localization of blepharismin photosensors and identification of a photoreceptor complex mediating...

Photochemistry and Photobiology, 1997, 65(5): 91 5-921

Localization of Blepharismin Photosensors and Identification of a Photoreceptor Complex Mediating the Step-up Photophobic Response of the Unicellular Organism, Blepharisma

Tatsuomi Matsuoka*I, Mie Satol, Mitsuru Maeda2, Hideo Naoki2, Takaharu Tanaka2 and Hiyoshizo Kotsuki3 'Department of Biology, Kochi University, Kochi, Japan; 2Bio-Organic Chemistry Laboratory, Suntory Limited, Osaka, Japan and 3Department of Chemistry, Kochi University, Kochi, Japan

Received 9 October 1996; accepted 5 February 1997

ABSTRACT

Photosensitivity for the step-up photophobic response of Blepharisma is localized in the anterior 115 of the cell body. Blepharismin pigment, which is believed to be a photoreceptor pigment mediating the step-up photophobic response of the cells, was separated into five types of blepharismin (BL-1, -2, -3, -4 and -5). Blepharismin-1, -3, -4 and -5 were localized in the posterior 415, while BL-2 was located over the entire cell body; the anterior end, which is the photosensitive region, contained only BL-2. The results indicate that a functional photoreceptor pigment mediating the step-up photophobic response is BL-2. Hydroxylapatite chromatography revealed that BL-2 was bound to a 200 kDa membrane protein. We concluded that a photoreceptor mediating the step-up photophobic response was a BL-2/200 kDa protein complex.

INTRODUCTION

Blepharisma japonicum cultured under dark condition has a pink-colored pigment called blepharismin (l), contained in the pigment granules located just beneath the plasma membrane (1.2). The cells of Blepharisma show a step-up photophobic response (temporal backward swimming or rotating movement induced by a sudden increase in light intensity) in response to a sudden increase in light intensity (3.4). Such a photoresponse helps the cells in avoiding strongly illumi- nated regions and to accumulate in shaded regions (3). The cells of Bfepharisma can also detect the direction of a light source in a trial-and-error manner during rotating movement and orient away from the light source (5,6) (Fig. 1). In this case, the step-up photophobic response also plays an impor- tant role. To orient against the light source, the cell requires its photosensitivity to be localized in the anterior end (7) (see Fig. 1).

When the pink-colored cells are exposed to light in the presence of oxygen, the cells turn blue and the absorption

*To whom correspondence should be addressed at: Department of Biology, Kochi University, Kochi 780, Japan. Fax: 0888-44-8356.

0 1997 American Society for Photobiology 003 1-865397 $5.00+0.00

spectrum of the pigment shifts 15-20 nm toward the red (1). Such blue-colored cells are also photosensitive and the action spectrum of the response also shifts 15-20 nm toward the red (8,9).

Blepharismin is believed to function as the photoreceptor pigment mediating the step-up photophobic response, based on several lines of evidence (2.8-1 3). Photosensitivity for the step-up photophobic response of Blepharisma is localized in the anterior 1/5 of a cell body (7), despite that the photoreceptor pigment (blepharismin) mediating the photoresponse seems to be homogeneously distributed all over the entire cell surface. This inconsistency that is remained un- solved for 10 years has sometimes suggested that blepharismin may not be a photosensor for the step-up photophobic response. Recently, we have revealed that Blepharisma has five types of blepharismin pigments (pink forms). Therefore, we expected that these pigments might be functionally differentiated in the photosensory system and that some of the pigments mediating the step-up photophobic response might be localized in the anterior end. In the present study, we show that the anterior end of the cell contains only blepharismin-2 (BL-2)t and conclude that this might be the primary photoreceptor pigment mediating the step-up photophobic response.

Previously, we have isolated a blepharismin-200 kDa membrane protein complex as a candidate for the photoreceptor complex mediating the step-up photophobic response (2.14). However, we have not determined which types of blepharismins are associated with this 200 kDa protein. The present study also aims at the isolation of the membrane protein bound with BL-2.

In the pink forms of Blepharisma, a large amount of the pigment tends to be dissociated from the proteins when the photoreceptor complex is solubilized with detergent (14). In contrast, in the blue form of Blepharisma, a large amount of blepharismin (blue form) remained associated with membrane proteins when the hydrophobic components were solubilized with detergent. In the present study, therefore, we

tAbbreviarions: BL- 1, 2, etc., blepharismin-1, -2, etc.; CBB, Coom- assie brilliant blue staining; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; TLC, thin-layer chromatography.

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1 II 1 1

Figure 1. A schematic diagram showing how Blepharisma orients away from a light source. Blepharisma shows a temporal backward swimming (1-3) in response to a relative increase in light intensity, followed by a rotating movement (3-5’) due to ciliary reversal. The cell continues ciliary reversal while a photosensitive region localized in the anterior end is irradiated by light (1-3). The photosensitive anterior end is then shaded by the posterior region; thereby, ciliary reversal is ceased and forward swimming is regained (4). If the cell failed to orient away from a light source (5’). further rotating movement is elicited, because the photosensitive anterior region is again exposed to light. Redrawn from Matsuoka (6).

exclusively employed the blue form of the cells to isolate a pigment-protein complex and thereby revealed that BL-2 was bound to the 200 kDa membrane protein.

MATERIALS AND METHODS Cell culture. Blepharisma japonicum was cultured at 23°C in the dark in 0.1 % cereal leaves infusion containing bacteria (Enterobac- ter aerogenes) as food. The bacteria, which were supplied by the Institute for Fermentation, Osaka, Japan (IFO) were cultured on 1.5% agar plates containing 0.5% polypeptone, 1 % meat extract and 0.5% NaCI. Cultured cells were collected by gentle centrifugation (150 g, 1 min) and resuspended in a saline solution containing 5 mM Tris-HCI (pH 7.4). 1 mM CaC12 and 1 mM KCI and then kept at 23-25°C overnight.

Microsurgical operation of cells. The cytostome of Blepharisma cells employed in this work is located at the position of 3/5 from the anterior end. Based on the mark of cytostome, we cut the cells into the fragments with a fine glass needle under a dissecting mi- croscope. Each fragment was collected with a fine glass pipette and suspended in acetone within 30 rnin after microsurgical operation. To obtain 2000 fragments took a whole day.

Preparation of pigment granules. The cells that had been suspended in a saline solution containing 5 mM Tris-HCI (pH 7.4). 1 mM CaCI, and 1 mM KCI were collected by centrifugation (150 g, 1 min) and transferred in the cooled (2°C) saline solution. After gentle pipetting, the cells and cell debris were sedimented by centrifugation (ISOg, 5 min). The supernatant containing pigment gran-

ules was decanted and subjected to centrifugation (8000 g, 10 min) to sediment the pigment granules.

Extraction and preparation of free pigment. To extract the pigment, the cells or fragments were collected and then suspended in acetone. After 1 min extraction, the cells or fragments were rese- dimented (8000 g, 1 rnin), and the supernatant containing extracted pigment was decanted. The pigment preparation was dried with a rotary evaporator (Rotavapor, Sibata) prior to applying on thin-layer chromatograpy (TLC) plates or a column for HPLC (Hitachi 655 liquid chromatograph system).

Purification and assay of free pigment. The pigment was dissolved in 30% acetonitrile solution to apply on a column (COS- MOSIL 5‘218-300, 4.6 X 150 mm; Nakarai) for a reverse-phase HPLC with acetonitrile gradient or dissolved in acetone for applying normal-phase TLC plates with silica gel 60 F2s4 (Merck) or silica gel 60A LK6F (Whatmann). In TLC, the pigment was developed with a mixture of ethyl acetate and acetone (4: 1, 3: 1 or 2: I , vol/vol). All procedures of pigment preparation and TLC were carried out under dim light (below 0.05 W/m2). The absorption spectra of the pigments were determined with a Hitachi 220A spectrophotometer.

Extraction of pigment from the samples eluted by hydroxvlapatite chromatography. In order to extract the pigment from fraction I1 (see Fig. 7). eluted sample was highly concentrated with a rotary evaporator (Rotavapor, Sibata), a large amount of acetone ( 100-fold volume) was added and then cooled at -30°C. Cooled sample was centrifuged (8000 g, 10 min) to decant supernatant containing pigment, followed by complete drying with an evaporator. Dried sample was dissolved in 1 mL of acetone, 0.5 mL of chloroform was added and then the mixture was cooled at -30°C to produce precipitates. The precipitates were removed through a membrane filter (Chro- matodisk 13 N, Kurabo; 0.45 p,m pore size). The sample was dried with a rotary evaporator and dissolved in acetone.

Isolation of pigment-protein complex. The cells (blue form) were frozen once at -20°C and remelted to disrupt cell structure. The disrupted cell debris was sedimented (8000 g, 10 min) and water- soluble components (supernatant) were removed. In order to solu- bilize blepharismin-membrane protein complexes, the cell fragment pellet was resuspended in a solution containing 20 mM sodium cholate and 50 mM phosphate buffer (pH 7.4) and kept at 4°C overnight. After incubation, the cell debris was sedimented (8000 g, 10 min) and the colored supernatant was decanted. The buffer solution in which pigment-protein complexes were solubilized was replaced by a 10 mM sodium cholate, 100 mM NaCI, 50 mM phosphate buffer (pH 7.4) solution by adding an equal volume of solution containing 50 mM phosphate buffer (pH 7.4) and 200 mM NaCI. Prior to applying on a column, the sample was centrifuged (8000 g, 10 min) to remove the precipitates. The sample was eluted at 4°C under darkness at a flow rate of 20 mL/h on a 22 cm hydroxylapatite column equilibrated in a buffer containing 10 mM sodium cholate, 50 mM phosphate buffer (pH 7.4) and 100 mM NaCI. The fractions were collected using a fraction collector (model 221 10, Bio-Rad) and monitored by absorbance at 280 and 605 nm. The absorbance of the fractions was determined with a Hitachi spectrophotometer 220A.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE). The eluted fractions were analyzed by SDS-PAGE accord- ing to Laemmli (15). Prior to SDS-PAGE, the buffer solution in the fractions was replaced by a solution containing 60 mM sodium cholate and 10 mM Tris-HC1 (pH 7.4) through 10 kDa cutoff membrane filter (Millipore UFPl LGC BK) and then incubated at 4°C overnight. After incubation, a three-fold volume of sample buffer containing 2% SDS, 30 mM Tris-HCI (pH 6.8), 10% glycerol was added and then incubated at 23°C for 24-48 h. Gels were run at 150 V, stained for 1-2 h with 0.2% Coomassie brilliant blue (CBB) R-250 in a 45% (voVvol) methanol, 10% (vollvol) glacial acetic acid solution. For standard proteins, a molecular standards kit (Bio-Rad) was employed.

RESULTS Molecular structure and localization of the pink form of blepharismin Thin-layer chromatography of the blepharismin pigment (pink form) resulted in two spots (see Fig. 5) . When the

Photochemistry and Photobiology, 1997, 65(5) 91 7

I I - Pink form

Blue form Intact BL-, 2

0 10 20 30 40 50 60 70 Retention time (min)

Figure 2. Elution profiles of five types of pink blepharismins contained in cell fragments by a reverse-phase HPLC with a 30-100% acetonitrile gradient. (A) Intact cells. The amplitude of peak 5 (BL-5) sometimes fluctuated. (B) Anterior 115 fragments. ( C ) An- terior half fragments. (D) Posterior 1/5 fragments. The cells were cut into fragments with a fine needle and collected to be suspended in acetone within 30 min. For an assay, 2000 fragments were used. Blepharismin was extracted with acetone, dried and then dissolved in 30% acetonitrile prior to applying a column.

mixture of pigment preparations extracted from the two colored spots on the TLC plate was analyzed on reverse-phase HPLC, five pink-colored components (BL- 1, 2, 3 ,4 , 5) were isolated (see. Fig. 2A). The absorption spectra of the pigments were almost identical in the visible range (data not shown), indicating that the blepharismin molecules have a common chromophore structure. Recently, the determination of the molecular structure of blepharismin pigments has been carried out by Song and Lenci's group (16) and our research group independently. Our results indicate that the chromophore structure of blepharismins was common (data not shown).

Localization of the pink form of blepharismin pigments

The fact that the photosensitivity for the step-up photophobic response is localized in the anterior end (7) of the cell body suggests that the photoreceptor pigment mediating the pho-

350 450 550 650 Wavelength (nm)

Figure 3. Absorption spectra of crude pink (solid line) and blue (dashed line) forms of blepharismin dissolved in acetone. The absorption spectra were normalized with respect to the maximum in the visible range. In order to obtain blue pigment, the pink-colored cells were irradiated by white light (5 Wlrn') for 5-10 h in the presence of excess O?.

toresponse is possibly localized in the anterior end. In order to determine the pigment localization, pigments extracted from the fragments obtained by microsurgical operation were analyzed on reverse-phase HPLC (Fig. 2). The anterior 1/5 contained only BL-2 while the anterior 1/3 (data not shown), the anterior half, the posterior half (data not shown) or the posterior one-fifth contained five types of blepharismin (Fig. 2). The distribution of blepharismin pigments is drawn schematically in Fig. 9 based on the results shown in Fig. 2. The fact that the anterior end contains only BL-2 indicates that BL-2 may be the primary photoreceptor pigment for the step-up photophobic response.

Localization of blue forms of blepharismin

The absorption spectra of pink and blue forms of crude blepharismins extracted with acetone are shown in Fig. 3. We failed to purify blue forms of blepharismin by reverse-phase HPLC with an acetonitrile gradient. We tried, therefore, to separate blue-colored pigments by TLC. By using TLC plates with silica gel 60FZs4 (Merck), the blue form of pigment was separated into two spots (Fig. 4, right figure; lane 2); in this case, the lower spot (labeled b) including the origin does not seem to be a single component. The absorption spectrum (Fig. 4b) of the upper spot labeled a showed an absorption maximum at 599 nm; hence the pigment is called blue-599 in this paper. The absorption spectrum of the lower spot (labeled b) is also shown in Fig. 4c, indicating an absorption maximum at 614 nm; the pigment is, therefore, called blue-614. The absorption spectrum of a mixture of these two components is shown in Fig. 4a, showing an absorption maximum at 599 nm and a shoulder at 614 nm.

The absorption spectra of the crude blue form of blepharismin extracted from intact cells, posterior half and anterior 1/3 fragments showed a maximum at 599 nm and a shoulder at 614 nm (Fig. 4d-f), indicating that these cells and fragments contained two types of blue forms (blue-599 and blue- 614). By contrast, the absorption spectrum of blue pigment obtained from the anterior 1/5 fragments did not have any shoulder at 614 nm, and the spectrum agreed with that of an isolated blue-599. Thin-layer chromatography demonstrated that the anterior 1/5 contained only blue-599 (Fig. 4; right, lane 4), while the posterior 415 contained blue-599 and blue-614 (Fig. 4; right, lane 3). These results indicate that

91 8 Tatsuomi Matsuoka et a/.

a

b

C

w m l r p n (m Figure 4. The TLC assays (right photographs) and absorption spectra (left figure) of blue forms (blue-599 or blue-614) of blepharismin extracted from the cell fragments. Right photographs: The TLC assays of pigment extracted from pink-colored intact cells (lane I) , blue-colored intact cells (lane 2). blue-colored posterior 4/5 fragments (lane 3) and blue-colored anterior 115 fragments (lane 4). The spots labeled a and b indicate blue-599 and blue-614, respectively. The Rf values of pink blepharismins (lane 1) are 0.25 (upper spot) and 0.19 (lower spot); upper and lower spots contained two types of pink blepharismins (BL-4, -5 ) and three types of the pink forms (BL-I, -2, -3). respectively (data not shown). The Rf values of the blue form (lane 2) are 0.29 (spot a) and 0.0-0.1 (spot b). Small points at the bottom of the lanes (lanes 1, 3, 4) are pencil marks of the origin. The extracted and concentrated pigment preparations were applied on TLC plates with silica gel 60 F,,, (merck) and developed with a mixture of ethyl acetate and acetone (4: 1, vol/vol). Absorption spectra (left figure): (a) Mixture of two types of blue forms (3.1 kg of blue-599 and 1.2 pg of blue-614/mL) separated by TLC (see right photograph, lane 2). The spectrum has an absorption maximum at 599 nm with a shoulder at 614 nm. (b) Blue-599 (3.1 kg/mL). (c) Blue-614 (1.2 kg/mL). (d-g) Absorption spectra of blue blepharismins extracted from intact cells (d), posterior half fragments (e), anterior 1/3 fragments (0 and anterior 115 fragments (g). The pigments were dissolved in acetone for absorption spectrum determination.

blue-599 is distributed over the entire cell surface, whereas blue 614 is localized near 4/5 from the posterior end. In the pink form of Blepharisma, the anterior end of cells contains only BL-2 (Fig. 2). On the other hand, in the blue form of the cells, the anterior end contains only blue-599 (Fig. 4). This means that blue-599 is derived from BL-2, which is a primary photoreceptor pigment.

The pink form of Blepharisma contains five types of pigments, whereas in the blue form of the cells, only two types of the pigments are isolated. Isolation of the blue form of blepharismin on TLC plates with silica gel 60FZs4 (Merck) usually results in an upper spot (blue-599) and a tailed lower spot (blue-614), implying that the lower spot contains several types of the pigments. Therefore, we tried to isolate the pigments further (blue-599 and blue-614) using a different type of TLC plate with silica gel 60A LK6F (Whatmann) and a different composition of eluent. In this assay, TLC of blue-599 resulted in a single spot (Fig. 5 ; right, lane 2), whereas blue-614 was separated into four spots (Fig. 5 ; right, lane 3a-d) or at least three spots (Fig. 5 ; lane 3a-c). In this case, a spot labeled d included the origin. The BL-1, -3, -4 and -5 possibly correspond to these spots (a-d). The absorp-

Figure 5. The TLC assays (right photograph) of blue forms using different plates with silica gel 60A LK6F (Whatmann) and absorption spectra (left figure) of four types (right photograph, lane 3a-d) of blue forms obtained by TLC of blue-614. Right photograph: The TLC assays of pigment extracted from pink-colored intact cells (lane I ) , blue-599 (lane 2) and blue-614 (lane 3) isolated by TLC as de- scribed in Fig. 4. The spots containing pigments isolated by TLC (using TLC plates with silica gel 60 F2,,) were scraped and pigments were extracted with acetone. Concentrated pigments were applied on a TLC plate with silica gel 60A LK6F (Whatmann) and developed with a mixture of ethyl acetate and acetone (3:1, vol/vol). Left figure: Absorption spectra labeled a-d correspond to isolated subtypes (right photograph, lane 3a-d) of blue-614, respectively. (a') An absorption spectrum of a further purified subtype a. The pigment extracted from a spot labeled a (right photograph, lane 3) was developed with a mixture of ethyl acetate and acetone (2:l. vol/vol) to remove contaminating red-colored pigment.

tion spectra of pigments obtained from these spots (Fig. 5a- d) are shown in Fig. 5 (left figure). The spectra have a common absorption maximum at 614 nm. However, spot a seemed to be overlapped with a spot containing red-colored pigment that is possibly an intermediate produced during transformation of blepharismin. The pigment on spot a was, therefore, analyzed again by TLC using a different of eluent composition (ethyl acetate/acetone, Ul), and the red pigment component was removed (Fig. 5, left figure; a').

Isolation of blue-599 (BL-2)-binding membrane protein

Hydrophobic components of blue form of the cells solubilized with sodium cholate were analyzed by hydroxylapatite chromatography, and two colored fractions (containing blepharismin) were obtained (Fig. 6A). The TLC-purified free pigments (blue-599 and blue-614) were also analyzed on the same column under identical conditions (Fig. 6B). Both free pigments eluted in fraction I, indicating that fraction I in Fig. 6A might contain free pigments dissociated from membrane proteins. On the other hand, the pigment eluted in fraction I1 is probably bound to membrane protein, because free pigments are never eluted in this fraction. The absorption spectra of the native sample eluted in fractions I (Fig. 7a) and of free pigment extracted from this fraction with acetone (Fig. 7c) indicate that two components (blue-599 and blue- 614) are contained in fraction I. On the other hand, the absorption spectra of native sample (Fig. 7b) and acetone-extracted free pigment (Fig. 7d) showed that fraction II might contain only blue-599. The TLC of the free pigment extract-

Photochemistry and Photobiology, 1997, 65(5) 91 9

0 Blue599 0 81ue-614

2 M iosphate bufler I

" 1 0 20 40 80 80

FracHon nunber

Figure 6. Hydroxylapatite chromatography of detergent-solubilized components obtained from blue-colored Blepharisrna cells (upper) and TLC-isolated free pigments (lower). Upper figure: Fractions were monitored by absorbance at 280 (0) and 605 nm (A). Inset: The SDS-PAGE of the fractions obtained by chromatography. Lane (M), marker proteins. Figures labeled on the top of the lanes correspond to the fraction numbers. The samples were analyzed on a 10% polyacrylamide gel. Lower figure. Fractions were monitored by absorbance at 599 (0) and 614 nm (0). Prior to applying the sample (0.5 mL), the column packed with hydroxylapatite was equilibrated in a 0.05 M phosphate buffer (pH 7.4) containing 10 mM sodium cholate and 100 m M NaCI. The sample was eluted at a flow rate of 20 mLh.

ed from fraction I1 demonstrated that this fraction contained only blue-599 (Fig. 7; inset, lane 4).

In the procedure for removing salts contained in the elution buffer, the pigment sample was dissolved in the mixture of acetone and chloroform (see Materials and Methods). In this procedure, blue-6 14 is possibly precipitated because po- larity of blue-614 is greater than blue-599. Therefore, we confirmed that blue-6 14 was completely dissolved in the mixture of acetone and chloroform (2:l. vol/vol).

The SDS-PAGE of each fraction elucidated that fraction I1 exclusively contained 200 kDa protein (Fig. 6A, inset). The photosensitive anterior end of the cell contains only blue-599, which is probably derived from BL-2 (see Fig. 9). These results indicate that blue-599 (BL-2) is bound to a 200 kDa membrane protein to form a primary photoreceptor complex mediating the step-up photophobic response. We have also tried to isolate the pigment (pink form)-protein complex on a hydroxylapatite column using detergent-solubilized sample obtained from pink form of Blepharisma. Un- fortunately, BL-2-protein complex could not be isolated

Figure 7. Absorption spectra of fractions I (a) and I1 (b) eluted by hydroxylapatite chromatography (Fig. 6) and those of acetone-extracted pigments from fractions 1 (c) and I1 (d). Inset photograph: A TLC assay of pigment species contained in fraction I1 (lane 4). For comparison, standard pigments isolated by TLC (using TLC plate with silica gel 60 F,,,) were applied on lanes 1-3. Lane 1, pink blepharismin; lane 2, blue-599; lane 3, blue-614.

from free pigments that had been dissociated from the proteins.

Assay of membrane proteins contained in the pigment granules

The SDS-PAGE of detergent-solubilized components obtained from isolated pigment granules resulted in two thick bands corresponding to a molecular mass of 200 kDa and 46 kDa and smear bands around 30 kDa (Fig. 8). In SDS- PAGE (CBB staining) of the samples obtained by hydroxylapatite chromatography, a band corresponding to a molecular mass of 46 kDa protein could not be detected (Fig. 6A, inset). In order to find out which fraction contains the 46 kDa protein, a gel obtained by an identical procedure was silver-stained (data not shown). The result showed that the 46 kDa protein occurred in fraction I. The 46 kDa protein may be partially adsorbed to hydroxylapatite so that a band

Figure 8. The SDS-PAGE of detergent-solubilized components obtained from isolated blue-colored pigment granules.

920 Tatsuomi Matsuoka et a/.

BL- 2 (Blue-599)

BL- 1, 3, 4, 5 (Blue-61 4)

Figure 9. A schematic diagram showing the distribution of blepharismins along the cell body.

corresponding to 46 kDa is not detected by staining with CBB.

DISCUSSION The step-up photophobic response of Blepharisma is caused only when a light stimulus was applied to the anterior end of the cell (7). The fact that the anterior end contains only BL-2 (blue-599) means that the photoreceptor pigment mediating the step-up photophobic response is BL-2 (blue-599). Light stimulation of the posterior portion does not cause such a photoresponse (7), although BL-2 also is located in the posterior region (Figs. 2 and 9). Our speculation to ex- plain such a localization of photosensitivity is as follows: BL-2 may be a photoreceptor pigment for generating excitable signals that lead to membrane depolarization (genera- tion of depolarizing photoreceptor potential) (1 7). while other types (BL-1, -3, -4 and -5) of blepharismins or some of them possibly generate inhibitory signals to cancel excitable signals generated by BL-2. In Blepharisma, depolarizing photoreceptor Ca channels (18) are possibly linked to the BL-2 photoreceptor system, and hypothetical hyperpolariz- ing K photoreceptor channels are possibly linked to BL-1, -3, -4, -5 or some of them. If so, photoreceptor Ca channels linked to BL-2 may be distributed over the entire cell surface, while photoreceptor K channels may be localized in the posterior 415. In Paramecium, a density gradient of mechanoreceptor Ca channels responsible for membrane depolarization exists from the anterior toward the posterior end, while the gradient of mechanoreceptor K channels responsible for membrane hyperpolarization occurs from the posterior to the anterior end (19). The distribution of photoreceptor channels of Blepharisma seems to be different from that of Paramecium mechanoreceptor channels.

In Fig. 9, a density gradient of BL-1, -3, -4 and -5 is drawn to occur from the posterior toward the anterior end. However, we have no evidence for such a density gradient of such a pigment distribution. In addition, a density gradient of BL-2 possibly occurs from the anterior toward the posterior end, although BL-2 is drawn to be distributed equally over the entire cell body. The present study revealed only a relative amount of blepharismins. Further work will involve

determining, by means of an immunocytochemical or mi- crospectrophotometrical technique, a density gradient of blepharismins.

The absorption spectra of subtypes of blue-614 are identical with each other in the visible range (Fig. 5) , indicating that the chromophore structure of these pigments may be common. The difference of Rf values between these subtypes in TLC (Fig. 5) might reflect differences of functional groups of the pigment structure. On the other hand, the absorption spectrum of blue-599 is different from those of the subtypes of blue-614 in the visible range (see Figs. 4 and 5) , suggesting that the chromophore structure of blue-599 may be different from that of blue-614. The result that R, values between blue-599 and blue-6 14 are conspicuously different (see Figs. 4 and 5) also suggests that the chromophore structure may be different.

Blue-614 was separated into three or four components (Fig. 5; right, lane 3); spot d is overlapped with the orgin and too small, so it may be difficult to identify it as one of four components. Judging from the results that blue-614 is located in the portion except for the anterior 115, it is likely that these components correspond to BL-1, -3, -4 and -5. The correspondence between three or four components of blue-614 and four types (BL-1, -3, -4 and -5) of the pink form of blepharismin has not been elucidated directly because the purified free pink form of blepharismin cannot be transformed to blue forms by light irradiation in the presence of 02. Presumably, the proteins to which blepharismin is bound or certain intracellular components are required for the phototransformation of the pigment.

In the pink form of blepharismin, the absorption spectra of the different subtypes are identical in the visible range because the chromophore of the pigment molecule is common (data not shown). On the other hand, the absorption spectra of blue forms (blue-599 and blue-614) are different from each other and from the spectrum of the pink form. It has been demonstrated, in addition, that hydroxyl radicals are generated from the pink form by light irradiation in the presence of O2 (20), and that in the absence of O,, the in vitro photoresponse (photobleaching) of pink forms of blepharismin is inhibited (13). These results suggest that the common chromophore structure of pink forms may be transformed to two different structures of the blue-colored chromophore by photooxidation. It is likely that such a different transformation of chromophore structure might reflect a difference of the proteins to which blepharismin pigments are bound: blue-599 (BL-2) is bound to the 200 kDa protein, while blue-614 (BL-1, -3, -4 and -5) to other unidentified proteins. A 46 kDa protein or proteins corresponding to a molecular mass of 27-35 kDa (Fig. 8), which possibly involve a blepharismin-binding protein with an apparent molecular mass ranging between 35 and 38 kDa suggested by Gioffre ef al. (21), are candidates for the pigment-binding protein.

The quinone pigment (22) (stentorin) contained in the ciliated protozoan Stentor coeruleus cells, which is believed to be a photoreceptor for the step-up photophobic response (23,24), resembles blepharismins (pink forms) in its molecular structure. In Stentor, two types of pigment-protein complexes (stentorins I and 11) have been isolated (25). It is presumed that these photoreceptor complexes are function-

ally differentiated as in Blepharisma. Stentorin I1 can be eluted with 0.2 M phosphate buffer in hydroxylapatite chromatography and is an extremely large complex with a molecular mass greater than 500 kDa (25). Judging from these results, the stentorin I1 photoreceptor complex seems to re- semble the BL-2 (blue-599)/200 kDa complex of the Ble- pharisma photoreceptor because a BL-2/200 kDa complex is eluted with 0.2 M phosphate buffer in hydroxylapatite chromatography with a stepwise phosphate gradient (Fig. 6), and the molecular mass of a pigment-200 kDa protein complex without SDS treatment is also greater than 500 kDa (unpublished).

Acknowledgements-We thank Mr. Y. Kawasaki and Mr. M. Mat- sushita for technical assistance in the HPLC analysis of blepharismin and Mr. Y. Kato and Ms. E. Kodera for technical assistance during isolation of the photoreceptor complexes by hydroxylapatite chromatography. We also thank Prof. P . 3 . Song of the University of Nebraska for suggesting hydroxylapatite chromatography for isolat- ing blepharismin-binding proteins and Prof. Lenci of CNR Istituto di Biofisica for valuable comments on this work. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan (no. 08640869) and was also supported financially by the Sumitomo Foundation, Suntory Limited, and the Japan Science Society (Sasakawa Scientific Research Grant).

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localization of blepharismin photosensors and identification of a photoreceptor complex mediating...

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