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Originally published In Press as doi:10.1074/jbc.M313801200 on December 29, 2003

J. Biol. Chem., Vol. 279, Issue 11, 10408-10412, March 12, 2004
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Recombinant Phospholipase C{zeta} Has High Ca2+ Sensitivity and Induces Ca2+ Oscillations in Mouse Eggs*

Zen Kouchi{ddagger}§, Kiyoko Fukami¶, Tomohide Shikano{ddagger}, Shoji Oda{ddagger}, Yoshikazu Nakamura¶||, Tadaomi Takenawa||, and Shunichi Miyazaki{ddagger}

From the {ddagger}Department of Physiology, Tokyo Women's Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan, ||Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan, Laboratory of Genome and Biosignal, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

Received for publication, December 17, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Sperm-specific phospholipase C{zeta} (PLC{zeta}) is known to induce intracellular Ca2+ oscillations and subsequent early embryonic development when expressed in mouse eggs by injection of RNA encoding PLC{zeta} (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development 129, 3533-3544). The present study addressed characteristics of purified mouse PLC{zeta} protein that was synthesized using the baculovirus/Sf9 cell expression system. Microinjection of recombinant PLC{zeta} protein into mouse eggs induced serial Ca2+ spikes quite similar to those produced by the injection of sperm extract, probably because of repetitive Ca2+ release from the endoplasmic reticulum caused by continuously produced inositol 1,4,5-trisphosphate. Recombinant PLC{delta}1 also induced Ca2+ oscillations, but a 20-fold higher concentration was required compared with PLC{zeta}. In the enzymatic assay of phosphatidylinositol 4,5-bisphosphate hydrolyzing activity in vitro at various calcium ion concentrations ([Ca2+]), PLC{zeta} exhibited a significant activity at [Ca2+] as low as 10 nM and had 70% maximal activity at 100 nM [Ca2+] that is usually the basal intracellular calcium ion concentration level of cells. On the other hand, the activity of PLC{delta}1 increased at a [Ca2+] between 1 and 30 µM. EC50 was 52 nM for PLC{zeta} and 5.7 µM for PLC{delta}1. Thus, PLC{zeta} has an ~100-fold higher Ca2+ sensitivity than PLC{delta}1. The ability of purified PLC{zeta} protein to induce Ca2+ oscillations qualifies PLC{zeta} as a proper candidate of the mammalian egg-activating sperm factor. Furthermore, such a high Ca2+ sensitivity of PLC activity as PLC{zeta} that can be active in cells at the resting state is thought to be an appropriate characteristic of the sperm factor, which is introduced into the ooplasm upon sperm-egg fusion, triggers Ca2+ release first, and maintains Ca2+ oscillations.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Fertilized mammalian eggs exhibit repetitive transient rises in [Ca2+]i1 referred to as Ca2+ oscillations because of repeated Ca2+ release from the ER mainly through the InsP3 receptor (1). The [Ca2+]i rises are a pivotal signal for egg activation and are responsible for early embryogenesis (2, 3). Accumulated evidence suggests that Ca2+ oscillations are induced by cytosolic sperm factor introduced into the ooplasm upon sperm-egg fusion (2, 4). Therefore, the identification of the Ca2+ oscillation-inducing sperm factor, which is the egg-activating sperm factor, is currently being studied as a central subject to elucidate the mechanisms of fertilization. Recently, Saunders et al. (5) have reported a novel type of PLC (the enzyme that produces InsP3 and diacylglycerol from membrane PtdInsP2), PLC{zeta}, which is specifically expressed in mammalian sperm. The injection of RNA-encoding PLC{zeta} into mouse eggs causes Ca2+ oscillations and subsequent early embryonic development by expressed PLC{zeta} at an estimated level comparable with the content in a single sperm (5). The Ca2+ oscillation-inducing activity of sperm extract (4, 6) is lost when pretreated with an antibody against PLC{zeta} (5). Thus, PLC{zeta} is considered a strong candidate of the sperm factor. To assure this possibility, it is primarily necessary to examine whether purified PLC{zeta} protein induces Ca2+ oscillations in the egg.

It has been shown that PLC{beta}1 (7), -{gamma}1 (8, 9), -{gamma}2 (8), -{delta}1 (10), and -{delta}4 (7) are expressed in mammalian sperm and that recombinant PLC{beta}1, -{gamma}1, -{gamma}2, and -{delta}1 failed to cause Ca2+ release in the ooplasm (11). PLC{zeta} is the smallest PLC identified to date, lacking the N-terminal PH domain (Fig. 1A) (5) that is found in all isoforms of PLC{beta}, -{gamma}, and -{delta} and is the site for interaction with membrane phospholipids (12). Because PLC{zeta} as well as PLC{delta} lacks a regulatory domain such as the G protein-binding site of PLC{beta} or the SH domain of PLC{gamma} for phosphorylation by tyrosine kinase, the activation mechanism of PLC{zeta} and PLC{delta} is unknown. Therefore, it is also necessary to access how PLC{zeta} undergoes the active state for production of InsP3. Here we first show that recombinant PLC{zeta} protein induces Ca2+ oscillations in mouse eggs and that PLC{zeta} possesses an extremely high Ca2+ sensitivity in the PtdInsP2 hydrolyzing activity to be active even at the resting state of cells.



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  		FIG. 1.
SDS-polyacrylamide gel electrophoresis of purified PLC{zeta} and PLC{delta}1. A, schematic illustration of the domain features of PLC{zeta}, s-PLC{zeta}, and PLC{delta}1. X, X domaon; Y, Y domain; C2, C2 domain; aa, amino acids. B, SDS-polyacrylamide gel electrophoresis of purified mouse PLC{zeta} and PLC{delta}1 expressed in Sf9 cells. PLC{zeta} (100 ng) and PLC{delta}1 (100 ng) were separated on 10% SDS-polyacrylamide gel and subjected to silver staining. M, marker. Arrowheads indicate His-tagged PLC{zeta} and PLC{delta}1.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Cloning of PLC{zeta} and PLC{delta}1—cDNA encoding full-length PLC{zeta} (GenBankTM accession number AF435950 [GenBank] ) was cloned from a cDNA library originated from mouse testis mRNAs. PLC{zeta} cDNA was amplified by PCR using Pfu polymerase and the following primers: forward, 5'-GAC AAGCGGCCCAGATCATG-3'; internal forward primer involving an EcoRI site, 5'-GGAATTCATATGGAAAGCCAACTTCATGAG-3'; reverse, 5'-CTAACGCGTCAGTTACATGCG-3'; internal reverse primer involving an XbaI site, 5'-GTCTAGATTACTCTCTGAAGTACCAAACATAAATAAAC-3'. cDNA of PLC{delta}1 (GenBankTM accession number AF133125 [GenBank] ) derived from the mouse testis was amplified using 5'-GGAATTCATATGGACTCCGGTCGGGAC-3' involving an EcoRI site and 5'-GCTCTAGA TTAGTCCTGGATGGAGATCTTC-3' involving an XbaI site. PLC{zeta} and PLC{delta}1 were subcloned into pBluescript II SK(+) (Stratagene) using EcoRI and XbaI sites. A PLC{zeta} variant lacking three EF-hand domains (GenBankTM accession number AK006672 [GenBank] ), designated as s-PLC{zeta}, was cloned using the same primers as those for PLC{zeta} except the internal forward primer, 5'-GGAATTCATATGGAGATCGATCACTCTGATTC-3'. A mutant PLC{zeta} lacking three EF-hand domains and a catalytic X domain, PLC{zeta}{Delta}EFX, was constructed using 5'-GGAATTCATATGGAGATCGATCACTCTGATT-3' involving an EcoRI site, 5'-GTCTAGATTACTCTCTGAAGTACCAAACATAAATAAAC-3' involving an XbaI site, 5'-GTGGGTACCTTATCTGAAACCCACGAG-3' involving a KpnI site, and the reverse primer. The correct nucleotide sequences of PLC{zeta} (1), s-PLC{zeta}, PLC{delta}1 (10), and PLC{zeta}{Delta}EFX were confirmed using a BigDye Terminator Sequencing kit (Applied Biosystems).

Synthesis and Purification of PLC{zeta} and PLC{delta}1—Recombinant PLC{zeta} or PLC{delta}1 was prepared by a baculovirus expression system (13). Using EcoRI and XbaI sites, the open reading frames of PLC{zeta}, s-PLC{zeta}, PLC{delta}1, and PLC{zeta}{Delta}EFX were subcloned into a baculovirus expression vector, pFastBac HT (Invitrogen), which contains an N-terminal His tag sequence. The recombinant baculoviruses were generated by transformation into DH10Bac, and the transformants containing a PLC-inserted bacmid were selected by blue/white screening (see Instruction Manual by Invitrogen). Sf9 cells were grown at 28 °C in Sf900 serum-free medium (Invitrogen) containing 5% fetal bovine serum. The cells (1 x 106 cells/ml) were infected with baculoviruses and incubated for 3 days. The infected cells (3 liters for PLC{zeta} and 300 ml for PLC{delta}1) were lysed with a French pressure cell in a lysis buffer (120 mM KCl, 10 mM imidazole, 0.2 mM phenylmethanesulfonyl fluoride, 2 µg/ml pepstatin, 5 µg/ml leupeptin, and 20 mM HEPES/KOH, pH 7.5). The lysate was centrifuged at 15,000 x g for 20 min, and the supernatant filtrated through a 0.22-µm filter was applied to nickel-chelating Sepharose (Amersham Biosciences). The column was washed with a wash buffer (400 mM KCl, 50 mM imidazole, and 20 mM HEPES/KOH, pH 7.5), and protein was eluted with an elution buffer (400 mM KCl, 300 mM imidazole, and 20 mM HEPES/KOH, pH 7.5). Purified PLC was dialyzed against an intracellular buffer (120 mM KCl and 20 mM HEPES/KOH, pH 7.5). The final solution was designated as PLC solution.

Measurement of PLC Activity—The PLC activity was assayed by hydrolysis of PtdInsP2 (13) by mixing 10 µl of PLC solution with 30 µl of Ca2+ buffer and 10 µl of phospholipid micelle solution. The final concentration was 50 µM phosphatidylethanolamine (Sigma), 40 µM PtdInsP2 (Sigma), 1 µCi/ml (~100 nM) [3H]PtdInsP2 (PerkinElmer Life Sciences), 50 mM HEPES (pH 7.0), 100 mM KCl, 2 mM EGTA, 1 nM to 100 µM Ca2+, and 0.5 mg/ml bovine serum albumin. Ca2+ buffers of various [Ca2+] were prepared by EGTA/CaCl2 mixture (14). The reaction mixture containing 25,000 dpm of [3H]PtdInsP2 was incubated at 37 °C for 5 min, and the reaction was stopped by adding 2 µm of chloroform:methanol (2:1, v/v). Radioactive InsP3 was extracted by adding 0.5 ml of 1 N HCl (13), and radioactivity in the upper aqueous phase was measured for 1 min in a liquid scintillation counter. PLC for assay was used at the concentration that produced a maximal [3H]InsP3 level of ~4,000 dpm at a certain range of [Ca2+]. Under these conditions, [3H]InsP3 formation was linear as the function of time and enzyme concentration.

Preparation of Eggs—Mature eggs were obtained from the oviducts of B6D2F1 female mice superovulated by intraperitoneal injection of gonadotropins (see Ref. 6 for details). The eggs were collected into M2 medium supplement with bovine serum albumin (4 mg/ml) (6). The eggs were loaded with the Ca2+-sensitive fluorescent dye fura-2 acetoxymethyl ester (5 µM) (Molecular Probes Inc.) for 8 min at 37 °C. The eggs were then transferred to a plastic dish mounted on an inverted fluorescence microscope and heated at 30-32 °C.

Injection of PLC and [Ca2+]i Measurement—PLC solution was injected into the eggs through a glass micropipette (15). The injected amount was 5 pl (egg volume, ~200 pl). [Ca2+]i was measured by a conventional Ca2+ imaging method using an image processor (Argus 200, Hamamatsu Photonics) (see Ref. 15 for details). Images were acquired every 20 s for 30-40 min by applying 340- and 380-nm lights and measuring fluorescence (F) of fura-2 at 510 nm. Data were processed to calculate the fluorescence ratio F340/F380.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
PLC{zeta} and PLC{delta}1 with N-terminal His tags were purified up to a major single band shown by silver staining (Fig. 1B). The molecular mass including the His tag was ~78 and ~90 kDa, consistent with the full-length PLC{zeta} (647 amino acids) (5) and PLC{delta}1 (756 amino acids) (16), respectively.

Exogenously applied PLC was predicted to produce InsP3 continuously in the eggs and, thereby, cause repetitive Ca2+ release from the endoplasmic reticulum. Microinjection of recombinant PLC{zeta} into the mouse eggs did induce Ca2+ oscillations (Fig. 2A, n = 10 eggs). The first Ca2+ transient lasting for 4-5 min was followed by sharp Ca2+ spikes at intervals of 2-3 min. The interspike interval was progressively prolonged. The pattern is very similar to that of Ca2+ oscillations induced by injection of sperm extract (2, 6), although Ca2+ oscillations of lower frequency occur in eggs fertilized with a single spermatozoon (17). The minimal PLC{zeta} concentration in the injection solution for induction of Ca2+ oscillations was ~60 µg/ml, and that found in the egg was calculated to be 1.5 µg/ml. The total amount injected was calculated as 300 fg/egg. It has been shown that low frequency Ca2+ oscillations similar to those at fertilization are produced after injection of PLC{zeta} RNA by expressed PLC{zeta} of 45-75 fg/egg estimated from densitometric calibration using an antibody against PLC{zeta} (5). The difference in the effective amount of PLC{zeta} for induction of Ca2+ oscillations might be derived from that in some modification of the PLC{zeta} molecule in the cell to a more active form. Injection with a 2-fold lower PLC{zeta} (0.75 µg/ml in the egg) produced only a single Ca2+ transient after a time lag of ~2 min (Fig. 2B, n = 3). This Ca2+ response pattern is usually observed upon injection of diluted sperm extract (data not shown). The critical PLC{zeta} concentration for induction of a single Ca2+ release was between 15 and 30 µg/ml in the injection solution (Fig. 2, B and C).



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  		FIG. 2.
Changes of [Ca2+]i in mouse eggs in response to injection of PLC{zeta} or s-PLC{zeta}. The ordinate is the fluorescence ratio of fura-2 in the egg excited by 340- and 380-nm lights and reflects [Ca2+]i. The vertical arrows indicate the time of injection. A, Ca2+ oscillations induced by the injection of 60 µg/ml PLC{zeta} (1.5 µg/ml in the egg). B, a single Ca2+ spike induced by 30 µg/ml PLC{zeta}. C and D,no[Ca2+]i change upon injection of 15 µg/ml PLC{zeta} and 1.1 mg/ml s-PLC{zeta} (28 µg/ml in the egg), respectively. The concentration in the egg was calculated from the injection volume (~5 pl) and egg volume (200 pl).

 
A protein similar to but shorter than PLC{zeta} (s-PLC{zeta}) is expressed in the mouse testis (GenBankTM accession number AK006672 [GenBank] ). s-PLC{zeta} lacks 110 amino acid residues from the N terminus corresponding to the EF1, EF2, and EF3 domains but is identical to PLC{zeta} in EF4 and the succeeding region (Fig. 1A). The s-PLC{zeta} gene has not been found in the mouse genome, and s-PLC{zeta} is probably a splicing variant of PLC{zeta}. Recombinant s-PLC{zeta} elicited no Ca2+ spike at the concentration 37-fold higher than that of PLC{zeta} (Fig. 2D), indicating that the EF domain(s) is significant in the induction of Ca2+ oscillations.

Recombinant PLC{delta}1, which possesses basically similar domain features to those of PLC{zeta} except the PH domain (Fig. 1A), induced Ca2+ oscillations at the concentration of 1.2 mg/ml in the injection solution (30 µg/ml in the egg) (Fig. 3A, n = 6) and only a few Ca2+ transients at 0.8 mg/ml (Fig. 3B, n = 3). Thus, an ~20-fold higher concentration was required for induction of Ca2+ oscillations compared with PLC{zeta}. PLC{delta}1 induced no Ca2+ response at 0.6 mg/ml (Fig. 3C) or at the lower concentration range at which PLC{zeta} induced Ca2+ oscillations (data not shown).



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  		FIG. 3.
Changes of [Ca2+]i in response to injection of PLC{delta}1. A, Ca2+ oscillations induced by injection of 1.2 µg/ml PLC{delta}1 (30 µg/ml in the egg). B, three Ca2+ spikes induced by 0.8 µg/ml PLC{delta}1. C,no[Ca2+]i change upon injection of 0.6 µg/ml PLC{delta}1. The vertical arrows indicate the time of injection.

 
The PLC activity (PtdInsP2 hydrolyzing activity) of PLC{zeta} in vitro was measured at [Ca2+] between 10-9 and 10-4 M in comparison with that of PLC{delta}1 (Fig. 4). No PLC activity was detected for PLC{zeta}{Delta}EFX at any [Ca2+] (Fig. 4B), indicating that the samples from Sf9 cells had no intrinsic PLC activity. The assay was performed using the amount of PLC that was capable of generating maximal [3H]InsP3 of ~4,000 dpm, which corresponded to 0.32 nmol of InsP3, under the condition that retained the linearity of InsP3 formation to the reaction time and the enzyme concentration during incubation for 5 min. The amount used was 50 ng for PLC{zeta} and 1 ng for PLC{delta}1 in 50 µl of reaction mixture corresponding to 1 µg/ml and 0.02 µg/ml, respectively. This concentration of PLC{zeta} was in the same range as the intracellular concentration of injected PLC{zeta} that induced Ca2+ release (Fig. 2, A and B). The specific activity of PLC{zeta} was 1.3 µmol/mg/min at 1 µM Ca2+ and that of PLC{delta}1 was 65 µmol/mg/min at 30 µM Ca2+ (Fig. 4). The specific activity was 50-fold higher for PLC{delta}1. Nevertheless, PLC{zeta} induced Ca2+ oscillations at 20-fold lower concentrations than PLC{delta}1 (Figs. 2A and 3A). This implies that PLC{zeta} has a 1000-fold higher efficiency than PLC{delta}1 in induction of Ca2+ oscillations in the egg when calculated on the basis of the specific PLC activity in vitro under the assumption of comparable dependence on [Ca2+].



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  		FIG. 4.
The dependence of PtdInsP2-hydrolyzing activity of PLC{zeta} and PLC{delta}1 on [Ca2+]. The values of the specific activity (µmol of InsP3 produced from [3H]PtdInsP2/min/mg PLC) are presented as mean ± S.D. in three experiments. A, PLC{delta}1 (0.02 µg/ml in 50 µl of reaction mixture). B, PLC{zeta} (1 µg/ml) and PLC{zeta}{Delta}EFX (50 µg/ml). M, molar.

 
To address the advantageous characteristics of PLC{zeta} for induction of Ca2+ oscillations, the dependence of PLC activity on [Ca2+] was examined in vitro. The PLC activity of recombinant PLC{delta}1 was substantially recognized at 1 µM Ca2+ (Fig. 4A). The activity steeply increased between 1 and 30 µM Ca2+ and attained a saturation level at 30 µM Ca2+. The [Ca2+] for half-maximal PLC activity, EC50, was obtained by fitting a curve to the data using the Hill equation. It was calculated as 5.7 µM (Hill constant, 1.7). In contrast, the PLC activity of recombinant PLC{zeta} was significantly recognized at [Ca2+] as low as 10 nM and reached a maximum at 1 µM Ca2+ (Fig. 4B). EC50 was 52 nM (Hill constant, 0.9), that is, ~100-fold lower than that for PLC{delta}1. It should be noted that PLC{zeta} had 70% activity of the maximal level at 100 nM Ca2+, which is the resting [Ca2+]i level in mouse eggs (6, 18) as well as somatic cells (19). This implies that PLC{zeta} can be active even at the resting state of cells. PLC{zeta} has the highest Ca2+ sensitivity among PLC isoforms identified and characterized to date (20-24).

[Ca2+]i increases from 100 nM up to 500-1000 nM in each Ca2+ spike during Ca2+ oscillations in fertilized mouse eggs (25). In Fig. 4B, this [Ca2+] range does not involve a steep Ca2+ dependence, which may be favorable for a positive feedback and pulsatile rise of the PLC activity. The Ca2+ dependence of PLC{zeta} as a whole formed a bell-shaped curve (Fig. 4B), which may cause oscillatory changes in the PLC activity associated with Ca2+ oscillations. However, this seems to be insignificant, because the PLC{zeta} activity substantially decreased at [Ca2+] as high as 10 µM. Ca2+ oscillations could occur on the basis of Ca2+ dependence of the InsP3 receptor (1), even if any oscillatory change of PLC activity is absent. They are produced by artificial supply of InsP3 at a sustained low level in the ooplasm (26). A single injection of a nonhydrolysable agonist of InsP3, adenophostin B, into the mouse egg produces long-lasting Ca2+ oscillations (18).

The present study demonstrated two critical properties of PLC{zeta} using purified recombinant PLC{zeta} protein: the high Ca2+ oscillation-inducing activity in the egg and the high Ca2+ sensitivity in the PtdInsP2 hydrolyzing activity. These properties may rely on the N-terminal EF-hand domains of PLC{zeta}, because s-PLC{zeta} lacking three EF-hand domains was incapable of inducing Ca2+ oscillations (Fig. 2D) and exhibited significant Pt-dInsP hydrolyzing activity only when [Ca2+] was over 1 µM (data not shown). The EF domains of PLC{zeta} might have a high affinity to Ca2+. The higher Ca2+ sensitivity of PLC{zeta} is likely to give the ability to trigger Ca2+ release in cells at the resting state. The two properties of PLC{zeta} described above are appropriate for a Ca2+ oscillation-inducing sperm factor, because the sperm factor is thought to be introduced into the ooplasm upon sperm-egg fusion, first triggering Ca2+ release and then maintaining Ca2+ oscillations (2, 4). It has been shown that boar sperm extract possesses one-third of the maximal PtdInsP2 hydrolyzing activity at 100 nM Ca2+ (27). Thus, sperm-specific PLC{zeta} is a strong candidate of the mammalian egg-activating sperm factor. There might be an inactivation mechanism for PLC{zeta} activity in the sperm before it is introduced into the ooplasm.

PLC{delta}1 possessed the much higher enzymatic activity in vitro, but had the much lower Ca2+ oscillation-inducing activity in vivo compared with PLC{zeta}. PLC{delta}1 has been shown to be expressed in mouse immature germ cells, spermatogonia, but not detected in differentiated spermatids and spermatozoa (10). Thus, PLC{delta}1 is unlikely to be the sperm factor. The superiority of PLC{zeta} to PLC{delta}1 in the Ca2+ oscillation-inducing activity is thought to be derived from the much higher Ca2+ sensitivity (~100-fold difference in EC50). However, the large difference in the efficiency of inducing Ca2+ oscillations is not interpreted only in terms of Ca2+-dependent PLC activity in vitro and suggests an additional advantage of PLC{zeta}. There might be an egg factor that promotes the activation of PLC{zeta} or special target membranes for PLC{zeta} in the ooplasm. Further studies are necessary to determine whether PLC{zeta} is the physiological sperm factor at fertilization as well as to elucidate the activation and modification mechanism of PLC{zeta} on the basis of the molecular structure.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AK006672 [GenBank] .

* This work was supported by a grant-in-aid for general scientific research (Category B) (to S. M.) from the Japan Ministry of Education, Science, Sports, and Culture. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed. Tel.: 81-3-5269-7414; Fax: 81-3-5269-7414; E-mail: zkouchi{at}research.twmu.ac.jp.

1 The abbreviations used are: [Ca2+]i, intracellular [Ca2+]; [Ca2+], calcium ion concentration; PLC, phospholipase C; PLC{zeta}{Delta}EFX, PLC{zeta} lacking three EF-hand domains and catalytic X domain; PtdInsP2, phosphatidylinositol 4,5-bisphosphate; InsP3, inositol 1,4,5-trisphosphate; PH domain, pleckstrin homology domain; s-PLC{zeta}, short form of PLC{zeta}. Back


   ACKNOWLEDGMENTS
 
We thank Drs. T. Awaji, H. Shirakawa, and S. Mitani for valuable advice and discussion throughout the present study.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

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