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• W1970061319 abstract "“Soluble” adenylyl cyclase (sAC) is a widely expressed source of cAMP in mammalian cells that is evolutionarily, structurally, and biochemically distinct from the G protein-responsive transmembrane adenylyl cyclases. In contrast to transmembrane adenylyl cyclases, sAC is insensitive to heterotrimeric G protein regulation and forskolin stimulation and is uniquely modulated by bicarbonate ions. Here we present the first report detailing kinetic analysis and biochemical properties of purified recombinant sAC. We confirm that bicarbonate regulation is conserved among mammalian sAC orthologs and demonstrate that bicarbonate stimulation is consistent with an increase in the V max of the enzyme with little effect on the apparent K m for substrate, ATP-Mg2+. Bicarbonate can further increase sAC activity by relieving substrate inhibition. We also identify calcium as a direct modulator of sAC activity. In contrast to bicarbonate, calcium stimulates sAC activity by decreasing its apparentK m for ATP-Mg2+. Because of their different mechanisms, calcium and bicarbonate synergistically activate sAC; therefore, small changes of either calcium or bicarbonate will lead to significant changes in cellular cAMP levels. “Soluble” adenylyl cyclase (sAC) is a widely expressed source of cAMP in mammalian cells that is evolutionarily, structurally, and biochemically distinct from the G protein-responsive transmembrane adenylyl cyclases. In contrast to transmembrane adenylyl cyclases, sAC is insensitive to heterotrimeric G protein regulation and forskolin stimulation and is uniquely modulated by bicarbonate ions. Here we present the first report detailing kinetic analysis and biochemical properties of purified recombinant sAC. We confirm that bicarbonate regulation is conserved among mammalian sAC orthologs and demonstrate that bicarbonate stimulation is consistent with an increase in the V max of the enzyme with little effect on the apparent K m for substrate, ATP-Mg2+. Bicarbonate can further increase sAC activity by relieving substrate inhibition. We also identify calcium as a direct modulator of sAC activity. In contrast to bicarbonate, calcium stimulates sAC activity by decreasing its apparentK m for ATP-Mg2+. Because of their different mechanisms, calcium and bicarbonate synergistically activate sAC; therefore, small changes of either calcium or bicarbonate will lead to significant changes in cellular cAMP levels. adenylyl cyclase soluble AC truncated isoform of sAC transmembrane AC in vitro fertilization media glutathione S-transferase Two types of adenylyl cyclase (AC)1 are ubiquitously expressed in mammalian cells, a well characterized gene family of transmembrane ACs (tmACs) and the recently discovered “soluble” AC (sAC). The tmACs are plasma membrane bound, and their activities are regulated by G proteins in response to extracellular stimuli such as neurotransmitters and hormones (reviewed in Ref. 1Taussig R. Gilman A.G. J. Biol. Chem. 1995; 270: 1-4Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). In contrast, sAC is associated with various intracellular organelles, including mitochondria, centrioles, mitotic spindle, mid-bodies, and nuclei (2Zippin J.H. Chen Y. Nahirney P. Kamenetsky R. Fischman D.A. Levin L.R. Buck J. FASEB J. 2003; 17: 82-84Crossref PubMed Scopus (244) Google Scholar). sAC activity is modulated by bicarbonate (3Chen Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Science. 2000; 289: 625-628Crossref PubMed Scopus (690) Google Scholar) and, as shown in this report, by Ca2+; regulation by these intracellular signaling molecules suggests that sAC mediates cAMP-dependent responses to intrinsic cellular changes (4Wuttke M.S. Buck J. Levin L.R. JOP. 2001; 2: 154-158PubMed Google Scholar,5Zippin J.H. Levin L.R. Buck J. Trends Endocrinol. Metab. 2001; 12: 366-370Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The catalytic mechanism of tmACs has been determined from biochemical and crystallographic studies. tmACs convert ATP to cAMP using two-metal catalysis where one ion acts as a free metal and the other coordinates ATP in the active site (6Zimmermann G. Zhou D. Taussig R. J. Biol. Chem. 1998; 273: 19650-19655Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 7Tesmer J.J. Sunahara R.K. Johnson R.A. Gosselin G. Gilman A.G. Sprang S.R. Science. 1999; 285: 756-760Crossref PubMed Scopus (279) Google Scholar). Its activators, Gαssubunit or forskolin, stimulate tmACs by allosteric modulation of the active site (8Tesmer J.J. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google Scholar, 9Hurley J.H. J. Biol. Chem. 1999; 274: 7599-7602Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). More than 25 years ago, when soluble AC activity was first discovered, it was predicted to be molecularly distinct from tmACs because its activity appeared to be dependent on the presence of the divalent cation, Mn2+, and it was insensitive to forskolin and G protein regulation (10Braun T. Dods R.F. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 1097-1101Crossref PubMed Scopus (165) Google Scholar, 11Gordeladze J.O. Andersen D. Hansson V. J. Clin. Endocrinol. Metab. 1981; 53: 465-471Crossref PubMed Scopus (13) Google Scholar, 12Rojas F.J. Patrizio P. Do J. Silber S. Asch R.H. Moretti-Rojas I. Endocrinology. 1993; 133: 3030-3033Crossref PubMed Scopus (16) Google Scholar). These differential properties enabled purification (13Buck J. Sinclair M.L. Levin L.R. Methods Enzymol. 2002; 345: 95-105Crossref PubMed Scopus (10) Google Scholar) and cloning of sAC from rat testis (14Buck J. Sinclair M.L. Schapal L. Cann M.J. Levin L.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 79-84Crossref PubMed Scopus (426) Google Scholar). The sAC gene is indeed molecularly distinct from tmACs; it possesses no transmembrane domains, and its catalytic domains are more closely related to those of cyanobacterial ACs than to those from other eukaryotic ACs. The purified soluble AC exhibited ∼10-fold lower affinity for substrate ATP relative to tmACs (tmACK m for ATP-Mn2+ is ∼ 100 μm, whereas purified rat testis sAC K m for ATP-Mn2+ is ∼ 1 mm), and the activity of the heterologously expressed, cloned sAC gene product is insensitive to forskolin or G proteins (14Buck J. Sinclair M.L. Schapal L. Cann M.J. Levin L.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 79-84Crossref PubMed Scopus (426) Google Scholar). Soluble AC, as the predominant, if not only, source of cAMP in sperm, was predicted to be responsible for the cAMP changes induced by seminal and oviductal fluids (and mimicked by in vitro fertilization (IVF) media) necessary for fertilization of an egg, capacitation, hyperactivated motility, and acrosome reaction (15Visconti P.E. Westbrook V.A. Chertihin O. Demarco I. Sleight S. Diekman A.B. J. Reprod. Immunol. 2002; 53: 133-150Crossref PubMed Scopus (290) Google Scholar, 16Kulanand J. Shivaji S. Andrologia. 2001; 33: 95-104Crossref PubMed Scopus (58) Google Scholar, 17Lee M.A. Storey B.T. Biol. Reprod. 1986; 34: 349-356Crossref PubMed Scopus (135) Google Scholar, 18Okamura N. Tajima Y. Soejima A. Masuda H. Sugita Y. J. Biol. Chem. 1985; 260: 9699-9705Abstract Full Text PDF PubMed Google Scholar). Two essential components of defined IVF media are bicarbonate and calcium, and we previously demonstrated that sAC is directly stimulated by physiological levels of the bicarbonate anion (3Chen Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Science. 2000; 289: 625-628Crossref PubMed Scopus (690) Google Scholar). The role of calcium in IVF media is less clear because of contradicting reports detailing Ca2+ modulation of sperm cyclase (19Hyne R.V. Garbers D.L. Biol. Reprod. 1979; 21: 1135-1142Crossref PubMed Scopus (83) Google Scholar, 20Garbers D.L. Tubb D.J. Hyne R.V. J. Biol. Chem. 1982; 257: 8980-8984Abstract Full Text PDF PubMed Google Scholar, 21Stengel D. Hanoune J. Ann. N. Y. Acad. Sci. 1984; 438: 18-28Crossref PubMed Scopus (16) Google Scholar). We performed kinetic analyses on purified recombinant 48-kDa truncated human sAC protein (sACt) fused to GST. This truncated protein corresponds to a splice variant of the sAC gene (22Jaiswal B.S. Conti M. J. Biol. Chem. 2001; 21: 21Google Scholar) that consists almost exclusively of the sAC catalytic domains and corresponds to the native isoform originally purified from testis cytosol (14Buck J. Sinclair M.L. Schapal L. Cann M.J. Levin L.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 79-84Crossref PubMed Scopus (426) Google Scholar). We identify a synergistic interaction between bicarbonate and calcium ions, where bicarbonate functions to increase theV max of the enzyme whereas calcium increases its affinity for substrate ATP-Mg2+. ATP, chlorpromazine, and LaCl3 were purchased from Sigma; all tissue culture reagents were from Invitrogen and [α32P]ATP and [3H]cAMP were from PerkinElmer Life Sciences. Human sACt was subcloned using gateway cloning technology (Invitrogen) into a baculovirus expression vector utilizing the polyhedron promoter to generate an N-terminal glutathione S-transferase (GST) fusion protein. Recombinant GST·human sACt-expressing baculovirus was produced in adherent SF9 cells (Bac-to-Bac baculovirus expression systems, Invitrogen), and identity of the resultant fusion protein was confirmed by Western blotting and enzymatic activity. Hi-Five cells, grown to a density of ∼1.0 × 106 cells per ml, were infected with GST·human sACt baculovirus. After 48 h, cells were pelleted, resuspended in lysis buffer (phosphate-buffered saline with 1 mm EDTA, pH 7.4, 1 mm dithiothreitol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mmphenylmethylsulfonyl fluoride), and lysed by sonication. The cell lysate was cleared by centrifugation at 17,500 × g for 60 min at 4 °C, and the supernatant was applied to a glutathione-Sepharose 4B column (Amersham Biosciences). The GST·human sACt fusion protein was eluted with glutathione elution buffer (10 mm reduced glutathione, 50 mmTris-HCl, pH 8.0, 10 μg/ml aprotinin, 10 μg/ml leupeptin). GST·human sACt was further purified by gel filtration over Superdex 200 HR 10/30 column (Amersham Biosciences); sAC-containing fractions were stored in 50% glycerol at −20 °C. Coomassie Blue-stained SDS-PAGE reveals one predominant band corresponding to GST·human sACt and a minor contaminant corresponding to GST alone (Fig. 1, inset). Multiple independent assays comparing the GST·sACt fusion protein with cleaved and re-purified sACt confirmed that the GST fusion does not affect enzymatic activity or kinetic parameters of sAC (data not shown). Cyclase assays were performed in 100 μl of total reaction volume using ∼100 ng of purified GST·human sACt fusion protein in the presence of 50 mmTris-HCl, pH 7.5, substrate [α-32P]ATP, and either MnCl2, MgCl2, and/or CaCl2 as indicated. Reactions were incubated at 30 °C for 30 min unless otherwise noted and were stopped by adding 200 μl of 2% SDS. [32P]cAMP generated by the reaction was recovered using the two-column method (23Levin L.R. Han P.L. Hwang P.M. Feinstein P.G. Davis R.L. Reed R.R. Cell. 1992; 68: 479-489Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 24Levin L.R. Reed R.R. J. Biol. Chem. 1995; 270: 7573-7579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). For kinetic analysis, sAC activity was assayed as a function of varying ATP-Mn2+ or ATP-Mg2+ in the presence of excess MnCl2, MgCl2, or CaCl2. ATP was preincubated with MnCl2 or MgCl2, and serial dilutions were prepared; assays were started by addition of sAC protein. Kinetic analyses were performed using the program EnzymeKinetics v 1.11 (Trinity Software, Plymouth, NH). Adenylyl cyclases require a divalent cation for catalytic activity. Historically, soluble adenylyl cyclase activity found in testis has been assayed in the presence of Mn2+ (10Braun T. Dods R.F. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 1097-1101Crossref PubMed Scopus (165) Google Scholar); little or no in vitro activity was detected when Mg2+, Ca2+, or Co2+ was used as the sole divalent (19Hyne R.V. Garbers D.L. Biol. Reprod. 1979; 21: 1135-1142Crossref PubMed Scopus (83) Google Scholar, 25Braun T. J. Cyclic Nucleotide Res. 1975; 1: 271-281PubMed Google Scholar, 26Magnus O. Brekke I. Abyholm T. Purvis K. Arch. Androl. 1990; 24: 159-166Crossref PubMed Scopus (18) Google Scholar). The K m for ATP-Mn2+ of soluble AC activity from testis cytosol has been reported to be 1–2 mm (11Gordeladze J.O. Andersen D. Hansson V. J. Clin. Endocrinol. Metab. 1981; 53: 465-471Crossref PubMed Scopus (13) Google Scholar, 21Stengel D. Hanoune J. Ann. N. Y. Acad. Sci. 1984; 438: 18-28Crossref PubMed Scopus (16) Google Scholar, 27Braun T. Methods Enzymol. 1991; 195: 130-136Crossref PubMed Scopus (21) Google Scholar). We now demonstrate that recombinant human sAC has similar activity, displaying a K m of 0.8 mm ATP-Mn2+(Fig. 1). We recently described direct stimulation of rat sAC by the bicarbonate anion (3Chen Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Science. 2000; 289: 625-628Crossref PubMed Scopus (690) Google Scholar). Bicarbonate activation of sAC is thought to be at the center of fertilization-related processes that occur in all mammalian sperm, and we now confirm that purified human sAC is also stimulated by bicarbonate (Fig. 2 A). Similar to rat sAC, in the presence of 10 mm ATP human sAC was stimulated up to 30-fold with a half-maximal effect (EC50) of ∼11 mm NaHCO3. Bicarbonate stimulates sAC activity in the presence of magnesium, and because manganese is not found at the millimolar concentrations necessary to support sAC activity inside cells, magnesium represents the more physiologically relevant cation. Therefore, all subsequent experiments were performed using Mg2+-ATP as substrate. In the absence of bicarbonate, activity of recombinant human sAC was inhibited at high ATP-Mg2+ concentrations. This substrate inhibition was relieved by the addition of bicarbonate (Fig.2 B). At 0 mm NaHCO3, the onset of inhibition is at [ATP-Mg2+] > 6 mm; at 15 mm NaHCO3 the onset is shifted to [ATP-Mg2+] > 13 mm and, at 50 mmNaHCO3, substrate inhibition is virtually abated. The data indicate that, in addition to relieving the inhibition observed at high substrate concentrations, bicarbonate was not altering the apparentK m for ATP-Mg2+; rather it was increasing the V max of sAC (Fig. 2 B). These 2-fold effects contribute to the observed 30-fold stimulation of activity (Fig. 2 A). Similar effects were observed with purified recombinant rat sAC; bicarbonate increased theV max of the enzyme and abated substrate inhibition (data not shown). Previously published studies of crude soluble AC activity revealed an elevated K m for ATP-Mg2+ (12–16 mm) relative to ATP-Mn2+ (1–2 mm) (21Stengel D. Hanoune J. Ann. N. Y. Acad. Sci. 1984; 438: 18-28Crossref PubMed Scopus (16) Google Scholar). We were unable to determine a true K m for ATP-Mg2+ in the presence of NaHCO3 because even at the highest NaHCO3 (80 mm) and substrate (up to 30 mm ATP-Mg2+) concentrations used, sAC activity did not plateau (Fig. 2 C). We could only conclude from non-linear regression analysis and Eadie-Hofstee plots that the apparent K m for ATP-Mg2+ was greater than 10 mm, consistent with published reports using crude soluble AC activity (21Stengel D. Hanoune J. Ann. N. Y. Acad. Sci. 1984; 438: 18-28Crossref PubMed Scopus (16) Google Scholar). Like bicarbonate, CaCl2 (1.7–3 mm) is an essential component ofin vitro fertilization media, and Ca2+ has been implicated along with bicarbonate in activation of sperm cyclases (12Rojas F.J. Patrizio P. Do J. Silber S. Asch R.H. Moretti-Rojas I. Endocrinology. 1993; 133: 3030-3033Crossref PubMed Scopus (16) Google Scholar,15Visconti P.E. Westbrook V.A. Chertihin O. Demarco I. Sleight S. Diekman A.B. J. Reprod. Immunol. 2002; 53: 133-150Crossref PubMed Scopus (290) Google Scholar, 16Kulanand J. Shivaji S. Andrologia. 2001; 33: 95-104Crossref PubMed Scopus (58) Google Scholar, 19Hyne R.V. Garbers D.L. Biol. Reprod. 1979; 21: 1135-1142Crossref PubMed Scopus (83) Google Scholar). We added CaCl2 to bicarbonate-stimulated sAC and found enzymatic activity increased synergistically (Fig.3 A). CaCl2activation was dose-dependent with an EC50∼750 μm (Fig. 3 B). Interestingly, the dose response to NaHCO3 was unaffected by calcium; the EC50 for NaHCO3 remained ∼11 mmin the presence or absence of CaCl2 (Fig.3 C). As shown above, bicarbonate stimulated sAC activity by alleviating ATP inhibition and by increasing V max with little effect on apparent K m. In contrast, kinetic analysis revealed that calcium had no effect on ATP inhibition and little effect on V max (Fig. 3 E) but stimulated sAC activity by decreasing its apparent K m for ATP-Mg2+ (Fig. 3 D). Addition of CaCl2 causes a dramatic shift in the apparentK m of sAC for ATP-Mg2+ from greater than 10 mm (Fig. 2 B) to less than 1 mm(Fig. 3 D). Overlaying the kinetic curves (Fig.3 E) illustrates how the effects of NaHCO3 and CaCl2 differ; NaHCO3 activates sAC by increasing V max, whereas CaCl2increases its affinity for substrate. Coomassie Blue staining of purified human sAC did not reveal any substantial contaminating proteins (Fig. 1,inset), suggesting that calmodulin was not mediating the effect of calcium on sAC. To confirm that calmodulin was not involved, we found that adding exogenous bovine calmodulin did not have any affect on Ca2+-dependent sAC activity (data not shown) and inhibiting any potential calmodulin contamination using two independent calmodulin inhibitors, chlorpromazine or LaCl3, had no effect on calcium-stimulated sAC activity (Fig.4). Therefore, we conclude that the effect of CaCl2 on sAC activity is because of direct binding of Ca2+, making sAC the first mammalian adenylyl cyclase to be stimulated by calcium directly. Specific tmAC isoforms are known to be stimulated by Ca2+, but they require calmodulin for the modulatory effect (23Levin L.R. Han P.L. Hwang P.M. Feinstein P.G. Davis R.L. Reed R.R. Cell. 1992; 68: 479-489Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 28Cali J.J. Zwaagstra J.C. Mons N. Cooper D.M. Krupinski J. J. Biol. Chem. 1994; 269: 12190-12195Abstract Full Text PDF PubMed Google Scholar, 29Choi E.-J. Xia Z. Storm D.R. Biochemistry. 1992; 31: 6492-6498Crossref PubMed Scopus (205) Google Scholar, 30Tang W.J. Krupinski J. Gilman A.G. J. Biol. Chem. 1991; 266: 8595-8603Abstract Full Text PDF PubMed Google Scholar, 31Wei J. Wayman G. Storm D.R. J. Biol. Chem. 1996; 271: 24231-24235Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 32Wayman G.A. Wei J. Wong S. Storm D.R. Mol. Cell. Biol. 1996; 16: 6075-6082Crossref PubMed Scopus (68) Google Scholar, 33Cooper D.M.F. Mons N. Karpens J.W. Nature. 1995; 374: 421-424Crossref PubMed Scopus (557) Google Scholar, 34Cooper D.M. Karpen J.W. Fagan K.A. Mons N.E. Adv. Second Messenger Phosphoprot. Res. 1998; 32: 23-51Crossref PubMed Scopus (74) Google Scholar). Soluble AC activity was first characterized in sperm and testis in the presence of MnCl2 (10Braun T. Dods R.F. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 1097-1101Crossref PubMed Scopus (165) Google Scholar). Activity had been described in the presence of other divalents, such as Co2+, Ca2+, and Mg2+, but Mn2+-dependent activity was always significantly higher (19Hyne R.V. Garbers D.L. Biol. Reprod. 1979; 21: 1135-1142Crossref PubMed Scopus (83) Google Scholar, 25Braun T. J. Cyclic Nucleotide Res. 1975; 1: 271-281PubMed Google Scholar, 26Magnus O. Brekke I. Abyholm T. Purvis K. Arch. Androl. 1990; 24: 159-166Crossref PubMed Scopus (18) Google Scholar). We purified the 48-kDa isoform of human sAC fused to GST and confirmed that its K m for ATP-Mn2+ (∼0.8 mm) was indistinguishable from the value obtained for sAC activity purified from rat testis (14Buck J. Sinclair M.L. Schapal L. Cann M.J. Levin L.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 79-84Crossref PubMed Scopus (426) Google Scholar) and matched the values reported for soluble cAMP-producing activity in testis and sperm from a variety of mammals (11Gordeladze J.O. Andersen D. Hansson V. J. Clin. Endocrinol. Metab. 1981; 53: 465-471Crossref PubMed Scopus (13) Google Scholar, 21Stengel D. Hanoune J. Ann. N. Y. Acad. Sci. 1984; 438: 18-28Crossref PubMed Scopus (16) Google Scholar, 27Braun T. Methods Enzymol. 1991; 195: 130-136Crossref PubMed Scopus (21) Google Scholar). These data confirm that the cloned sAC gene is responsible for the activities described in testis and sperm, and the kinetic parameters determined here correlate well with values established using native enzyme. However, it should be remembered that the kinetic analyses described in this study were performed on a heterologously expressed and purified enzyme; the in vivo properties of native sAC may differ because of post-translational modifications or interactions with regulatory proteins. Soluble AC appears to be ubiquitously expressed (2Zippin J.H. Chen Y. Nahirney P. Kamenetsky R. Fischman D.A. Levin L.R. Buck J. FASEB J. 2003; 17: 82-84Crossref PubMed Scopus (244) Google Scholar, 35Sinclair M.L. Wang X.Y. Mattia M. Conti M. Buck J. Wolgemuth D.J. Levin L.R. Mol. Reprod. Dev. 2000; 56: 6-11Crossref PubMed Scopus (107) Google Scholar), and cells throughout the body do not possess concentrations of Mn2+necessary to support sAC activity. We recently demonstrated that the ATP-Mg2+-dependent activity of purified rat sAC was stimulated by bicarbonate. Among mammalian adenylyl cyclases, bicarbonate regulation was unique to sAC; tmACs were unaffected by NaHCO3 addition (3Chen Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Science. 2000; 289: 625-628Crossref PubMed Scopus (690) Google Scholar). Here we demonstrate that the human ortholog of sAC is also responsive to bicarbonate stimulation. The half-maximal effect (∼11 mm NaHCO3) is slightly lower for human sAC than the EC50 reported (25 mm NaHCO3) for rat sAC (3Chen Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Science. 2000; 289: 625-628Crossref PubMed Scopus (690) Google Scholar). Although both EC50 for bicarbonate are within the physiological range (24–26 mm in most extracellular fluids and 10–15 mm inside cells), the difference might reflect species variation, or it could reflect differences between the two preparations; rat sAC was purified as a His6·sAC fusion protein (3Chen Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Science. 2000; 289: 625-628Crossref PubMed Scopus (690) Google Scholar), whereas this study uses a GST fusion protein. Bicarbonate can increase sAC activity in two ways; besides increasing enzyme velocity, bicarbonate also relieves the substrate inhibition observed at high ATP-Mg2+ concentrations. Therefore, the low (<5 mm) bicarbonate concentration in the epididymis (36Rodriguez-Martinez H. Ekstedt E. Einarsson S. Int. J. Androl. 1990; 13: 238-243Crossref PubMed Scopus (77) Google Scholar, 37Levine N. Marsh D.J. J. Physiol. 1971; 213: 557-570Crossref PubMed Scopus (291) Google Scholar), where sperm are stored awaiting ejaculation, would further limit sAC activity in resting spermatozoa by allowing substrate inhibition. Upon ejaculation, the higher HCO3− (25 mm) found in seminal and prostatic fluids (which induces sperm motility and initiates capacitation) would lead to large, rapid increases in cAMP because of the increased V maxof sAC and relief from substrate inhibition. Calcium (1.7–3 mm) is required in IVF media for capacitation, hyperactivated motility, and the acrosome reaction in sperm (15Visconti P.E. Westbrook V.A. Chertihin O. Demarco I. Sleight S. Diekman A.B. J. Reprod. Immunol. 2002; 53: 133-150Crossref PubMed Scopus (290) Google Scholar, 38Breitbart H. Mol. Cell. Endocrinol. 2002; 187: 139-144Crossref PubMed Scopus (174) Google Scholar). It is thought that Ca2+ is able to enter sperm via voltage-dependent and cyclic nucleotide gated calcium channels (39Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Crossref PubMed Scopus (281) Google Scholar) as well as the putative cation channel CatSper (40Ren D. Navarro B. Perez G. Jackson A.C. Hsu S. Shi Q. Tilly J.L. Clapham D.E. Nature. 2001; 413: 603-609Crossref PubMed Scopus (720) Google Scholar). Previous reports demonstrated that detergent-dispersed adenylyl cyclase from guinea pig sperm membranes was activated by CaCl2 (0.1–1 mm) in the presence of 5 mm MgCl2 (19Hyne R.V. Garbers D.L. Biol. Reprod. 1979; 21: 1135-1142Crossref PubMed Scopus (83) Google Scholar), and adenylyl cyclase from human epididymal sperm membranes was activated by 50 mm CaCl2 and 50 mmNaHCO3 (12Rojas F.J. Patrizio P. Do J. Silber S. Asch R.H. Moretti-Rojas I. Endocrinology. 1993; 133: 3030-3033Crossref PubMed Scopus (16) Google Scholar). However, the molecular identity of the AC in these preparations remained unclear. In this report we confirm that calcium directly stimulates sAC activity and that calcium functions independently from calmodulin to increase the affinity of sAC for its substrate ATP-Mg2+. Regulation by calcium, which is capable of supporting catalytic activity in the absence of Mg2+ (19Hyne R.V. Garbers D.L. Biol. Reprod. 1979; 21: 1135-1142Crossref PubMed Scopus (83) Google Scholar, 25Braun T. J. Cyclic Nucleotide Res. 1975; 1: 271-281PubMed Google Scholar, 26Magnus O. Brekke I. Abyholm T. Purvis K. Arch. Androl. 1990; 24: 159-166Crossref PubMed Scopus (18) Google Scholar), suggests that sAC, like tmACs, utilizes two metals in its active site; however, the active center of sAC would function best with different metals. It is possible that Ca2+, which lowers the apparentK m for substrate ATP-Mg2+, would be better at coordinating ATP, while Mg2+ would serve as the catalytic metal. The EC50 for CaCl2 stimulation appears to be high (∼750 μm). However, it is important to keep in mind that CaCl2 concentrations reported in this study do not reflect free Ca2+ concentrations. sAC activity would be responsive to the transiently elevated Ca2+ concentrations found during acrosome reaction and sperm motility, or sAC could be located near the pore of Ca2+ channels, such as the cyclic nucleotide gated ion channels (39Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Crossref PubMed Scopus (281) Google Scholar), where it could mediate the cAMP regulation of channel opening. A microdomain consisting of sAC and cyclic nucleotide gated calcium channels could explain observed cAMP/calcium oscillations in cells (as proposed in Ref. 33Cooper D.M.F. Mons N. Karpens J.W. Nature. 1995; 374: 421-424Crossref PubMed Scopus (557) Google Scholar). Because of the synergy between calcium and bicarbonate, even small intracellular changes in calcium or subtle changes in intracellular pH and/or carbon dioxide, which will be in equilibrium with bicarbonate, will result in significant changes of cellular cAMP. We thank Drs. Yanqiu Chen, Carmen Dessauer, Randi Silver, and Donald Fischman and members of the Levin/Buck laboratory for constructive advice and critical reading of the manuscript, Dr. Mime Kobayashi for cDNA preparation, and two anonymous reviewers for improvements to the manuscript." @default.
• W1970061319 created "2016-06-24" @default.
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• W1970061319 creator A5024790869 @default.
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• W1970061319 date "2003-05-01" @default.
• W1970061319 modified "2023-10-01" @default.
• W1970061319 title "Kinetic Properties of “Soluble” Adenylyl Cyclase" @default.
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