cDNA encoding a novel phosphodiesterase (PDE) was isolated from a human fetal lung cDNA library and designated PDE10A. The deduced amino acid sequence contains 779 amino acids, including a putative cGMP binding sequence in the amino-terminal portion of the molecule and a catalytic domain that is 16 -47% identical in amino acid sequence to those of other PDE families. Recombinant PDE10A transfected and expressed in COS-7 cells hydrolyzed cAMP and cGMP with K m values of 0.26 and 7.2 M, respectively, and V max with cGMP was almost twice that with cAMP. Of the PDE inhibitors tested, dipyridamole was most effective, with IC 50 values of 1.2 and 0.45 M for inhibition of cAMP and cGMP hydrolysis, respectively. cGMP inhibited hydrolysis of cAMP, and cAMP inhibited cGMP hydrolysis with IC 50 values of 14 and 0.39 M, respectively. Thus, PDE10A exhibited properties of a cAMP PDE and a cAMP-inhibited cGMP PDE. PDE10A transcripts were particularly abundant in the putamen and caudate nucleus regions of brain and in thyroid and testis, and in much lower amounts in other tissues. The PDE10A gene was located on chromosome 6q26 by fluorescent in situ hybridization analysis. PDE10A represents a new member of the PDE superfamily, exhibiting unique kinetic properties and inhibitor sensitivity.Cyclic nucleotides cAMP and cGMP are well known as second messengers and regulate many functions in various tissues (1-4). Intracellular cAMP and cGMP concentrations are controlled via stimulation of adenyl and guanyl cyclases in response to extracellular signaling and their degradation by cyclic nucleotide phosphodiesterases (PDEs), 1 respectively. Many kinds of PDEs are involved in the metabolism of cyclic nucleotides. Based on their amino acid sequence homology, biochemical properties, and inhibitor profiles, seven PDE families have been recognized in mammalian tissues (5, 6). PDE1 is Ca 2ϩ / calmodulin-dependent, hydrolyzing both cAMP and cGMP. PDE2 is stimulated by cGMP and hydrolyzes cAMP and cGMP.PDE3 is cGMP-inhibited. PDE4 is cAMP-specific and rolipramsensitive. PDE5 is cGMP-specific. PDE6 is a photoreceptor cGMP PDE. PDE7 is cAMP-specific and rolipram-insensitive. Very recently, cDNAs encoding two kinds of novel PDEs were isolated from humans and mice (7-11). One is cAMP-specific (PDE8), and the other is cGMP-specific (PDE9). PDE7 and the two latter PDEs are insensitive to the nonspecific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). cDNA cloning of these novel PDEs was done by an approach using bioinformatics. A search of data bases of expressed sequence tags (ESTs) was performed using parts of PDE sequences such as the catalytic domain. The approach was shown to be effective for the isolation of novel PDE cDNAs.Genome sequencing projects are progressing in many organisms, providing us information of a variety of pathways involved in the cyclic nucleotide metabolism necessary to maintain life. In Caenorhabditis elegans, which is a small soil nematode found in temperate regions, there are five pairs of autosomal chromosomes and a...

cDNAs encoding a novel phosphodiesterase, phosphodiesterase 11A (PDE11A), were isolated by a combination of reverse transcriptase-polymerase chain reaction using degenerate oligonucleotide primers and rapid amplification of cDNA ends. Their catalytic domain was identical to that of PDE11A1 (490 amino acids) reported during the course of this study. However, the cDNAs we isolated had N termini distinct from PDE11A1, indicating two novel N-terminal variants of PDE11A. PDE11A3 cDNA encoded a 684-amino acid protein including one complete and one incomplete GAF domain in the Nterminal region. PDE11A4 was composed of 934 amino acids including two complete GAF domains and shared 630 C-terminal amino acids with PDE11A3 but had a distinct N terminus containing the putative phosphorylation sites for cAMP-and cGMP-dependent protein kinases. PDE11A3 transcripts were specifically expressed in testis, whereas PDE11A4 transcripts were particularly abundant in prostate. Recombinant PDE11A4 expressed in COS-7 cells hydrolyzed cAMP and cGMP with K m values of 3.0 and 1.4 M, respectively, and the V max value with cAMP was almost twice that with cGMP. Although PDE11A3 showed the same K m values as PDE11A4, the relative V max values of PDE11A3 were approximately one-sixth of those of PDE11A4. PDE11A4, but not PDE11A3, was phosphorylated by both cAMPand cGMP-dependent protein kinases in vitro. Thus, the PDE11A gene undergoes tissue-specific alternative splicing that generates structurally and functionally distinct gene products. Cyclic nucleotide phosphodiesterases (PDEs)1 metabolize cAMP and cGMP, which are second messengers regulating many functions in various cells and tissues. Based on their amino acid sequence homology, biochemical properties, and inhibitor profiles, many kinds of PDEs have been identified in mammalian tissues (1-3). The PDE1 family is Ca 2ϩ /calmodulindependent and hydrolyzes both cAMP and cGMP. PDE2 is stimulated by cGMP and hydrolyzes cAMP and cGMP, while PDE3 is cGMP-inhibited. The cAMP-specific and rolipram-sensitive PDEs belong to the PDE4 family. PDE5 is a cGMPbinding, cGMP-specific PDE. The photoreceptor cGMP PDEs are in the PDE6 family. PDE7 is cAMP-specific and rolipraminsensitive. PDE8 is a cAMP-specific PDE, and PDE9 is a cGMP-specific PDE (3-8). Recently, we revealed a new member of the PDE group, PDE10A, which hydrolyzes both cAMP and cGMP (9). Some of these PDEs constitute subfamilies encoded by distinct genes. In each PDE family, alternative splice variants have been reported (1, 10, 11). In many cases, different gene products and alternative splice variants in each PDE family show different expression patterns in tissues and different subcellular localization (1,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). PDEs encoded by alternatively spliced mRNAs have been reported to differ in their regulation by some kinases including cAMP-dependent protein kinase (cAK) and cGMP-dependent kinase (cGK) and associated proteins (19, 23). Thus, cyclic nucleotide levels are controlled by a complex system. Each ...

This is the first report of molecular characterization of a novel cyclic nucleotide PDE (phosphodiesterase), isolated from the human malaria parasite Plasmodium falciparum and designated PfPDE1. PfPDE1 cDNA encodes an 884-amino-acid protein, including six putative transmembrane domains in the N-terminus followed by a catalytic domain. The PfPDE1 gene is a single-copy gene consisting of two exons and a 170 bp intron. PfPDE1 transcripts were abundant in the ring form of the asexual blood stages of the parasite. The C-terminal catalytic domain of PfPDE1, produced in Escherichia coli, specifically hydrolysed cGMP with a K(m) value of 0.65 microM. Among the PDE inhibitors tested, a PDE5 inhibitor, zaprinast, was the most effective, having an IC50 value of 3.8 microM. The non-specific PDE inhibitors IBMX (3-isobutyl-1-methylxanthine), theophylline and the antimalarial chloroquine had IC50 values of over 100 microM. Membrane fractions prepared from P. falciparum at mixed asexual blood stages showed potent cGMP hydrolytic activity compared with cytosolic fractions. This hydrolytic activity was sensitive to zaprinast with an IC50 value of 4.1 microM, but insensitive to IBMX and theophylline. Furthermore, an in vitro antimalarial activity assay demonstrated that zaprinast inhibited the growth of the asexual blood parasites, with an ED50 value of 35 microM. The impact of cyclic nucleotide signalling on the cellular development of this parasite has previously been discussed. Thus this enzyme is suggested to be a novel potential target for the treatment of the disease malaria.

Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the sea hare (Aplysia kurodai) were screened and characterized to determine how the sea hare obtains glucose from sea lettuce (Ulva pertusa). Four endo-ß-1,4-glucanases (21K, 45K, 65K, and 95K cellulase) and 2 ß-glucosidases (110K and 210K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-ß-glucoside, 4MU–ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K ß-glucosidase. Kinetic analysis of the inhibition of 210K ß-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase. Our results indicate that 45K cellulase and 210K ß-glucosidase are the core components of the sea hare digestive system for efficient production of glucose from sea lettuce. These findings contribute important new insights into the development of biofuel processing biotechnologies from seaweed.

cGMP-dependent protein kinase (cGK) is a major cellular receptor of cGMP and plays important roles in cGMP-dependent signal transduction pathways. To isolate the components of the cGMP/cGK signaling pathway such as substrates and regulatory proteins of cGK, we employed the yeast two-hybrid system using cGK-I␣ as a bait and isolated a novel male germ cell-specific 42-kDa protein, GKAP42 (42-kDa cGMP-dependent protein kinase anchoring protein). Although the N-terminal region (amino acids 1-66) of cGK-I␣ is sufficient for the association with GKAP42, GKAP42 could not interact with cGK-I␤, cGK-II, or cAMP-dependent protein kinase. GKAP42 mRNA is specifically expressed in testis, where it is restricted to the spermatocytes and early round spermatids. Endogenous cGK-I is co-immunoprecipitated with anti-GKAP42 antibody from mouse testis tissue, suggesting that cGK-I physiologically interacts with GKAP42. Immunocytochemical observations revealed that GKAP42 is localized to the Golgi complex and that cGK-I␣ is co-localized to the Golgi complex when coexpressed with GKAP42. Although both cGK-I␣ and -I␤, but not cAMP-dependent protein kinase, phosphorylated GKAP42 in vitro, GKAP42 was a good substrate only for cGK-I␣ in intact cells, suggesting that the association with kinase protein is required for the phosphorylation in vivo. Finally, we demonstrated that the kinase-deficient mutant of cGK-I␣ stably associates with GKAP42 and that binding of cGMP to cGK-I␣ facilitates their release from GKAP42. These findings suggest that GKAP42 functions as an anchoring protein for cGK-I␣ and that cGK-I␣ may participate in germ cell development through phosphorylation of Golgi-associated proteins such as GKAP42.

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cGMP-dependent protein kinase (cGK) is a major intracellular receptor of cGMP and is implicated in several signal transduction pathways. To identify proteins that participate in the cGMP/cGK signaling pathway, we employed the yeast two-hybrid system with cGK Ialpha as bait. cDNAs encoding slow skeletal troponin T (skTnT) were isolated from both mouse embryo and human skeletal muscle cDNA libraries. The skTnT protein interacted with cGK Ibeta but not with cGK II nor cAMP-dependent protein kinase. The yeast two-hybrid and in vitro binding assays revealed that the N-terminal region of cGK Ialpha, containing the leucine zipper motif, is sufficient for the association with skTnT. In vivo analysis, mutations in cGK Ialpha, which disrupted the leucine zipper motif, were shown to completely abolish the binding to skTnT. Furthermore, cGK I also interacted with cardiac TnT (cTnT) but not with cardiac troponin I (cTnI). Together with the observations that cTnI is a good substrate for cGK I and is effectively phosphorylated in the presence of cTnT in vitro, these findings suggest that TnT functions as an anchoring protein for cGK I and that cGK I may participate in the regulation of muscle contraction through phosphorylation of TnI.

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