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101.
Phosphorylation and activation of phospholipase D1 by protein kinase C in vivo: determination of multiple phosphorylation sites. 总被引:5,自引:0,他引:5
Y Kim J M Han J B Park S D Lee Y S Oh C Chung T G Lee J H Kim S K Park J S Yoo P G Suh S H Ryu 《Biochemistry》1999,38(32):10344-10351
Protein kinase C (PKC) is an important regulator of phospholipase D1 (PLD1). Currently there is some controversy about a phosphorylation-dependent or -independent mechanism of the activation of PLD1 by PKC. To solve this problem, we examined whether PLD1 is phosphorylated by PKC in vivo. For the first time, we have now identified multiple basal phophopeptides and multiple phorbol myristate acetate (PMA) induced phosphopeptides of endogenous PLD1 in 3Y1 cells as well as of transiently expressed PLD1 in COS-7 cells. Down regulation or inhibition of PKC greatly attenuated the PMA-induced phosphorylation as well as the activation of PLD1. In the presence of PMA, purified PLD1 from rat brain was also found to be phosphorylated by PKCalpha in vitro at multiple sites generating seven distinct tryptic phosphopeptides. Four phosphopeptides generated in vivo and in vitro correlated well with each other, suggesting direct phosphorylation of PLD1 by PKCalpha in the cells. Serine 2, threonine 147, and serine 561 were identified as phosphorylation sites, and by mutation of these residues to alanine these residues were proven to be specific phosphorylation sites in vivo. Interestingly, threonine 147 is located in the PX domain and serine 561 is in the negative regulatory "loop" region of PLD1. Mutation of serine 2, threonine 147, or serine 561 significantly reduced PMA-induced PLD1 activity. These results strongly suggest that phosphorylation plays a pivotal role in PLD1 regulation in vivo. 相似文献
102.
Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex 下载免费PDF全文
Bacteriophage T4 deoxycytidylate hydroxymethylase (EC 2.1.2.8), a homodimer of 246-residue subunits, catalyzes hydroxymethylation of the cytosine base in deoxycytidylate (dCMP) to produce 5-hydroxymethyl-dCMP. It forms part of a phage DNA protection system and appears to function in vivo as a component of a multienzyme complex called deoxyribonucleoside triphosphate (dNTP) synthetase. We have determined its crystal structure in the presence of the substrate dCMP at 1.6 A resolution. The structure reveals a subunit fold and a dimerization pattern in common with thymidylate synthases, despite low (approximately 20%) sequence identity. Among the residues that form the dCMP binding site, those interacting with the sugar and phosphate are arranged in a configuration similar to the deoxyuridylate binding site of thymidylate synthases. However, the residues interacting directly or indirectly with the cytosine base show a more divergent structure and the presumed folate cofactor binding site is more open. Our structure reveals a water molecule properly positioned near C-6 of cytosine to add to the C-7 methylene intermediate during the last step of hydroxymethylation. On the basis of sequence comparison and crystal packing analysis, a hypothetical model for the interaction between T4 deoxycytidylate hydroxymethylase and T4 thymidylate synthase in the dNTP-synthesizing complex has been built. 相似文献
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106.
A nodule-specific dicarboxylate transporter from alder is a member of the peptide transporter family 下载免费PDF全文
Jeong J Suh S Guan C Tsay YF Moran N Oh CJ An CS Demchenko KN Pawlowski K Lee Y 《Plant physiology》2004,134(3):969-978
Alder (Alnus glutinosa) and more than 200 angiosperms that encompass 24 genera are collectively called actinorhizal plants. These plants form a symbiotic relationship with the nitrogen-fixing actinomycete Frankia strain HFPArI3. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen, but this metabolite exchange in actinorhizal nodules has not been well defined. We isolated an alder cDNA from a nodule cDNA library by differential screening with nodule versus root cDNA and found that it encoded a transporter of the PTR (peptide transporter) family, AgDCAT1. AgDCAT1 mRNA was detected only in the nodules and not in other plant organs. Immunolocalization analysis showed that AgDCAT1 protein is localized at the symbiotic interface. The AgDCAT1 substrate was determined by its heterologous expression in two systems. Xenopus laevis oocytes injected with AgDCAT1 cRNA showed an outward current when perfused with malate or succinate, and AgDCAT1 was able to complement a dicarboxylate uptake-deficient Escherichia coli mutant. Using the E. coli system, AgDCAT1 was shown to be a dicarboxylate transporter with a K(m) of 70 microm for malate. It also transported succinate, fumarate, and oxaloacetate. To our knowledge, AgDCAT1 is the first dicarboxylate transporter to be isolated from the nodules of symbiotic plants, and we suggest that it may supply the intracellular bacteria with dicarboxylates as carbon sources. 相似文献
107.
The potential role of green tea polyphenol (GtPP) in preserving the human saphenous vein was investigated under physiological conditions. The vein segments were incubated for 1, 3, 5, 7 and 14 days, either after 4h of treatment with 1.0mg/ml GtPP or in the presence of GtPP at the same concentration. After incubation, the endothelial cell viability, endothelial nitric oxide synthase (eNOS) expression and the vein histology were evaluated. When the veins were not treated with GtPP, the viability of the endothelial cells was significantly reduced with the progress in the culture time, and none of the cells expressed eNOS after 5 days. Furthermore, severe histological changes and structural damage were observed in the non-treated veins. In contrast, incubating the veins after 4h of GtPP treatment significantly prevented these phenomena. The cellular viability of the GtPP-treated vein was approximately 64% after 7 days, and eNOS expression was maintained up to 40%, compared to that of the fresh vein. The histological observations showed that the vasculature was quite similar to that of the fresh vein. When incubated with GtPP, the vein could also be preserved for 1 week under physiological conditions retaining both its cellular viability (61%) and eNOS expression level (45%) and maintaining its venous structure without any morphological changes. These results demonstrate that GtPP treatment may be a useful method for preserving the HSV. 相似文献
108.
Rho J Shin JH Song JW Park MR Kee SJ Jang SJ Park YK Suh SP Ryang DW 《Journal of microbiology (Seoul, Korea)》2004,42(2):80-86
Pulsed-field gel electrophoresis (PFGE) typing was applied to the epidemiological investigation of 21 Candida tropicalis isolates collected from urine specimens of 11 patients and one healthcare worker, in an intensive care unit (ICU) over a 4-month period. Seventeen epidemiologically unrelated strains from 14 patients were also tested to determine the discriminatory power of PFGE. PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG), using two restriction enzymes (BssHII and SfiI). The EK pattern was the same in all 38 isolates, while REAG using SfiI separated the isolates into nine types. However, 16 different PFGE types were identified by REAG with BssHII, and the same results were obtained when the results of both REAG tests were combined. In serial urinary isolates from 10 patients, all strains from each patient had the same PFGE pattern. While the epidemiologically unrelated strains from 14 patients consisted of 13 different PFGE types, the 20 isolates from the 11 ICU patients fell into only two PFGE types (types C1 and C2), and these apparently originated from the two different outbreaks. All strains of type C1 (n = 12) were isolated from six patients, between November 1999 and January 2000, and all of the type C2 strains (n=8) were isolated from five patients, during January and February 2000. This study shows two consecutive clusters of C. tropicalis candiduria in an ICU, defined by PFGE typing, and also demonstrates that a PFGE typing method using BssHII is perhaps the most useful method for investigating C. tropicalis candiduria. 相似文献
109.
6,7-Dichloroquinoline-5,8-dione (1) was reacted with a number of 2-aminopyridine derivatives. Of the several possible products of this reaction, 4a,10,11-triazabenzo[3,2-a]fluorene-5,6-dione (6), produced by condensation and rearrangement, was obtained as the major product, and its structure was subsequently unambigously determined by X-ray crystallographic study. Ortho-quinones were produced via nucleophilic substitution at position C7, which was unexpected, considering that para-quinones were produced via C6 substitution in the reaction between compound 1 and ethyl acetoacetate in our previous work. Such unexpected nucleophilic substitution at C7 provides an effective, yet simple route, to the preparation of biologically active ortho-quinone derivatives. 相似文献
110.
The enzyme HemK (or PrmC) is one of the first identified methyltransferases that modify glutamine. It methylates the highly conserved GGQ motif in class I release factors (RF1 and RF2) in Escherichia coli. HemK from Thermotoga maritima was over-expressed and crystallized in the presence of S-adenosylmethionine at 296 K using ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.5 A resolution from a native crystal. The crystal is orthorhombic, belonging to the space group I222 (or I2(1)2(1)2(1)), with unit-cell parameters of a = 104.24, b = 118.73, and c = 146.62 A. Two (or three) monomers of recombinant HemK are likely to be present in the crystallographic asymmetric unit, giving a V(M) of 3.62 A3 Da(-1) (or 2.41 A3 Da(-1)), with a solvent content of 62.7% (or 44.0%). 相似文献