Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.4.1.14 (SPS)
 813 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Analysis of Ag specificity of TRC-gamma delta+ T cells in humans has been hampered by the fact that cloned lines of these cells expanded in IL-2 generally display high NK-like cytotoxic activity. A TCR-gamma delta+ CTL clone, isolated in IL-4, strongly lysed a specific stimulator cell, the EBV-transformed cell line JY, but failed to lyse K562 and other target cells sensitive for NK cell activity. Subsequent culture of this clone (CD124) in IL-2 induced high cytotoxic activity against the NK sensitive target cells. K562 cells were unable to induce the secretion of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester [(BLT)-serine esterase] or influx of Ca2+ ions in clone CD124 cultured in either IL-4 or IL-2. In contrast, JY cells induced high BLT-serine esterase secretion and an increase of cytosolic Ca2+ levels. By using a combination of a 51Cr-release assay and a BLT-serine esterase secretion assay, the reactivity of clone CD124 against a limited number of target cells was analyzed. CD124 which expresses HLA-A2 and -B7, recognized an Ag shared by JY (HLA-A2; B7; C blank; DR4,6) and one haplotype expressed by the cell line SPS (HLA-A1; B14; Cw6; DR4). The only specificity shared by SPS and JY was HLA-DR4. However, clone CD124 failed to lyse 5 other HLA-DR4+ target cells. The cytotoxic activity of clone CD124 was inhibited by the class I MHC specific mAb W6/32 and the anti-beta 2m mAb A88, but not, or only marginally, by the anti HLA-DQ mAb SPV-L3 or the anti-HLA-DR mAb 135. These data strongly suggest that clone CD124 recognizes a class I MHC Ag different from HLA-A, -B, or -C.
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PMID:Antigen-specific, but not natural killer, activity of T cell receptor-gamma delta cytotoxic T lymphocyte clones involves secretion of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase and influx of Ca2+ ions. 247 1

Sucrose-phosphate synthase (SPS; EC 2.4.1.14) is regulated in part by reversible protein phosphorylation. When dephospho-SPS is partially purified from illuminated spinach leaves and incubated with [gamma-32P]ATP the enzyme is phosphorylated by a copurifying protein kinase. In this report, 32P-phosphopeptides from tryptic digests of in vitro phosphorylated SPS were purified by metal-ion affinity chromatography and reversed-phase high-performance liquid chromatography. Three distinct 32P-phosphopeptides were resolved. Edman sequencing of the major phosphopeptide (which contained > 80% of the total 32P) identified the amino acid sequence as Ile-Ser-Ser(P)-Val-Glu-Met-Met-Asp-Asn-Trp-Ala-Asn-Thr-Phe-Lys. This sequence corresponds to residues 156 to 170 of the deduced amino acid sequence of spinach SPS [Klein, R. R., Crafts-Brandner, S. J., and Salvucci, M. E. (1993) Planta 190, 498-510, and Sonnewald, U., Quick, W. P., MacRae, E., Krause, K.-P., and Stitt, M. (1993) Planta 189, 174-181]. Identification of the phosphoseryl residue was accomplished by manual Edman sequencing. The two other phosphopeptides, which each contained less than 10% of the total 32P, were not sequenced. An Escherichia coli expressed, 26-kDa fragment of SPS which contains the major phosphorylation site was a substrate for the protein kinase which copurifies with SPS. Two-dimensional peptide mapping analysis of this fragment showed the major phosphopeptide was present but not the other site(s), suggesting that other peptides are derived from a site other than Ser158. These results provide additional indirect evidence for the presence of multiple phosphorylation sites in SPS.
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PMID:Identification of the major regulatory phosphorylation site in sucrose-phosphate synthase. 827 10

Sucrose-6(F)-phosphate phosphohydrolase (SPP; EC ) catalyzes the final step in the pathway of sucrose biosynthesis and is the only enzyme of photosynthetic carbon assimilation for which the gene has not been identified. The enzyme was purified to homogeneity from rice (Oryza sativa L.) leaves and partially sequenced. The rice leaf enzyme is a dimer with a native molecular mass of 100 kDa and a subunit molecular mass of 50 kDa. The enzyme is highly specific for sucrose 6(F)-phosphate with a K(m) of 65 microM and a specific activity of 1250 micromol min(-1) mg(-1) protein. The activity is dependent on Mg(2+) with a remarkably low K(a) of 8-9 microM and is weakly inhibited by sucrose. Three peptides from cleavage of the purified rice SPP with endoproteinase Lys-C showed similarity to the deduced amino acid sequences of three predicted open reading frames (ORF) in the Arabidopsis thaliana genome and one in the genome of the cyanobacterium Synechocystis sp. PCC6803, as well as cDNA clones from Arabidopsis, maize, and other species in the GenBank database of expressed sequence tags. The putative maize SPP cDNA clone contained an ORF encoding a 420-amino acid polypeptide. Heterologous expression in Escherichia coli showed that this cDNA clone encoded a functional SPP enzyme. The 260-amino acid N-terminal catalytic domain of the maize SPP is homologous to the C-terminal region of sucrose-phosphate synthase. A PSI-BLAST search of the GenBank database indicated that the maize SPP is a member of the haloacid dehalogenase hydrolase/phosphatase superfamily.
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PMID:Purification, molecular cloning, and sequence analysis of sucrose-6F-phosphate phosphohydrolase from plants. 1105 Jan 82

The activity and allosteric properties of plant phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) are controlled posttranslationally by specific reversible phosphorylation of a strictly conserved serine residue near the N-terminus. This up/down-regulation of PEPC is catalyzed by a dedicated and highly regulated serine/threonine (Ser/Thr) kinase (PEPC-kinase) and an opposing type-2A Ser/Thr phosphatase (PP2A). In marked contrast to PEPC-kinase, the PP2A holoenzyme from photosynthetic tissue has been virtually unstudied to date. In the present investigation, we have partially purified and characterized the native form of this PP2A from illuminated leaves of maize (Zea mays L.), a C4 plant, using maize [32P]PEPC as substrate. Various conventional chromatographic matrices, together with thiophosphorylated C4 PEPC-peptide and microcystin-LR affinity-supports, were exploited for the enrichment of this PP2A from soluble leaf extracts. Biochemical and immunological results indicate that the C4-leaf holoenzyme is analogous to other eukaryotic PP2As in being a approximately 170-kDa heteromer comprised of a core PP2Ac-A heterodimer (approximately 38- and approximately 65-kDa subunits, respectively) complexed with a putative, approximately 74-kDa B-type regulatory/targeting subunit. This heterotrimer lacks any strict substrate specificity in that it dephosphorylates C4 PEPC, mammalian phosphorylase a, and casein in vitro. This activity is independent of free Me2+, insensitive to levamisole and the Inhibitor-2 protein that targets PP1, activated by several polycations such as protamine and poly-L-lysine, and highly sensitive to inhibition by microcystin-LR and okadaic acid (IC50 approximately 30 pM), all of which are diagnostic features of yeast and mammalian PP2As. In addition, this C4-leaf PP2A holoenzyme (i) is inhibited in vitro by physiological concentrations of certain C4 PEPC-related metabolites (L-malate, PEP, glucose 6-phosphate, but not the activator glycine) when either 32P-labeled maize PEPC or rabbit muscle phosphorylase a is used as substrate, suggesting a direct effect on this Ser/Thr phosphatase; and (ii) displays, at best, only modest light/dark effects in vivo on its apparent molecular mass, component core subunits and activity against C4 PEPC, in marked contrast to the opposing activity of PEPC-kinase in C4 and Crassulacean acid metabolism leaves. This report represents one of the few studies of a heteromeric PP2A holoenzyme from photosynthetic tissue that dephosphorylates a known target enzyme in plants, such as PEPC, sucrose-phosphate synthase or nitrate reductase.
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PMID:Partial purification and biochemical characterization of a heteromeric protein phosphatase 2A holoenzyme from maize (Zea mays L.) leaves that dephosphorylates C4 phosophoenolpyruvate carboxylase. 1150 60

In the present contribution we report on a novel route to synthesize 2D-polyaniline (2D-PAN) on sulfonated-poly(styrene) (SPS) templates by allowing first monomer assembly followed by chemical oxidation to achieve polymerization. We show that Aplysia neurons grown on 2D-PAN exhibit an unusual growth pattern and adhesion to this conducting substrate that is manifested by the formation of giant lamellipodia. The lamellipodial domains are characterized by small gap between the plasma membrane and the 2D-PAN substrate (ca. 30 nm) and actin rich skeleton resembling the skeleton of growth cones. This behavior is characteristic to uniform substrates containing only 2D-PAN. However, in patterned substrates containing additionally poly(L-lysine) Aplysia neurons prefer to extend new neurites on the poly(L-lysine) domains.
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PMID:Electrically conductive 2D-PAN-containing surfaces as a culturing substrate for neurons. 1564 68

Histone lysine methylation is an evolutionally conserved modification involved in determining chromatin states associated with gene activation or repression. Here we report that the Arabidopsis SET domain group 8 (SDG8) protein is a histone H3 methyltransferase involved in regulating shoot branching. Knockout mutations of the SDG8 gene markedly reduce the global levels of histone H3 trimethylation at lysines 9 and 36 as well as dimethylation at lysine 36. The sdg8 mutants produce more shoot branches than wild-type plants. The expression of SPS/BUS (supershoot/bushy), a repressor of shoot branching, is decreased in sdg8 mutants, while UGT74E2 (UDP-glycosyltransferase 74E2), a gene associated with increased shoot branching, is up-regulated in sdg8 mutants. The altered expression of SPS/BUS and UGT74E2 correlates with changed histone H3 methylation at these loci. These results suggest that SDG8 regulates shoot branching via controlling the methylation states of its target genes.
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PMID:The histone methyltransferase SDG8 regulates shoot branching in Arabidopsis. 1860 72

Regulated proteolysis serves as a mechanism to control cellular processes. The SPS (Ssy1-Ptr3-Ssy5) sensor in yeast responds to extracellular amino acids by endoproteolytically activating transcription factors Stp1 and Stp2 (Stp1/2). The processing endoprotease Ssy5 is regulated via proteasomal degradation of its noncovalently associated N-terminal prodomain. We find that degradation of the prodomain requires a conserved phosphodegron comprising phosphoacceptor sites and ubiquitin-accepting lysine residues. Upon amino acid induction, the phosphodegron is modified in a series of linked events by a set of general regulatory factors involved in diverse signaling pathways. First, an amino acid-induced conformational change triggers phosphodegron phosphorylation by the constitutively active plasma membrane-localized casein kinase I (Yck1/2). Next the prodomain becomes a substrate for polyubiquitylation by the Skp1/Cullin/Grr1 E3 ubiquitin ligase complex (SCF(Grr1)). Finally, the modified prodomain is concomitantly degraded by the 26S proteasome. These integrated events are requisite for unfettering the Ssy5 endoprotease, and thus Stp1/2 processing. The Ssy5 phosphoacceptor motif resembles the Yck1/2- and Grr1-dependent degrons of regulators in the Snf3/Rgt2 glucose-sensing pathway. Our work defines a novel proteolytic activation cascade that regulates an intracellular signaling protease and illustrates how general signaling components are recruited to distinct pathways that achieve conditional and specific signaling outputs.
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PMID:A phosphodegron controls nutrient-induced proteasomal activation of the signaling protease Ssy5. 2165 27

The Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney is a key determinant of Na(+) balance. Disturbances in NCC function are characterized by disordered volume and blood pressure regulation. However, many details concerning the mechanisms of NCC regulation remain controversial or undefined. This is partially due to the lack of a mammalian cell model of the DCT that is amenable to functional assessment of NCC activity. Previously reported investigations of NCC regulation in mammalian cells have either not attempted measurements of NCC function or have required perturbation of the critical without a lysine kinase (WNK)/STE20/SPS-1-related proline/alanine-rich kinase regulatory pathway before functional assessment. Here, we present a new mammalian model of the DCT, the mouse DCT15 (mDCT15) cell line. These cells display native NCC function as measured by thiazide-sensitive, Cl(-)-dependent (22)Na(+) uptake and allow for the separate assessment of NCC surface expression and activity. Knockdown by short interfering RNA confirmed that this function was dependent on NCC protein. Similar to the mammalian DCT, these cells express many of the known regulators of NCC and display significant baseline activity and dimerization of NCC. As described in previous models, NCC activity is inhibited by appropriate concentrations of thiazides, and phorbol esters strongly suppress function. Importantly, they display release of WNK4 inhibition of NCC by small hairpin RNA knockdown. We feel that this new model represents a critical tool for the study of NCC physiology. The work that can be accomplished in such a system represents a significant step forward toward unraveling the complex regulation of NCC.
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PMID:A new model of the distal convoluted tubule. 2271 90

Pregnancy is characterized by plasma volume expansion due to Na(+) retention, driven by aldosterone. The aldosterone-responsive epithelial Na(+) channel is activated in the kidney in pregnancy. In the present study, we investigated the aldosterone-responsive Na(+)-Cl(-) cotransporter (NCC) in mid- and late pregnant rats compared with virgin rats. We determined the abundance of total NCC, phosphorylated NCC (pNCC; pT53, pS71 and pS89), phosphorylated STE20/SPS-1-related proline-alanine-rich protein kinase (pSPAK; pS373), and phosphorylated oxidative stress-related kinase (pOSR1; pS325) in the kidney cortex. We also measured mRNA expression of NCC and members of the SPAK/NCC regulatory kinase network, serum and glucocorticoid-regulated kinase (SGK)1, total with no lysine kinase (WNK)1, WNK3, and WNK4. Additionally, we performed immunohistochemistry for NCC kidneys from virgin and pregnant rats. Total NCC, pNCC, and pSPAK/OSR1 abundance were unchanged in midpregnant versus virgin rats. In late pregnant versus virgin rats, total NCC and pNCC were decreased; however, pSPAK/OSR1 was unchanged. We detected no differences in mRNA expression of NCC, SGK1, total WNK1, WNK3, and WNK4. By immunohistochemistry, NCC was mainly localized to the apical region in virgin rats, and density in the apical region was reduced in late pregnancy. Therefore, despite high circulating aldosterone levels in pregnancy, the aldosterone-responsive transporter NCC is not increased in total or activated (phosphorylated) abundance or in apical localization in midpregnant rats, and all are reduced in late pregnancy. This contrasts to the mineralocorticoid-mediated activation of the epithelial Na(+) channel, which we have previously reported. Why and how NCC escapes aldosterone activation in pregnancy is not clear but may relate to regional differences in aldosterone sensitivity the increased K(+) intake or other undefined mechanisms.
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PMID:Renal NCC is unchanged in the midpregnant rat and decreased in the late pregnant rat despite avid renal Na+ retention. 2592 54

The ability to control the spatial distribution and temporal release of a therapeutic remains a central challenge for biomedical research. Here, we report the development and optimization of a novel substrate mediated therapeutic delivery system comprising of hyaluronic acid covalently functionalized liposomes (HALNPs) embedded into polyelectrolyte multilayer (PEM) platform via ionic stabilization. The PEM platform was constructed from sequential deposition of Poly-L-Lysine (PLL) and Poly(Sodium styrene sulfonate) (SPS) "(PLL/SPS)4.5" followed by adsorption of anionic HALNPs. An adsorption affinity assay and saturation curve illustrated the preferential HALNP deposition density for precise therapeutic loading. (PLL/SPS)2.5 capping layer on top of the deposited HALNP monolayer further facilitated complete nanoparticle immobilization, cell adhesion, and provided nanoparticle confinement for controlled linear release profiles of the nanocarrier and encapsulated cargo. To our knowledge, this is the first study to demonstrate the successful embedment of a translatable lipid based nanocarrier into a substrate that allows for temporal and spatial release of both hydrophobic and hydrophilic drugs. Specifically, we have utilized our platform to deliver chemotherapeutic drug Doxorubicin from PEM confined HALNPs. Overall, we believe the development of our HALNP embedded PEM system is significant and will catalyze the usage of substrate mediated delivery platforms in biomedical applications.
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PMID:Ionic Driven Embedment of Hyaluronic Acid Coated Liposomes in Polyelectrolyte Multilayer Films for Local Therapeutic Delivery. 2642 10


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