Akiko Fujiwara,* Eigoro Tazawa,** and Ikuo Yasumasu*

Transcription

1 J. Biochem. 109, (1991) Activating Effect of Light Irradiation at Various Wavelength on the Respiration in Sperm of the Echiuroid, Urechis unicinctus, in the Presence of Carbon Monoxide1 Akiko Fujiwara,* Eigoro Tazawa,** and Ikuo Yasumasu* * Department of Biology, School of Education, Waseda University, Shinjuku-ku, Tokyo 169; and **Biological Institute, Faculty of Literature and Science, Yokohama City University, Kanazawa-ku, Yokohama, Kanagawa 236 Received for publication, August 28, 1990 In sperm of the echiuroid, Urechis unicinctus, respiration in the presence of CO was reversibly augmented by light irradiation in an examined range of wavelengths between 350 and 650 nm. The respiratory rate of sperm in the presence of CO was enhanced by light irradiation in proportion to the light fluence rate. A sharp and large peak was obtained at the wavelength of 430 nm in the action spectrum of photo-activated respiration of sperm in the presence of CO. Broad and small peaks were also found at around 530 and 570 nm. This action spectrum is similar in its profile to the absorption spectrum of reduced cytochrome b. Absorption of photon energy by reduced b-type cytochrome probably activates the redox reaction of this cytochrome to enhance the respiratory rate. Photo-activated respiration in the presence of CO was inhibited by antimycin A and cyanide. In this respiratory system, an electron equivalent is probably transferred through the mitochondrial respiratory chain between cytochrome b and cytochrome c and finally to molecular oxygen in the reaction catalyzed by the CO-insensitive terminal oxidase. It has been reported that echiuroid eggs exhibit CO -insensitive, CN--sensitive respiration which is enhanced in its rate by white light irradiation (1-3). In echiuroid eggs, mitochondria have been reported to be responsible for this photo-activated CO-insensitive respiration (3). In the present study, it was found that echiuroid sperm also exhibit photo-activated CO-insensitive respiration. The activating effect of light irradiation on CO-insensitive respiration was examined in echiuroid sperm at various wavelengths, as a first step to characterize respiratory pigments which would be activated by the absorption of photon energy to result in an increase in the rate of CO-insensitive respiration. MATERIALS AND METHODS Estimation of Respiratory Rate-Respiratory rate of sperm was estimated by a polarographic method. Dry sperm, obtained from the isolated segmental organ of the echiuroid, Urechis unicinctus, were stored in an ice bath until use. A glass vessel containing 3.5 ml of sea water was closed by a stopper equipped with an oxygen electrode (Yellow Springs). Sea water used in the present study was filtered through a Millipore filter before use. Upon closing the glass vessel, bubbles of air were completely removed through a small hole in the stopper. Dry sperm in a volume of 100 p l was added to sea water in the closed vessel through a small hole in the stopper. The temperature of sea water in the vessel was maintained at 20 Ž by circulating 1 This study was carried out under the NIBB Cooperative Research Program for the Okazaki Large Spectrograph (89-511). Abbreviations: AMA, antimycin A; CN-, Na-cyanide; FCCP, carbox ylcyanide p-trifluoromethoxyphenylhydrazone; TMPD, tetramethyl p-phenylenediamine. temperature-controlled water through a water jacket sur rounding the vessel. Oxygen concentration in the sperm suspension, which was stirred with a magnetic stirrer, was polarographically estimated and recorded on a recorder (U-125MU Shimadzu, Kyoto). The respiratory rate was calculated on the basis of the decrease in oxygen concentra tion in sperm suspension, resulting from oxygen consump tion by sperm, and was expressed as nmol 0,/108 cells/ min. Cell numbers were estimated by microscopy using a hemocytometer. Exposure of Sperm to CO-Sea water was bubbled rather vigorously for 15 min with CO gas. The CO concen tration in thus bubbled sea water was indirectly monitored by estimating its 0, concentration. When sea water, having been bubbled with air, was again bubbled with CO gas for 15 min, the 02 concentration became less than 10% of the 02 concentration in air-bubbled sea water. The low concen tration of OZ in CO-bubbled sea water is not evidence of a high concentration of CO but indicates that 02 dissolved in sea water effectively diffuses into gas bubbles, so that the 02 concentration becomes low in the sea water. Thus, it was assumed that CO gas in the bubbles was dissolved in sea water at a similar rate to the diffusion rate of 02 from sea water to gas bubbles, to give a CO concentration close to saturation, when the 02 concentration is made quite low by CO-gas bubbling. For convenience, the CO concentration in sea water bubbled with CO gas for 15 min is taken here after as its saturated concentration. Unless otherwise specified, 3 ml of CO-bubbled sea water was added to 0.5 ml of air-bubbled sea water in the glass vessel (indicated as 86% of saturated concentration) and the vessel was closed with the stopper as quickly as possible. When the 02 concentration in this mixture of CO-bubbled and air-bub bled sea water was higher than 30% of the 02 concentration 486 J. Biochem.

2 Photo-Activation of CO-Insensitive Respiration 487 in air bubbled sea water, this mixture was not used for the estimation of CO-insensitive respiration. The concentra tion of CO was changed in some experiments by altering the mixing ratio of CO-bubbled, air-bubbled, and N2-bubbled sea water. N2-bubbled sea water was added in place of CO-bubbled sea water to avoid a change in 02 concentration in the mixture. Estimation of Respiratory Rate in the Presence of Inhibitors of Respiration-NaCN (10mM), oligomycin (5 mm), TMPD (5mM), and FCCP (0.1mM), dissolved in distilled water, were added in a volume of 35,u 1 each to 3.5 ml of sperm suspension in the closed glass vessel through the small hole in the stopper. Antimycin A (AMA: 5mM), dissolved in ethanol, was also added in a volume of 35 p 1 to 3.5 ml of sperm suspension. Isolation of Mitochondrial Fragments and Plasma Membrane Fraction-A suspension of dry sperm in 0.3 M sucrose containing 10mM EDTA and 50mM HEPES buffer ph 7.2 (homogenizing medium) was homogenized in a glass homogenizer by a motor-driven Teflon pestle. The middle part of the sperm, containing the mitochondrion, is known to be mechanically weak as compared with the head (nucleus) and the tail. Hence, the sperm homogenate probably contains mitochondrial fragments. The homog enate was centrifuged for 25 min at 2,500 x g. The result ant precipitate was diluted in the homogenizing medium and again centrifuged as above. This procedure was again repeated and the finally obtained pellet, found to contain mainly the heads (nuclei) and tails of sperm by dark-field microscopy, was used as the head and tail fraction. The supernatants thus obtained were mixed together and centrifuged at 10,000 x g for 30 min. The precipitate fraction thus obtained is designated hereafter as the mitochondrial fragment fraction. Plasma membrane frac tion was isolated from echiuroid sperm according to the procedure described for its isolation from sea urchin sperm by Podell and Vacquier (4). Judging from the high specific activity of 5 L-nucleotidase, the purity of the plasma membrane thus obtained was high in this fraction but the recovery was assumed to be about 10%, calculated on the basis of the activity of 5 L-nucleotidase in this membrane fraction and its whole activity in sperm. Enzyme Assays-The activity of cytochrome c oxidase was estimated at 20 Ž by a polarographic method (5). The reaction mixture (2.5 ml) was composed of 2 ml of 50mM phosphate buffer, ph 7.2, containing 10mM EDTA, 0.15 ml of 0.1 M ascorbate-na, 90ƒÊl of 2mM cytochrome c, 10 pl of 50mM TMPD, and 400ƒÊl of enzyme source. The reaction was started by adding the enzyme source. Oxygen concentration in the reaction mixture in a closed vessel was monitored with an oxygen electrode. The activity is ex pressed as nmol O2 consumed/108 cell eq./min. When the activity was estimated in the presence of CO, 50mM phosphate buffer, which was to be added to the reaction mixture, was vigorously bubbled with CO gas for 15 min. The activity of 5 L-nucleotidase was estimated by the method of Ipta (6). Inorganic phosphate liberated from AMP during 30 min incubation at 25 Ž was estimated by the method of Gomori (7). The activity is expressed as nmol P, liberated/mg protein/30 min. Protein amount was determined by the method of Lowry et al. (8), using bovine serum albumin as the standard. The activity of 5 L-nucleo tidase was not expressed per 108 cell eq., because the recovery of plasma membrane was quite poor Light Irradiation at Various Wavelengths-Light irradi ation was performed with the Okazaki Large Spectrograph at the National Institute for Basic Biology (NIBB), Okazaki. The light fluence rates were monitored by a photon density meter HK 1, custom made at the Institute for Physical and Chemical Research, Wako, Saitama. Neutral density filters were used to alter the fluence rate. Chemicals-Cytochrome c, NADH, NADPH, FCCP, antimycin A, and oligomycin were obtained from Sigma Chem., MO. Ascorbate-Na, TMPD, and NaCN were the products of Kanto Chem., Tokyo. RESULTS Figure 1 shows the relationship between the respiratory rate of sperm in the presence of CO and wavelength of light irradiation. The dashed line indicates the mean respiratory rate of echiuroid sperm in the presence of CO observed 1 min before light irradiation. The estimation of the respira tory rate in the presence of CO was made in a mixture of 0.5 ml of air-bubbled and 3 ml of CO-bubbled sea water. The respiratory rate of sperm in the absence of CO (thin line) was estimated in a mixture of 0.5 ml of air-bubbled and 3 ml of N,-bubbled sea water. The respiratory rate estimated in this mixture, containing 02 at a concentration as low as in a mixture of 3 ml of CO-bubbled and 0.5 ml of air Fig. 1. Relationship between the respiratory rate of sperm in the presence of CO and wavelength of light irradiation at the light fluence rate of 2.0 ~1015 photon/cm2/s. The rate of sperm respiration was estimated in a mixture of 0.5 ml of air-bubbled and 3 ml of CO-bubbled sea water. The respiratory rate under light irradiation in the presence of CO (v) was estimated 2 min after the initiation of irradiation. The rate at the fluence rate of 2 ~1015 photon/cm2/s was calculated on the basis of the relationship between the respiratory rate and the light fluence rate at each wavelength shown in Fig. 2. The rates of respiration in the absence of CO were also estimated under light irradiation (0) at the light fluence rates of 7.7 x 10" (350 nm), 6.5 ~1015 (400 nm), 9.9 ~1015 (430 nm), 1.1 ~1011 (450 nm), 1.0 ~1016 (500 nm), 1.1 ~1016 (530 nm), 1.0 ~1016 (570 nm), 9.7 ~1015 (600 nm), and 7.9 ~1015 photons/cm2/s (650 nm). The respiratory rate of sperm used in this experiment was 1.86±0.21 (SEM) nmol 02/108 cells/min in the absence of CO in the dark (thin line) and was the same as the rate under light irradiation ( ). The dotted line indicates the mean value of respiratory rate in the presence of CO 1 min before the light irradiation (in the dark). All values shown in the figure were obtained by a single experiment on one batch of sperm. Essentially the same action spectra were also obtained in 3 other experiments. Vol. 109, No. 3, 1991

3 488 A. Fujiwara et al. bubbled sea water, was very similar to the rate estimated in air-bubbled sea water. The respiratory rate in the presence of CO was slightly higher than the rate in its absence. In the absence of CO, light irradiation did not alter the respiratory rate of sperm even at the maximum fluence rate provided by the large spectrograph, but it enhanced the respiratory rate in its presence at all examined wave lengths (Fig. 1). The rates of respiration in the presence of CO under light irradiation, shown in Fig. 1, are those estimated 2 min after the initiation of irradiation at the light fluence rate of 2.0 x 1015 photon/cm2/s. The action spectrum of the activating effect of light irradiation on the respiration of sperm in the presence of CO (Fig. 1) shows a sharp and large peak at the wavelength of 430 nm and broad and small peaks at 530 and 570 nm in the examined range of wavelengths between 350 and 650 nm. The activating effect of light irradiation was minimum at wavelengths of nm. Figure 2 shows the relationships between the respiratory rate in the presence of CO and the light fluence rate at 400, 430, 460, 510, and 530 nm. The relationships at other wavelengths than those shown in Fig. 2 are omitted to simplify the figure. The respiratory rate of sperm in the presence of CO was enhanced by light irradiation at 460 and 510 nm in proportion to the light fluence rate (Fig. 2). The same result was observed on light irradiation at , , and nm (data not shown). The respira tory rates were also enhanced by light irradiation at 400, 430, and 530 nm in proportion to the light fluence rate at lower rates than about 5 ~1015, 3 ~1015, and 9 ~1015 photon/cm2/s, respectively. At higher fluence rates, the light irradiation at these wavelengths did not cause any further increase in the respiratory rate. At these high rates of light fluence, the activating effect of light irradiation thus reached a plateau. The plateau rates of respiration obtained at 400, 430, and 530 nm were almost the same, irrespective of wavelength. The plateau respiratory rates obtained at 410, 420, 440, 520, 540, and 570 nm (data not shown) were also very similar to those obtained at 400, 430, and 530 nm (Fig. 2). The rate of respiration obtained at the plateau of activating effect of light irradiation, is probably the highest that can be obtained by light irradiation. The respiratory rate of sperm in the dark in the presence of CO at 14 and 29% of its saturated concentration (defined in "MATERIALS AND METHODS") was almost the same as in its absence, but was slightly higher at concentrations between 43 and 86%, as shown in Fig. 3. The rate of sperm respiration under light irradiation in the presence of CO at 14 and 29% of its saturated concentration was also very similar to the rate in the dark and became higher in the presence of CO at about 43% of its saturated concentration. The rate under light irradiation steeply increased in rela tion to CO concentration between 29 and 86% of its saturated concentration. In this experiment (see also Figs. 4 and 5), the light irradiation was performed at 430 nm at the fluence rate of 9.9 x 1015 photon/cm2/s (the maximum fluence rate at 430 nm obtainable with the large spectro graph). CO is necessary for the CO-insensitive respiratory chain to exhibit a high respiratory rate under light irradia tion. It is likely that the photo-activated reaction is the rate-limiting one in the CO-insensitive respiratory chain and hence, the respiratory rate is probably low unless this reaction is activated by light irradiation. The rates of respiration in the dark estimated in the presence and absence of CO gradually changed in sperm, when dry sperm were stored in an ice bath. The respiratory rate of sperm kept for 1 h in an ice bath was almost the same as observed in fresh sperm just after isolation from the echiuroid body, and was 2.29±0.18 nmol 02/108 cells/ min. In fresh sperm, the rate became lower than 0.2 nmol 02/108 cells/min in the presence of CO. The respiratory rates of sperm 3 h after dry sperm isolation was 1.79 ± 0.23 and 2.53±0.33 nmol 02/108 cells/min in the absence and Fig. 2. The respiratory rate of sperm irradiated at various rates of light fluence in the presence of CO. The respiratory rates of sperm in the presence of CO, shown in the figure, are those obtained 2 min after the initiation of light irradiation at the wavelength of (0), 460 (A), 510 (u), or 530 (O) at various light fluence rates. The respiratory rate in the dark (U) is the mean of 5 experiments on one sperm batch ± SD (shown with a vertical bar). The CO concentra tion was the same as shown in Fig. 1. The values were obtained on the same batch of sperm as that used in the experiment shown in Fig. 1, Fig. 3. Effect of CO on the respiratory rate of sperm in the dark and under light irradiation. CO concentrations in sea water were adjusted by replacing CO-bubbled sea water with N2-bubbled sea water in a mixture of CO. and air-bubbled sea water (6: 1, v/v). For convenience, CO concentration in sea water bubbled with CO gas for 15 min is regarded as its saturated concentration and CO concentra tion in sperm suspension is expressed as a percentage of its saturated concentration (see "MATERIALS AND METHODS"). The light irradiation was performed at 430 nm at the light fluence rate of 9.8 x 1015 photon/cm2/s. Values are the respiratory rates under light irradiation (0) and in the dark (0), and are typical values from 3 experiments. The results shown in the figure were obtained with one sperm batch. J. Biochem.

4 Photo-Activation of CO-Insensitive Respiration 489 Fig. 5. Sensitivities of sperm respiration to antimycin A, CN-, and TMPD. Solid lines indicate tracings of change in the oxygen concentration in sperm suspension in the absence (A) and presence of CO (B-D). The CO concentration was the same as shown in Fig. 1. Downward arrows indicate the times of initiation of the light irradia tion at 430 nm at the light fluence rate of 9.9 x 1015 photon/cm2/s. Arrowheads labeled AMA, CN-, and TMPD indicate the times of adding 35 #1 stock solutions of antimycin A (5mM), CN (10mM), and TMPD (5mM) to 3.5 ml of sperm suspension, respectively. The values shown are the rates of sperm respiration expressed as nmol 02/ 108 cells/min. Fig. 4. Responses of sperm respiration to light irradiation. Solid lines show tracings of the decrease in oxygen concentration in sperm suspension in the absence (A) and in the presence of CO (B, C). The CO concentration was the same as shown in Fig. 1. Arrowheads labeled Sp indicate times of sperm addition. Downward and upward arrows show the times of initiation and termination of light irradia tion at the wavelength of 430 nm, at the light fluence rate of 9.9 x 1015 photon/cm2/s. Values in the figure are the rates of respiration expressed as nmol 02/108 cells/min. These tracings were representa tive ones from 5-8 experiments. presence of CO, respectively. These values were the means of 6 experiments ±SD on different batches of sperm. The respiratory rates of sperm in the absence and presence of CO were maintained at almost the same level as above up to at least 24 h after sperm isolation from the body. CO does not inhibit respiration in sperm stored for a long time. It has been reported that CO strongly blocks the respiration of echiuroid sperm (3). In this previous study, the estima tion of respiratory rate was done on fresh echiuroid sperm. The light irradiation at 430, 460, 530, and 570 nm in the presence of CO, at the highest fluence rates obtainable with the large spectrograph, made the respiratory rate in fresh sperm (stored for 1 h) as high as in sperm stored for 3-20 h in an ice bath, though the minimum light fluence rates to induce evident increase in the respiratory rate of fresh sperm in the presence of CO were somewhat higher than in stored sperm at all examined wavelengths (data not shown). It has been reported that white light irradiation does not enhance the respiratory rate of echiuroid sperm in the presence of CO. In this previous study, the light fluence rate, which was considerably lower than in the present study, was not sufficient to enhance the respiratory rate in the presence of CO in fresh sperm, which are less sensitive to light than stored ones. In the present study, the action spectrum for photo activation of respiration (Fig. 1) was obtained on one batch of sperm. It took about 12 h to obtain the action spectrum (Fig. 1), since at least 4 estimations of respiratory rate at various fluence rates should be made at each wavelength, as shown in Fig. 2. Hence, dry sperm stored in an ice bath for at least 3 h and at most for 18 h were used in the present study. Sperm thus stored for 3 and 18 h in an ice bath exhibited the same responses to CO either in the dark or under light irradiation. Furthermore, sperm stored in an ice bath for 48 h vigorously moved following dilution of the dry sperm in sea water and retained the capacity to fertilize eggs. Physiological functions are maintained in sperm stored for a long time. The action spectrum thus obtained for stored sperm of one batch was essentially the same as those obtained on for 9 batches of fresh sperm. Figure 4 shows tracings of the decrease in oxygen concentration in sperm suspension. In the absence of CO, light irradiation at 430 nm did not enhance the respiratory rate (A). In the presence of CO, the light irradiation induced a marked increase in the rate of respiration about 30 s after the initiation of irradiation and the rate de creased within 1 min after turning off the light (B, C). Sperm, which had been once irradiated to undergo an augmentation of respiration, again exhibited a marked increase in the respiratory rate following another light irradiation (C). Activation of respiration by light irradia tion occurs reversibly in echiuroid sperm. As shown in Fig. 5, the respiration in the presence of CO was inhibited by antimycin A and cyanide either under light irradiation (C, D) or in the dark (A, B). The inhibition of respiration by antimycin A under light irradiation, or in the dark, was almost completely canceled by adding TMPD and TMPD-restored respiration was blocked by CN (B, D). These results suggest that photo-activated respiration in the presence of CO results from AMA-sensitive electron Vol. 109, No. 3, 1991

5 490 A. Fujiwara et al. with the maximum rate of respiration in sperm under light irradiation (about 11 nmol 02/108 cell/min, as shown in Fig. 2) suggests that the reaction catalyzed by cytochrome c oxidase is not the rate-limiting reaction in the photo - activated respiratory system even in the presence of CO, though cytochrome c oxidase is strongly blocked by CO. In the plasma membrane fraction, isolated from echiu roid sperm by the method of Podell and Vacquier (4) for the isolation of plasma membrane from sea urchin sperm, the activity of 5 L-nucleotidase, a marker enzyme for plasma membrane, was found to be nmol P, liberated/mg protein/30 min. In sperm homogenate, the activity was 19.2 nmol P,/mg protein/30 min. In the mitochondrial fragment fraction and in the fraction containing heads and tails, the activities were 11.2 and 21.8 nmol P, /mg protein/ 30 min. Thus, the other fractions seem to contain quite small amounts of plasma membrane. In the plasma membrane fraction, no activity of cytochrome c oxidase was found and 02 consumption was undetectable in the Fig. 6. Effects of oligomycin and FCCP on sperm respiration. Solid lines show tracings of change in the oxygen concentration in sperm suspension in the absence (A) and the presence of CO (B-E). Downward and upward arrows indicate the times of initiation and termination of the light irradiation at 430 nm at the fluence rate of 9.9 x 10" photon/cm2/s. Arrowheads labeled oligo, FCCP, and CN - indicate the times of adding 35,u l stock solutions of 5mM oligomycin, 100ƒÊM FCCP, and 10mM NaCN to 3.5 ml of sperm suspension, respectively. The values shown are the respiratory rates of sperm expressed as nmol/o2/108 cells/min. transport to cytochrome c and finally to molecular oxygen in the reaction catalyzed by CN--sensitive and CO-insensi tive cytochrome c oxidase. Besides the sensitivity to CO, the sensitivities of this photo-activated respiratory system to antimycin A and CN are the same to those of the mitochondrial respiratory chain. A fraction containing 88.2% of the whole activity of cytochrome c oxidase in sperm homogenate was obtained and was designated as mitochondrial fragment fraction. The activity in this fraction was nmol 02 consumed/ 108 cell eq./min, and became undetectable in the presence of 0.1mM KCN. The other precipitate fraction containing the heads and tails exhibited CN--sensitive cytochrome c oxidase activity of 12.6 nmol 02/108 cell eq./min. In the post mitochondrial supernatant fraction, the activity of cytochrome c oxidase was undetectable. In the presence of CO at 80% of its saturated concentration, the activity was undetectable in the fraction containing the heads and tails as well as in the supernatant fraction, and it was 21.7 nmol 02/108 cell eq./min in the mitochondrial fragment fraction. CO at this high concentration caused about 92% inhibition of cytochrome c oxidase in this fraction. The cytochrome c oxidase activity in the presence of CO, however, seems to be enough to support the maximum rate of respiration under light irradiation in the presence of CO in sperm. Simple comparison of the activity of cytochrome c oxidase in the presence of CO (about 22 nmol 02 / 108 cell eq. /min) presence of TMPD and ascorbate as well as NADH or NADPH (final concentration: 5ƒÊM). Further studies are necessary to establish that there are no terminal oxidases other than mitochondrial cytochrome c oxidase in sperm. At present, however, it can be assumed that cytochrome c oxidase in mitochondria is the only terminal oxidase in the photo-activated respiratory system in echiuroid sperm. As shown in Fig. 6, oligomycin completely blocked the respiration in the presence of CO either in the dark (D) or under light irradiation (E). Oligomycin-induced inhibition of respiration was canceled by FCCP, an uncoupler of oxidative phosphorylation (D, E). Responses of respira tion to these compounds in the presence of CO were the same as in its absence (data not shown). Probably, the respiration in the presence and absence of CO is coupled to oxidative phosphorylation in the dark and under light irradiation. In the presence of CO, FCCP hardly enhanced the respiratory rate in the dark (B), as well as under light irradiation (C), and the respiration in sperm in the presence of FCCP increased following the initiation of light irradia tion (B) in the same manner as observed in the absence of FCCP (Fig. 4). In the absence of CO, the respiratory rate in sperm kept with FCCP was almost the same as observed in sperm kept without this compound and was hardly en hanced by light irradiation (A). These observations rule out the possibility that light irradiation uncouples the respira tion from oxidative phosphorylation in the presence of CO, to enhance the respiratory rate in sperm. DISCUSSION The action spectrum of the activating effect of light irradia tion in the presence of CO on the respiration of echiuroid sperm shows a sharp and large peak at the wavelength of 430 nm and broad and small peaks at 530 and 570 am. This action spectrum is very similar in its profile to the absorp tion spectrum of reduced cytochrome b. This similarity suggests that absorption of photon energy by reduced b-type cytochrome results in an alteration of its conforma tion, which makes it more active, and accelerates its redox reaction to cause an increase in the respiratory rate in sperm. This b-type cytochrome is not necessarily identical with cytochrome b itself, which mediates electron trans port from CoQ to cytochrome c in the mitochondrial J. Biochem.

6 Photo-Activation of CO-Insensitive Respiration 491 respiratory chain. In the ciliate, Paramecium tetraurelia, photo-reactivated b-type cytochrome, identified on the basis of its absorption spectrum in the presence of CO, is proposed to act as a terminal oxidase in the respiratory system, branching from the mitochondrial respiratory chain between flavoproteins and cytochrome b (9, 10). This terminal oxidase may catalyze a reaction similar to that in Escherichia coli (11, 12). In echiuroid sperm, however, the respiration in the presence of CO was completely blocked by AMA either under light irradiation or in the dark and the inhibition by AMA was reversed by TMPD. TMPD is known to accept an electron equivalent from reduced cytochrome b, as well as ascorbate, and to transport it to cytochrome c in the mitochondrial respiratory chain (13). TMPD-induced res piration in AMA-treated sperm is blocked by CN-. On the basis of these observations, it is assumed that the electron equivalent is transported in the span of the mitochondrial respiratory chain between cytochrome b and cytochrome c in echiuroid sperm either in the dark or under light irradia tion. It is likely that this photo-activated cytochrome b is not a terminal oxidase in echiuroid sperm. Probably, the electron equivalent transported to cytochrome c is finally accepted by 0, in the reaction catalyzed by CN sensitive cytochrome c oxidase. Isozymes of cytochrome c oxidase and an alternative terminal oxidase have been found in mitochondria (14, 15). Provided that sperm mitochondria might contain CO -insensitive cytochrome c oxidase as an isozyme of terminal oxidase, an increase of the rate of cytochrome c reduction due to photo-activation o cytochrome b would become apparent as an increase in the respiratory rate in the presence of CO. In the present study, however, only the activity of CO-sensitive cytochrome c oxidase was found in mitochondrial fraction. In the other subcellular fractions, such as plasma membrane fraction, no oxygen consumption suggesting the presence of other terminal oxidases was detectable. Thus, photo-activated respiration in the pres ence of CO should be concluded to result from electron transport to molecular oxygen in the reaction catalyzed by CO-sensitive cytochrome c oxidase in mitochondria. Cyto chrome c oxidase in sperm mitochondria was strongly but not completely inhibited by CO at high concentration. Even in the presence of CO at high concentrations, as examined, the activity of the CO-inhibited enzyme seems to be high enough to support respiration at the maximum rate ob served under light irradiation. The light irradiation, especially at the wavelength of 430 nm, induced an extraordinarily high rate of respiration in sperm in the presence of CO but did not cause any increase in its absence. CO seems to be indispensable for the redox reaction in this b-type cytochrome to be activated by light irradiation. It seems likely that CO binding to cytochromes, which inhibits their redox reactions, is released by light irradiation in the echiuroid sperm, resulting in a reactiva tion of respiration. In echiuroid sperm, however, the respiratory rate was hardly reduced by CO and became higher with increasing CO concentration. This observation suggests that CO concentration-dependently activates sperm respiration. The maximum rate of respiration in the presence of CO under light irradiation was markedly higher than in its absence. Even when light irradiation releases CO-binding in cytochromes to cancel CO-induced inhibition of respiration, it does not make the respiratory rate in the presence of CO higher than in its absence. Thus, the markedly high rate of respiration in the presence of CO under light irradiation does not seem to result only from light-induced reversal of CO-caused blockage of redox reactions. On the other hand, it seems probable that CO is utilized as an artificial electron donor from which an electron equivalent is to be transported to molecular oxygen through the mitochondrial respiratory chain in echiuroid sperm. It has been reported that CO is oxidizable and is converted to CO, in echiuroid eggs (1, 2, 16). At 14 and 29% of saturated CO concentration, the respiratory rate was almost the same as in its absence, and was hardly enhanced by light irradia tion. When the CO concentration is quite low, respiration seems to result from electron transport mainly from physiological electron donors. At CO concentrations above 43%, the respiratory rate increased with increasing concen tration slightly in the dark and evidently under light irradiation. Utilization of CO as an artificial electron donor in this respiratory system probably becomes apparent, especially under light irradiation, when the amount of CO becomes markedly higher than the amounts of physiologi cal donors. In the absence of CO, supply of electron equivalent from physiological electron donors is probably rate-limiting in this respiratory system, even when the redox reaction in b-type cytochrome is activated by light. It was found that oligomycin inhibited the respiration of sperm in the presence and absence of CO either in the dark or under light irradiation. This suggests that the respiration of sperm is coupled to oxidative phosphorylation even in the presence of CO. 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