Photochemistry and Photobiology Vol. 56, No. 3, pp. 399-402, 1992 Printed in Great Britain. All rights reserved

(M31-86SS/Y2 $05.00+0.00 Copyright 0 1992 Pergarnon Press Ltd

PHOTORECEPTOR PIGMENT IN Blepharisma: H' RELEASE FROM RED PIGMENT

TATSUOMI MATSUOKA'*, YASUNORI MURAKAMI' , TAKAHIRO FURUKOHRI' , MASAKI I S H I D A ~ and KOJI TANEDA'

'Department of Biology, Faculty of Science, Kochi University, Kochi 780, Japan and *Laboratories of Cell Biology, Faculty of Integrated Arts and Sciences, Hiroshima University,

Hiroshima 730, Japan

(Received 2 December 1991; accepted 7 February 1992)

Abstract-In faded cells of Blepharisma kept in a standard saline solution containing bacteria which had been cultured on agar plates containing glucose and polypepton, threshold light intensity for step- up photophobic response elevated. This result suggests that red pigment (blepharismin) contained in Blepharisma cells is involved in the step-up photophobic response. The pH of the aqueous solution of the red pigment was found to decrease when light was applied, indicating that the pigment releases H + in response to light stimulation. However, faded pigment preparation by light irradiation did not show pH decrease. In the living cells faded by light irradiation, threshold light intensity for the step- up photophobic response was raised. Results suggest that H' release from the red pigment induced by light irradiation might be responsible for the step-up photophobic response of the cells.

INTRODUCTION

The heterotrichous ciliate Blepharisma has red pig- ment called blepharismin (Giese, 1973). The ble- pharismin is much like hypericin (Sevenants, 1965; Giese, 1973) which is suggested to have composition C30Hlh-IHOR and structural formula hexahydroxy- naphthodianthrone (Brockmann, 1957). Blephar- isma cells show photodispersal (negative photo- accumulation) caused by step-up photophobic response (temporal ciliary reversal induced by a sudden increase in light intensity) and by changes in swimming velocity due to an absolute light intensity (Matsuoka, 1983a,b). Action spectrum for the step- up photophobic response resembles the absorption spectrum of red pigment (blepharismin) contained in a number of vesicles occurring in the vicinity of plasma membrane of a cell, suggesting that the red pigment is responsible for the step-up photophobic response (Scevoli et al., 1987). A related species, Stentor coeruleus, has similar pigment called stentor- in in vesicles whose color is blue (Mdler, 1962; Song, 1981). The stentorin is suggested to be a functional photoreceptor pigment controlling photo- behavior of s. coeruleus (Wood, 1976; Song et al., 1980; Song, 1981). In S. coeruleus, light irradiation caused pH decrease in cytoplasm of the cells, suggesting that H + release is responsible for con- trolling photobehavior (Song, 1981).

In the present study, we found that pH of an aqueous preparation of red pigment of Blepharisma decreased when light was applied, although that of faded pigment preparation did not. In living cells faded by light irradiation, threshold light intensity for the step-up photophobic response elevated. In

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*To whom correspondence should be addressed.

the present paper, we suggest that oxidation of the pigment caused by light irradiation is responsible for the step-up photophobic response of Blepharisma.

MATERIALS AND METHODS

Blepharisma japonicum was cultured in 100-fold diluted lettuce juice at 23°C. To obtain red pigment preparation, the cells were collected by low-speed centrifugation, and stored frozen. To extrude the pigment, the cell pellet (500 pL) was mixed with 500 pL of 0.1% Triton X-100. After incubation for 1 h, 4 mL of distilled water was added, and then large cell fragments were sedimented by centrifugation (12000 g for 10 min at 4°C). The super- natant was decanted and subjected to pH analysis. The pH of the pigment preparation was slightly acidic. Therefore, a small amount of NaOH was added to adjust to approxi- mately pH 7.0. During pH measurement, the preparation was stirred with a small magnet tip. This procedure was carried out under dim light conditions. The pH of the pigment preparation was measured by pH electrode (S815- I , Horiba, Japan) equipped with pH meter (M-8, Horiba, Japan) and recorder (EPR-231A, TOA Electronics Ltd., Japan). For measurement of absorbance of the pigment preparation, 0.5 mL of 50 mM Tris-HCI (pH 7.2) and 3.5 mL of distilled water were added to 1 mL of aqueous pigment solution prepared as mentioned above. The absorbance of the pigment was determined by using spectrophotometer 220A (Hitachi, Ltd. Japan).

For analyses of photoresponse of the cells of Blephar- isma, the cells were collected by low-speed centrifugation and rinsed in a standard saline solution containing 1 mM CaCI,, 1 mM KCI and 5 mM Tris-HCI (pH 7.2). In order to analyze photoresponse of thhe cells, videocassette recorder (BR-1100, Victor, Japan) and videocamera (CCD-XI, Shimazu, Japan) attached to microscope were employed. The intensity of white light was varied by neu- tral density filters. To eliminate effects of heat rays, IR absorbing filters were placed in front of light source. Tem- perature controlled water (23°C) was circulated beneath the chamber to maintain the temperature of the cell sus- pension constant (Matsuoka et al., 1990). The intensity of light was determined by a silicon photodiode photometer.

399

400 TATSUOMI MATSUOKA et al.

Figure 1. Light micrographs of cell surface of Blephar- isma, using Normarski differential interference optics. (A) a normal cell; (B) a cell bleached by culturing in a standard saline solution containing bacteria. In a bleached cell, rows

of pigment granules are irregular.

Faded cells of Blepharisma were obtained by culturing for 3-4 weeks in a standard saline solution containing bacteria (Enterobacter aerogenes) supplied by the Institute for Fer- mentation Osaka (IFO). Enterobacter was cultured on 1.5% agar plates containing 0.5% polypepton and 0.5% glucose.

For electron microscopy, the cells were fixed with a mixture of glutaraldehyde (2.5%) and OsO, (1%) in 50 mM cacodylate buffer (pH 7.2). After 10 min fixation, the cells were washed with 50 mM cacodylate buffer and dehydrated through a graded ethanol series for 10 min each. The cells were embedded in Spurr’s resin (Spurr, 1969) and incubated overnight at 70°C. Ultrathin sections were produced by a ultramicrotome (LKB 2088 Ultrotome V), stained with 1% aqueous solution of uranyl acetate for 10 min, and observed with a JEOL electron microscope (JEM-100s).

RESULTS AND DISCUSSION

When the cells of Blepharisma were cultured in a standard saline solution containing bacteria which had been cultured on agar plates containing glucose and polypepton, the cells became bleached. Photo- microscopic (Fig. 1) and electron microscopic (Fig. 2) observations revealed that the number of vesicles containing red pigment called blepharismin (Giese, 1973) was reduced in the bleached cells and that the vesicles were almost empty in the faded cells [Fig. 2(B)], although the vesicles were filled

Figure 2. Electron micrographs of cell surface. (A) a nor- mal cell; (B) a cell bleached by culturing in a standard saline solution containing bacteria. Arrowheads and arrows indicate pigment granules (vesicles) and electron-

dense structure occurring inside the vesicles.

with the pigment in normally cultured cells [Fig. 2(A)]. In the vesicles, the electron-dense struc- ture was observed at the portion connected to plasma membrane (Fig. 2).

Step-up photophobic response was compared between normally pigmented and faded cells (Fig. 3). In faded cells, the threshold light intensity for the step-up photophobic response was appar- ently raised (Fig. 3). This result indicates that the red pigment contained in the vesicles is responsible for the photoresponse.

Absorption spectrum of the red pigment extruded in an aqueous solution is shown in Fig. 4 (solid line) which agreed with that of blepharismin reported by Giese (1973); in visible range, three peaks (581,540 and 491 nm) appeared.

The pH of the aqueous solution of the red pig- ment was found to decrease when white light (lo3 W/mz) was applied [Fig. 5(A)]. Presumably, H+ is released from the red pigment as a result of photooxidation. Giese (1973) has reported that the red pigment is oxidized by light irradiation. The present result is consistent with previous reports (Giese, 1973). When the pigment preparation was irradiated (lo3 W/mz) for 30 min, the pigment sol- ution faded. As shown in Fig. 4 (dashed line), three peaks in the absorption spectrum of bleached prep- aration disappeared. The pH of the bleached pig-

Photoreceptor in Blepharisma 40 1

100 A z - P u)

a 50

O L , I 1 I I l l l l l

0.5 1.0 5.0 10 W. m-2

-

-

Figure 3. Step-up photophobic response in normal (closed circles) and bleached cells (open circles) which were cul- tured in a standard saline solution containing bacteria. The degree of the photophobic response was expressed as the percentage of the total number ( n = 50-100 cells) showing response in 5 s. Points and attached bars indicate the means of 3 identical measurements and SE. Abscissa, white light intensity. Prior to light stimulation, the cells were adapted to background dim light (0.3 W m-z). Prior to light stimulation, the cells were adapted to background dim light of 0.3 W m-2 for 1 min. Abscissa, white light

intensitv.

Figure 4. Absorption spectra of aqueous pigment solutions containing 5 mM Tris-HCI (pH 7.2) and 0.002% Triton X-100. Solid and dashed lines indicate the absorption spec- trum of normal red pigment and that of bleached pigment

by 30-min irradiation with white light (lo3 W m-z).

/ - *

Figure 5. The pH change of an aqueous pigment solution containing 0.002% Triton X-100 induced by light irradiation. (A) red pigment solution; (B) bleached p i g ment by 30 min irradiation of white light (10’ W m-*).

402 TATSUOMI MATSUOKA er al.

In S. coeruleus, H + release from stentorin photo- receptor pigment is suggested to be involved in step- up photophobic and negative phototaxis of the cells (Song, 1981; Walker et al . , 1981). In the present study, red pigment blepharismin (Giese, 1973) con- tained in Blepharisma cells may also release H+ in response to light. It is likely that the mechanism of photoreception in Blepharisma resembles that of S. coeruleus. When red-colored cells of Blepharisma are exposed to light under excess 0 2 conditions, the red pigment changes to a blue-colored pigment called oxyblepharismin (Giese, 1973). The absorp- tion spectrum of oxyblepharismin (Giese, 1973) resembles that of the blue-green colored pigment (stentorin) contained in S. coeruleus (Song, 1981; Walker et al . , 1979). These facts imply that these pigments are structurally related to each other.

In Blepharisma, a yellow pigment is implied to be a functional photoreceptor mediating the step-up photophobic response (Kraml and Marwan, 1983). However, the absorption spectrum of blepharismin and action spectra for the step-up photophobic response of the cells strongly suggest that blephar- ismin is responsible for the response (Matsuaka et a l . , in preparation). This may lead to the idea that both of these pigments are possibly involved in the same photoresponse. In the present study, pH decrease of pigment preparation occurred in response to light stimulation. If the yellow pigment is contaminated in the present red pigment prep- aration, the yellow pigment may induce the pH change. Judging from the absorption spectrum of the pigment preparation employed in the present study (Fig. 4), however, the preparation seems to contain exclusively red pigment (blepharismin), because a 420 nm peak that is an absorption maximum of the yellow pigment (Kraml and Mar- wan, 1983) is not seen in the absorption spectrum of the preparation. The fact that fading of blephar- ismin (Fig. 4) is accompanied by lack of pH decrease [Fig. 5(B)] also indicates that the pH decrease of the preparation is due to the red pig- ment (blepharismin).

The pH changes and fading of color of the pig- ment are irreversible, indicating that the present pigment preparation is photooxidized irreversibly by light exposure. It is likely that, in intact cells, such a photoreaction of the pigment is irreversible. However, the intact cells containing the pigment faded by light irradiation recover their normal red form in.1-2 days in darkness. Presumably, the red pigment is regenerated in some pathways in vivo. The present preparation of pigment, therefore, may lack some factors to regenerate the red form, because the faded pigment does not recover its red color even when kept for 1-2 days in darkness.

In the present study, the step-up photophobic

response did not completely disappear in faded cells (Fig. 3), although the vesicles were almost empty [Fig. 2(B)]. This implies that other photoreceptors such as the rhodopsin-like substances which occur in another ciliate, Paramecium bursaria (Tokioka et al . , 1991; Nakaoka et a f . , 1991) may also be involved in the response. The present red pigment prep- aration is not pure, so other components contami- nated in the preparation may cause the pH decrease observed. Therefore, further examinations will involve purifying the photoreceptor pigment. Acknowledgement-This work was supported by Sasakawa Scientific Research Grant of the Japan Science Society.

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