4 results
Changes in the activities of protein phosphatase type 1 and type 2A in sea urchin embryos during early development
- Manabu Kawamoto, Akiko Fujiwara, Shin-ichi Kuno, Ikuo Yasumasu
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Serine/threonine protein phosphatases expected to participate in the process of signal transduction, cell movement such as cell division and gene expression (Kinoshita et al., 1990; Healy et al., 1991; Mayer-Jaekel et al., 1993; Mumby & Walter, 1993), are classified into type 1 (PP1), type 2A (PP2A), type 2B and type 2C in mammalian cells. PP1 and PP2A are known to be strongly inhibited by okadaic acid (OA) (Tachibana et al., 1981; Bialojan Takai, 1988), a polyether fatty acid isolated from the marine sponge Halicondria okadai (Haystead et al., 1989). OA is also known to inhibit PP2A at lower concentrations than that to block PP1 in mammalian cells, but does not inhibit the activities of other phosphatase species (Ishihara et al., 1989).
The p-nitrophenyl phosphate (pNPP) splitting activity in the extract obtained from eggs of the sea urchin Hemicentrotus pulcherrimus was found to be inhibited by OA and calyculin A (CLA), potent inhibitors of PP1 and PP2A. OA-sensitive phosphatases are known to catalyse pNPP splitting (Takai & Mieskes, 1991), in the same manner as other OA-insensitive phosphatases.
Four peaks of the pNPP splitting activity were obtained by QAE-Toyopearl chromatography in the extract of sea urchin eggs. In two of these four peaks, pNPP splitting reactions were strongly inhibited by OA and CLA at quite low concentration. High sensitivities of the pNPP splitting reaction to OA and CLA in these two peaks suggest that pNPP splitting results from the reaction catalysed by PP2A. The molecular masses of proteins exhibiting OA-sensitive pNPP splitting activities in these two peaks were found to be about 160 kDa by Superdex 200HR, and were similar to that of mammalian PP2A trimeric holoenzyme. By immunoblot analyses with anti-human PP2A catalytic subunit antibody, an immunoreactive 36 kDa protein was found by SDS-PAGE in a peak of OA-sensitive pNPP splitting activity obtained by QAE-Toyopearl chromatography. Sea urchin eggs have at least two PP2A-like enzymes with similar molecular masses to that of mammalian PP2A, and one of them contains human-type catalytic subunit.
Regulation of mitochondrial respiration in eggs and embryos of sea urchin
- Ikuo Yasumasu
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It is well known that sea urchin eggs, which exhibit quite a low rate of respiration before fertilisation, undergo a sudden increase in the rate of respiration followed by its gradual decrease in about a 15 min period after fertilisation (Ohnishi & Sugiyama, 1963; Epel, 1969), in which the respiration is mediated mainly by Ca2+-activated non-mitochondrial respiratory systems (Foerder et al., 1978; Perry & Epel, 1985a,b). During this short period the rate of mitochondrial respiration gradually increases (Yasumasu et al., 1988) and stabilises at a higher rate than before fertilisation (Warburg, 1908, 1910; Whitaker, 1933; Yasumasu & Nakano, 1963), when the respiration due to non-mitochondrial respiratory systems is turned off. The rate of mitochondrial respiration, once enhanced upon fertilisation, increases further in the period between hatching and the gastrula stage, without any changes in the number of mitochondria or the capacity of electron transport in the mitochondrial respiratory chain (Fujiwara & Yasumasu, 1997; Fujiwara et al., 2000). It is likely that the respiratory rate is reduced by regulation of electron transport in the mitochondrial respiratory chain and increases due to the release of electron transport from the regulation upon fertilisation and after hatching.
A marked increase in the respiratory rate after hatching is accompanied by an evident decrease in the ATP level without any change in the levels of ADP and AMP (Mita & Yasumasu, 1984). In isolated mitochondria, the rate of respiration, estimated in the presence of ADP at the same concentration as in embryos, is reduced by a high concentration of ATP as found in embryos before hatching but is not affected at a concentration as low as in gastrulae (Fujiwara & Yasumasu, 1997; Fujiwara et al., 2000) ATP at a high concentration probably blocks ATP release from mitochondria and consequently inhibits ADP uptake coupled to ATP release in the ATP/ADP translocation reaction in the mitochondrial membrane, causing a shortage of intra-mitochondrial ADP.
Morphogenesis of exogut isolated from vegetalised embryo of sea urchin
- Yasuyuki Kamata, Kazuyuki Endo, Hiroyuki Nozaki, Akiko Fujiwara, Ikuo Yasumasu
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It is well known that sea urchin embryos treated with lithium chloride (LiC1) develop to abnormally into vegetalised embryos, in which differentiation of ectodermal cells is inhibited. When embryos of the sea urchins, Hemicentrotus pulcherrimus and Anthocidaris crassispina were treated with 20 mM LiC1 from the 8-cell stage to the corresponding early gastrula stage, they developed to vegetalised embryos with a large exogut 45 h after fertilisation. In these vegetalised embryos, high activity of alkaline phosphatase (AP) was detected histochemically at the end of the exogut where it is attached to the embryo body. High activity of AP is known to be detected specifically in the gut of sea urchin pluteus larvae by the same procedure as used in this study. Hence, we concluded that this part of the exogut is composed of the cells which develop into the cells of the gut in normal development.
When exogut isolated from vegetalised embryos was cultured in the extract obtained from eggs or embryos, the end composed of the cells in which high AP activity was detected, expanded during culture and formed a large spherical structure about 24 h after the initiation of culture. The minimum concentration of extract to cause expansion of isolated exogut was 5 × 103 egg or embryo equivalent/ml ASW (artificial seawater). The extract boiled at 95 °C for 1 h also caused expansion of isolated exogut at the same concentrations as non-boiled extract. On the other hand, the extract obtained from eggs or embryos by chloroform–methanol extraction did not cause any expansion of exogut, but the aqueous phase, heat-dried and dissolved in ASW, induced expansion of isolated exogut.
Light-induced reactivation of movement in degenerated sperm of echiuroid, oyster and sea urchin
- Yasuyuki Kamata, Akiko Fujiwara, Ken Yamazaki, Eigoro Tazawa, Ikuo Yasumasu
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Previously, it has been reported that NADH cytochrome c reductase and succinate cytochrome c reductase, in which the redox reaction in cytochrome b is involved, are activated by light irradiation with peaks of photo-activation at wavelengths of 430, 530 and 570 nm corresponding to those in the absorption spectrum of reduced cytochrome b in mitochondria isolated from sperm of echiuroid, oyster and sea urchin (Tazawa et al., 1996). In sperm of these species, augmentation of respiration due to photo-activation of the cytochrome b reaction is observed only when the electron transport in this span of the mitochondrial respiratory chain is inhibited by carbon monoxide (Fujiwara et al., 1991; Yasumasu et al., 1991) or by a decrease in the amount of cytochrome b due to sperm ageing. In sperm cultured for a long time, the respiratory rate was very low and almost all sperm became immotile. In these sperm, respiration was reactivated by light irradiation at the wavelengths of 430, 530 and 570 nm (Fujiwara et al., 1999).
In the present study, photo-reactivation of movement in these somewhat degenerated sperm incubated for a long time was also found to occur, with peaks at the above-mentioned wavelengths. We concluded that photo-reactivation of movement in these sperm, in which the cytochrome b reaction probably became rate-limiting in the respiration chain, is supported by reactivation of respiration by light irradiation. On the other hand, though the ATP level decreased to a rather low level at about 30 min after the initiation of incubation in sperm of all species examined, sperm swam for more than 10 h, by which time the ATP level was quite low. Light irradiation induced reactivation of movement but did not alter the rather low ATP level in these sperm. Thus the ATP level does not seem to be responsible for making sperm immotile. In sperm treated with Triton X-100, movement was induced by adding ATP and Mg2+, even when many sperm had become immotile after a long incubation. Hence, capacity for movement does not seem to be reduced by a long incubation time. The movement of sperm treated with Triton X-100 was not activated bv light irradiation.