of the major axis rotational times.

Fluorescence lifetimes of ‘large’ (mol. wt. 120 000) and ‘small’ (mol. wt. 60 000) phytochromes isolated from oat and rye seedlings grown in the dark have been measured at 199 K and 298 K. Phytochrome model compounds have also been studied by phase modulation fluorometrically at 77 K for comparison with lifetime data for phytochrome. It was found that the fluorescence lifetime of ‘large’ phytochrome was significantly shorter than that of ‘small’ phytochrome and its chromophore models. The phytochrome chromophore of Pr form has been analyzed by fluorescence polarization, CD, and molecular orbital methods. The fluorescence excitation polarization of ‘small’ phytochrome and the chromophore model in buffer/glycerol mixture (3 : 1, v/v) at 77 K is very high (0.4) at the main absorption band and is negative (−0.1) and close to 0 in the near ultraviolet band, respectively. Analyses of the spectroscopic data suggest that the chromophore conformation of Pr and Pfr forms of phytochrome are essentially identical. The induced ellipticity of ‘large’ rye phytochrome in the blue and near ultraviolet regions was found to be significantly higher than that of ‘small’ phytochrome, indicating that the binding interaction between the phytochrome chromophore and apoprotein is much tighter in the former than in the latter. In addition, the excitation energy transfer does occur from Trp residue(s) to the chromophore in ‘large’ phytochrome but not in ‘small’ Pr. This illustrates one feature of the role played by the large molecular weight apoprotein in the binding site interactions and primary photoprocesses of Pr. Finally, a plausible model for the primary photoprocesses and the mechanism of phytochrome interactions triggered by the Pr → Pfr phototransformation have been proposed on the basis of the above results.

The photobinding of 5,7-dimethoxycoumarin to isolated adenovirus-type 2 DNA has been investigated with respect to the influence of the ionic environment, and varying molar ratios of DNA (p) : 5,7-dimethoxycoumarin. In particular, the ultraviolet radiation-induced covalent addition of 5,7-dimethoxycoumarin to adenovirus DNA was increased by reducing the concentration of Na + . The maximum photobinding of 5,7-[ 3 H]dimethoxycoumarin to adenovirus DNA under the given ionic condition was one 5,7-dimethoxycoumarin per 101 nucleotides. Moreover, restriction enzyme analysis of the 5,7-dimethoxycoumarin-DNA photoadduct versus unmodified viral DNA, suggested that the sequence d(A-T) is the preferential site for intercalation and subsequent photobinding of 5,7-dimethoxycoumarin. This susceptibility of d(A-T) sequences to 5,7-dimethoxycoumarin interaction has a corresponding influence on the survival of adenovirus because of the A-T-rich sequences that occur in some of the early gene regions of the adenovirus genome. Specifically, 5,7-dimethoxycoumarin per 800 nucleotides in adenovirus DNA reduced the surviving fraction of adenovirus to a value of 0.1 after DNA infectivity (transfection) into human 293 cells. Results suggest that 5,7-dimethoxycoumarin may be used for generating a limited ‘library’ of mutations in each of the five early gene regions of the adenovirus genome.

1. Live Stentor coeruleus exhibits a substantially red-shifted fluorescence maximum, corresponding to the anionic species of the photoreceptor chromophore. No change was observed in either the absorption or fluorescence excitation spectrum, indicating an efficient deprotonation of the photoreceptor pigment upon excitation by light. 2 Changes in external pH exhibit a dramatic effect on the pulmonary response of Stentor. Phototaxis is specifically inhibited at pH less than 6, with loss of photosensory perception which is restored when the pH is returned to pH greater than 6. 3. Fluorescence changes of 9-aminoacridine in suspensions of live Stentor indicate the generation of a pH gradient upon irradiation with light. Both pH gradient and phototaxis were inhibited by the addition of nigericin and p-tri-fluoromethoxy carbonyl cyanide phenylhydrazone (FCCP). 4. Incorporation of the Stentor photoreceptor protein in to artificial liposomes demonstrates the ability of the system to generate pH gradients across model membranes as monitored by the quenching of 9-aminoacridine fluorescence. The effect of external pH on net proton movement in the model system is strikingly similar to the pH dependent of the liver Stentor, thus lending support for transient proton flux being an important mode of light signal processing for photosensory transduction.

The suggestion, that the increase in the far-UV CD signal of the 124 kDa oat phytochrome upon phototransformation of the Pr to Pfr form is possibly due to the chromophore interaction with the N-terminus segment of the phytochrome protein in the Pfr from (Chai, Y.G., Song, P.S., Cordonnier, M.-M. and Pratt, L.H. (1987) Biochemistry 26, 4947–4952), has been investigated by measuring the circular dichroism in the absence and presence of exogenous tetrapyrrolic chromophores (bilirubin, biliverdin, chlorophyllin and hemin). Open tetrapyrrolic chromophores (bilirubin and biliverdin) did not have any significant effect on the phototransformability of the far-UV CD signal of the phytochrome, whereas closed tetrapyrroles (chlorophyllin and hemin) almost completely blocked the increase in the far-UV CD signal upon Pr to Pfr phototransformation. However, closed tetrapyrroles had no effect on the decrease in the CD signal upon Pfr to Pr photoconversion. Secondary structure analysis showed that the α-helix content of both Pr and Pfr forms of phytochrome (with 53 and 56% α-helical content, respectively) increased to 62% when a 50-fold molar excess of chlorophyllin was added to them separately. Spectral phototransformation of phytochrome was not affected in the presence of tetrapyrroles, except in the case of hemin. A 50-fold molar excess of hemin caused a significant bleaching of the Pfr form of phytochrome but not that of the Pr form. These results suggest that the chromophore-protein interaction is significantly altered during the phototransformation of phytochrome.

1. 1. On the basis of chromatographic and spectroscopic (absorption, fluorescence and its polarization, fluorescence lifetime, circular dichroism) characterization of the Stentor photoreceptor (stentorin) for photophobic response, the photoreceptor chromophore released from mild acid hydrolysis has been identified as hypericin. 2. 2. The native chromophore is apparently linked to a protein (65 K) containing Lys and several hydrophobic residues, which is soluble in acetone and n -pentane. The peptide-linked stentorin (I) chromophore exhibits circular dichroism in the visible region due to the induced optical activity provided by the peptide. 3. 3. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of a 38% fraction of the sucrose density centrifugation has resolved stentorin II proteins having molecular weights of 13 000, 16 000, 65 000 and 130 000. These proteins, as well as the acetone-soluble peptide, have been spectroscopically characterized with particular emphasis on their primary photoreactivity as the photophobic receptor of Stentor coeruleus . 4. 4. Irradiation of whole living Stentor in dilute buffer solutions induces a decrease in the pH of the medium. A strong dependence upon pH in the fluorescence spectra of both synthetic and native chromophores is also evident, showing a significant drop in the p K a of one or more hydroxyl groups in the excited state. A mechanism for the photophobic response, based on this lowering of the p K a as the primary photoprocess, has been discussed.