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1.
Identification and characterization of calmodulin-binding proteins in mammalian sperm flagella 总被引:2,自引:0,他引:2
A calcium and calmodulin-regulated cyclic nucleotide phosphodiesterase has been shown to be an integral component of both rat and bovine sperm flagella. The calcium-activated enzyme was inhibited by both trifluoperazine (ID50 = 10 microM) and [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid (EGTA), and the basal activity measured in the presence of EGTA was stimulated by limited proteolysis to that observed in the presence of calcium/calmodulin. 125I-Calmodulin binding to purified rat sperm flagella has been characterized and the flagellar-associated calmodulin-binding proteins identified by a combination of gel and nitrocellulose overlay procedures and by chemical cross-linking experiments using dimethyl suberimidate. 125I-Calmodulin bound to demembranated rat sperm flagella in a time- and concentration-dependent manner. At equilibrium, 30-40% of the bound 125I-calmodulin remains associated with the flagella after treatment with EGTA or trifluoperazine. The majority of the bound 125I-calmodulin, both the Ca2+-dependent and -independent, was displaced by excess calmodulin. A 67-kDa calmodulin-binding protein was identified by both the gel and nitrocellulose overlay procedures. In both cases, binding was dependent on Ca2+ and was totally inhibited by trifluoperazine, EGTA, and excess calmodulin. On nitrocellulose overlays, the concentration of calmodulin required to decrease binding of 125I-calmodulin by 50% was between 10(-10) and 10(-11) M. Limited proteolysis resulted in the total loss of all Ca2+-dependent binding to the 67-kDa polypeptide. Chemical cross-linking experiments identified a major calcium-dependent 125I-calmodulin:polypeptide complex in the 84-90-kDa molecular mass range and a minor complex of approximately 200 kDa. Immunoblot analysis showed that the major 67-kDa calmodulin-binding protein did not cross-react with polyclonal antibodies raised against either the calcium/calmodulin-regulated cyclic nucleotide phosphodiesterase or phosphoprotein phosphatase (calcineurin) from bovine brain. 相似文献
2.
D R Repaske J V Swinnen S L Jin J J Van Wyk M Conti 《The Journal of biological chemistry》1992,267(26):18683-18688
Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are expressed simultaneously in mammalian tissues. To identify and clone these PDEs, a polymerase chain reaction (PCR) strategy was developed using degenerate oligonucleotide primers designed to hybridize with highly conserved PDE DNA domains. Both known and novel PDEs were cloned from rat liver, the mouse K30a-3.3 lymphoma cell line, and a human hypothalamus cDNA library, demonstrating that these PCR primers can be used to amplify the cDNA of multiple PDE isozymes. One unique mouse PDE clone was found to encode a polypeptide identical with the corresponding portion of the bovine brain 63-kDa calmodulin-dependent PDE as reported in the companion article (Bentley, J. K., Kadlecek, A., Sherbert, C. H., Seger, D., Sonnenburg, W. K., Charbonneau, H., Novack, J. P., and Beavo, J. A. (1992) J. Biol. Chem. 267, 18676-18682). This mouse clone was used as a probe to screen a rat brain cDNA library for a full-length clone. The conceptual translation of the nucleotide sequence of the resulting rat clone has an open reading frame of 535 amino acids and maintains a high degree of homology with the bovine 63-kDa calmodulin-dependent PDE, indicating that this protein is likely to be the rat homolog of the 63-kDa calmodulin-dependent PDE. Expression of the full-length clone in Escherichia coli yielded a cGMP hydrolyzing activity that was stimulated severalfold by calmodulin. Northern blot analysis demonstrated that the mRNA encoding this PDE is highly expressed in rat brain and also in the S49.1 T-lymphocyte cell line. These data demonstrate that the PCR method described is a viable strategy to isolate cDNA clones of known and novel members of different families of PDE isozymes. Molecular cloning of these PDEs will provide valuable tools for investigating the roles of these isozymes in regulation of intracellular concentrations of the cyclic nucleotides. 相似文献
3.
Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL‐1 cardiac cell line
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Federica Campolo Alessandra Zevini Silvia Cardarelli Lucia Monaco Federica Barbagallo Manuela Pellegrini Marisa Cornacchione Antonio Di Grazia Valeria De Arcangelis Daniele Gianfrilli Mauro Giorgi Andrea Lenzi Andrea M. Isidori Fabio Naro 《Journal of cellular physiology》2018,233(1):325-337
Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2, and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N‐terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT‐PCR analysis showed that mPde5a1, mPde5a2, and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL‐1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL‐1 areas, and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy. 相似文献
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5.
Wang P Wu P Egan RW Billah MM 《Biochemical and biophysical research communications》2000,276(3):1271-1277
We have cloned a cDNA representing mouse phosphodiesterases (PDE) 7A1. The open reading frame encodes a protein of 482 amino acids with a predicted molecular mass of 55417. Like human PDE7A variants, mouse PDE7A1 and A2 are 5' splice variants from a common gene. The distinct N-terminal sequence of mouse PDE7A1 is highly homologous to the corresponding sequence of human PDE7A1 with a similarity of 98% but not to that of mouse PDE7A2 (with a similarity of 12%), and is more hydrophilic than that of mouse PDE7A2. Mouse PDE7A1 expressed in SF9 cells has been compared with human PDE7A1 under identical conditions. Mouse PDE7A1 has a Km for cAMP of 0.2 microM, an optimal pH of 7.5, an IC(50) value of 14 microM for 3-isobutyl-1-methylxanthine (IBMX), and is dependent on Mg(2+) for activity. All these characteristics are very similar to those of human PDE7A1. In mice, PDE7A1 is expressed in tissues of the immune system (lymph node, thymus, spleen, and blood leukocyte), testis, brain, kidney and lung but not in skeletal muscle, heart, embryo, or liver, while PDE7A2 is expressed in skeletal muscle, heart, embryo, and kidney, but not in the other tissues. This tissue distribution profile is very similar to that in humans, and hence suggests that PDE7A1 and 7A2 might play a similar role in different species. 相似文献
6.
Amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase from bovine heart 总被引:2,自引:0,他引:2
H L Trong N Beier W K Sonnenburg S D Stroop K A Walsh J A Beavo H Charbonneau 《Biochemistry》1990,29(44):10280-10288
The complete amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase (cGS-PDE) of bovine heart has been determined by analysis of five digests of the protein; placement of the C-terminal 330 residues has been confirmed by interpretation of the corresponding partial cDNA clone. The holoenzyme is a homodimer of two identical N alpha-acetylated polypeptide chains of 921 residues, each with a calculated molecular weight of 103,244. The C-terminal region, residues 613-871, of the cGS-PDE comprises a catalytic domain that is conserved in all phosphodiesterase sequences except those of PDE 1 from Saccharomyces cerevisiae and a secreted PDE from Dictyostelium. A second conserved region, residues 209-567, is homologous to corresponding regions of the alpha and alpha' subunits of the photoreceptor phosphodiesterases. This conserved domain specifically binds cGMP and is involved in the allosteric regulation of the cGS-PDE. This regulatory domain contains two tandem, internal repeats, suggesting that it evolved from an ancestral gene duplication. Common cyclic nucleotide binding properties and a distant structural relationship provide evidence that the catalytic and regulatory domains within the cGS- and photoreceptor PDEs are also related by an ancient internal gene duplication. 相似文献
7.
Bajpai M Fiedler SE Huang Z Vijayaraghavan S Olson GE Livera G Conti M Carr DW 《Biology of reproduction》2006,74(1):109-118
Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions. 相似文献
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L.‐C. L. Tsai G. C.‐K. Chan S. N. Nangle M. Shimizu‐Albergine G. L. Jones D. R. Storm J. A. Beavo L. S. Zweifel 《Genes, Brain & Behavior》2012,11(7):837-847
Phosphodiesterases (PDEs) are critical regulatory enzymes in cyclic nucleotide signaling. PDEs have diverse expression patterns within the central nervous system (CNS), show differing affinities for cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), and regulate a vast array of behaviors. Here, we investigated the expression profile of the PDE8 gene family members Pde8a and Pde8b in the mouse brain. We find that Pde8a expression is largely absent in the CNS; by contrast, Pde8b is expressed in select regions of the hippocampus, ventral striatum, and cerebellum. Behavioral analysis of mice with Pde8b gene inactivation (PDE8B KO) demonstrate an enhancement in contextual fear, spatial memory, performance in an appetitive instrumental conditioning task, motor‐coordination, and have an attenuation of age‐induced motor coordination decline. In addition to improvements observed in select behaviors, we find basal anxiety levels to be increased in PDE8B KO mice. These findings indicate that selective antagonism of PDE8B may be an attractive target for enhancement of cognitive and motor functions; however, possible alterations in affective state will need to be weighed against potential therapeutic value . 相似文献
10.
Calmodulin-dependent cyclic nucleotide phopshodiesterase (PDE1) has been extensively characterized and is a key enzyme involved in the complex interaction between cyclic nucleotide and Ca(2+) second-messenger systems. It is well established that PDE1 exists in different isozymes. For example, bovine brain tissue has two PDE1 isozymes (PDE1A2 and PDE1B1) whereas only one form (PDE1A1) is reported in bovine cardiac tissue. In this study, we report the cloning of two cDNA splice variants of PDE1: PDE1-small and PDE1-large, from bovine cardiac tissue. Their amino acid sequence similarity to PDE1 sequences from other mammalian species showed that all are very conserved, suggesting their importance in cellular functions. Interestingly, compared to other mammalian species, bovine PDE1A, PDE-small and PDE-large show a deletion at the C-terminal end of the catalytic domain of the gene. Although the significance of this deletion at this crucial location of the gene is not known, we have successfully over-expressed both PDE1-small and PDE1-large splice variants in E. coli and these splice variants are characterized in terms of Western blot, biotinylated calmodulin overlay and peptide mass fingerprinting. Results from these studies suggested that these two splice variants belong to the PDE1 superfamily. To our knowledge, this is the first report on cloning and characterization of these cDNA variants from bovine cardiac tissue. Since there are at least two isoforms of PDE1 in bovine heart tissue, this merits further in-depth study. 相似文献
11.
Takashi Miki Masato Taira Steven Hockman Fumio Shimada Jonathan Lieman Maria Napolitano David Ward Masanori Taira Hideichi Makino Vincent C. Manganiello 《Genomics》1996,36(3):476
Two distinct PDE3 [cyclic GMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE)] isoforms, cGIP1 and cGIP2, have been identified. Here we report cloning of the cDNA and gene encoding human (H)cGIP1 (classified as PDE3B). The cDNA encodes a protein of 1112 amino acids (123 kDa). Northern blots indicate that its mRNA is expressed in several adipose tissue depots. The human PDE3B gene is composed of 16 exons spanning more than 114 kb and was localized to chromosome 11p15 byin situhybridization. Exon/intron boundaries were determined, and genetic polymorphism, confirmed by single-strand conformational polymorphism of DNA from 25 healthy subjects, was demonstrated in exon 4 at nucleotide 1389 (A/G). Two polymorphic dinucleotide repeat sequences were identified in introns 5 and 12. 相似文献
12.
The accumulation of cyclic AMP in rat testis in response to FSH is much greater in immature rat compared to adult. Addition of 3-isobutyl-1-methylxanthine (MIX), a potent in vitro inhibitor of cyclic nucleotide phosphodiesterase activity, potentiates the effects of FSH but does not restore the cyclic AMP response of adult rat to that of immature rat testis. The apparent substrate affinities for cyclic AMP and cyclic GMP hydrolysis are similar in immature and mature testis. Developmental changes in cyclic AMP and cyclic GMP phosphodiesterase specific activities fail to account for the reduced responsiveness of adult testis to FSH. These results suggest a defect in synthetic rather than hydrolytic mechanisms in FSH-responsive cells. 相似文献
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14.
Establishment of an efficient purification method and further characterization of 32K calmodulin-binding protein in testis 总被引:1,自引:0,他引:1
A heat-stable 32K calmodulin-binding protein has been purified approximately 3,670-fold from porcine testis to apparent homogeneity as judged by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and polyacrylamide gel electrophoresis under native conditions. The purification employed calmodulin-Sepharose 4B affinity chromatography; elution was performed with a free Ca2+ gradient. This provided a simple and efficient procedure, and approximately 1.62 mg of pure heat-stable calmodulin-binding protein was obtained from 390 g of porcine testis with a yield of 47% in activity. The purified protein was asymmetric (f/fo = 1.89) and consisted of a single polypeptide of Mr = 32,000. It is a highly acidic protein (pI = 3.9) with a diffusion coefficient of 5.4 X 10(-7) cm2/s, a sedimentation coefficient of 1.43 S, and a Stokes radius of 39.5 A in its free form and 41.3 A in its complex form with calmodulin. The extent of inhibition of phosphodiesterase by the calmodulin-binding protein was affected by the order of addition of the agents to the reaction mixture. The extent of inhibition was maximal when phosphodiesterase was added last, while it was minimal when the calmodulin-binding protein was added last. This protein was indistinguishable from a heat-stable calmodulin-binding protein in rat testis (Ono, T., Koide, Y., Arai, Y., & Yamashita, K. (1984) J. Biol. Chem. 259, 9011-9016). 相似文献
15.
Timothy M Coskran Daniel Morton Frank S Menniti Wendy O Adamowicz Robin J Kleiman Anne M Ryan Christine A Strick Christopher J Schmidt Diane T Stephenson 《The journal of histochemistry and cytochemistry》2006,54(11):1205-1213
A monoclonal antibody directed against the amino terminal of rat phosphodiesterase 10A (PDE10A) was used to localize PDE10A in multiple central nervous system (CNS) and peripheral tissues from mouse, rat, dog, cynomolgus macaque, and human. PDE10A immunoreactivity is strongly expressed in the CNS of these species with limited expression in peripheral tissues. Within the brain, strong immunoreactivity is present in both neuronal cell bodies and neuropil of the striatum, in striatonigral and striatopallidal white matter tracks, and in the substantia nigra and globus pallidus. Outside the brain, PDE10A immunoreactivity is less intense, and distribution is limited to few tissues such as the testis, epididymal sperm, and enteric ganglia. These data demonstrate that PDE10A is an evolutionarily conserved phosphodiesterase highly expressed in the brain but with restricted distribution in the periphery in multiple mammalian species. 相似文献
16.
We have isolated and characterized rat cyclic nucleotide phosphodiesterase (PDE)11A, which exhibits properties of a dual-substrate PDE, and its splice variants (RNPDE11A2, RNPDE11A3, and RNPDE11A4). The deduced amino-acid sequence of the longest form of rat PDE11A splice variant, RNPDE11A4, was 94% identical with that of the human variant (HSPDE11A4). Rat PDE11A splice variants were expressed in a tissue-specific manner. RNPDE11A4 showed unique tissue distribution distinct from HSPDE11A4, which is specifically expressed in the prostate. Rat PDE11A splice variants were expressed in COS-7 cells, and their enzymatic characteristics were compared. Although the Km values for cAMP and cGMP were similar for all of them (1.3-1.6 and 2.1-3.9 microM, respectively), the Vmax values differed significantly (RNPDE11A4 > RNPDE11A2 > RNPDE11A3). Human PDE11A variants also displayed very similar Km values and significantly different Vmax values (HSPDE11A4 > HSPDE11A2 > HSPDE11A3 > HSPDE11A1). The Vmax values of HSPDE11A4 for cAMP and cGMP were at least 100 times higher than those of HSPDE11A1. These observations indicate unique characteristics of PDE11A splicing variants. 相似文献
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Sapiro R Tarantino LM Velazquez F Kiriakidou M Hecht NB Bucan M Strauss JF 《Biology of reproduction》2000,62(3):511-518
A cDNA encoding sperm antigen 6 (Spag6), the murine homologue of the Chlamydomonas reinhardtii PF16 protein-a component of the flagella central apparatus-was isolated from a mouse testis cDNA library. The cDNA sequence predicted a 55.3-kDa polypeptide containing 8 contiguous armadillo repeats with 65% amino acid sequence identity and 81% similarity to the Chlamydomonas PF1 protein. An antipeptide antibody generated against a C-terminal sequence recognized a 55-kDa protein in sperm extracts and localized Spag6 to the principal piece of permeabilized mouse sperm tails. When expressed in COS-1 cells, Spag6 colocalized with microtubules. The Spag6 gene was found to be highly expressed in testis and was mapped using the T31 radiation hybrid panel to mouse chromosome 16. Mutations in the Chlamydomonas PF16 gene cause flagellar paralysis. The presence of a highly conserved mammalian PF16 homologue (Spag6) raises the possibility that Spag6 plays an important role in sperm flagellar function. 相似文献
19.
The N-terminal regulatory region of the high affinity cAMP-specific phosphodiesterase, PDE7A1, contains two copies of the cAMP-dependent kinase (PKA) pseudosubstrate site RRGAI. In betaTC3 insulinoma cells, PDE7A1 co-localizes with PKA II in the Golgi-centrosome region. The roles PDE7A1 and its regulatory region play in cAMP signaling were examined by studying interactions with PKA subunits. PDE7A1 associates with the dissociated C subunit of PKA (C), but does not bind tetrameric PKA holoenzyme. High affinity binding of C by PDE7A1 inhibits kinase activity in vitro (IC50 = 0.5 nm). The domain containing PKA pseudosubstrate sites at the N terminus of PDE7A1 mediates complex formation with C. The PDE7A1 N-terminal repeat region inhibits C activity in CHO-K1 cells and also suppresses C dependent, cAMP-independent, physiological responses in yeast. Thus, PDE7A1 possesses a non-catalytic activity that can contribute to the termination of cAMP signals via direct inhibition of C. This study identifies a novel inhibitor of PKA and a non-catalytic affect of a cyclic nucleotide phosphodiesterase. 相似文献
20.
Calcium and cyclic nucleotides are second messengers that regulate the development and functional activity of spermatozoa. Calcium/calmodulin-dependent phosphodiesterases (CaM-PDEs) are abundant in testicular cells and in mature spermatozoa and provide one means by which calcium regulates cellular cyclic nucleotide content. We examined the spatial and temporal expression profiles of three knownCaM-PDE genes, PDE1A, PDE1B, and PDE1C, in the testis. In situ hybridization and immunofluorescent staining showed that both PDE1A and PDE1C are highly expressed but at different stages in developing germ cells. However, a very low hybridization signal of PDE1B exists uniformly throughout the seminiferous epithelium and the interstitium. More specifically, PDE1A mRNA is found in round to elongated spermatids, with protein expression in the tails of elongated and maturing spermatids. In contrast, PDE1C mRNA accumulates during early meiotic prophase and throughout meiotic and postmeiotic stages. Immunocytochemistry showed a diffuse, presumably cytosolic distribution of the expressed protein. The distinct spatial and temporal expression patterns of CaM-PDEs suggest important but different physiological roles for these CaM-PDEs in developing and mature spermatozoa. 相似文献