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1.
The genetic type of collagen was examined in both fresh and 2 to 4 month in vitro cultured human normal and osteoarthritic articular cartilage. Disc electrophoresis indicated that all cartilage samples examined contained only type II collagen, however CM cellulose chromatography revealed chromatographic differences between the normal and pathological tissues.  相似文献   
2.
l-Methionine gamma-lyase (EC 4.4.1.11, MGL_Pp) from Pseudomonas putida is a multifunctional enzyme, which belongs to the gamma-family of pyridoxal-5'-phosphate (PLP) dependent enzymes. In this report, we demonstrate that the three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method to an R-factor of 20.4% at 1.8 A resolution. Detailed information of the overall structure of MGL_Pp supplies a clear picture of the substrate- and PLP-binding pockets. Tyr59 and Arg61 of neighbouring subunits, which are strongly conserved in other gamma-family enzymes, contact the phosphate group of PLP. These residues are important as the main anchor within the active site. Lys240, Asp241 and Arg61 of one partner monomer and Tyr114 and Cys116 of the other partner monomer form a hydrogen-bond network in the MGL active site which is specific for MGLs. It is also suggested that electrostatic interactions at the subunit interface are involved in the stabilization of the structural conformation. The detailed structure will facilitate the development of MGL_Pp as an anticancer drug.  相似文献   
3.
Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts and is critical for proper dentin formation. DSPP is believed to be processed into only two structural/functional domains: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Here we report the isolation and characterization of a third domain of DSPP, designated dentin glycoprotein (DGP). DGP was isolated from a guanidine/EDTA extract of porcine tooth dentin by ion exchange, hydroxyapatite affinity, size exclusion, and RP-HPL chromatography. Endoproteinase lysine C digestion products of DGP were characterized by Edman sequencing and mass spectrometry. The porcine DGP backbone is the 81-amino acid segment of DSPP (Ser392 to Gly472) between the DSP and DPP domains. DGP has four phosphorylated serine residues (Ser453, Ser455, Ser457, and Ser462) and one glycosylated asparagine (Asn397). There are no other post-translational modifications. DGP is a stains-all positive protein with an apparent molecular mass on SDS-PAGE of 19 kDa, which is reduced by glycopeptidase A digestion to 16 kDa. A variety of glycans can be linked to Asn397. All are complex biantennary structures with a common N-linked pentasaccharide core (mannose3-N-acetylglucosamine2), most with a fucosyl residue on the innermost N-acetylglucosamine. The alpha1-3 and alpha1-6 arms are always galactose beta1-4 N-acetylglucosamine beta1-2 mannose, and either or both arms can be unsialidated or monosialidated. The calculated monoisotopic molecular masses of the different glycosylated forms of the DGP phosphoprotein are: unsialidated 10,523 and 10,670, monosialidated 10,815 and 10,961, and disialidated 11,106, and 11,252 Da, with the disialidated forms being the most abundant.  相似文献   
4.
Dentin sialophosphoprotein (DSPP) is critical for proper mineralization of tooth dentin, and mutations in DSPP cause inherited dentin defects. Dentin phosphoprotein (DPP) is the C-terminal cleavage product of DSPP that binds collagen and induces intrafibrillar mineralization. We isolated DPP from individual pigs and determined that its N-terminal and C-terminal domains are glycosylated and that DPP averages 155 phosphates per molecule. Porcine DPP is unstable at low pH and high temperatures, and complexing with collagen improves its stability. Surprisingly, we observed DPP size variations on SDS-PAGE for DPP isolated from individual pigs. These variations are not caused by differences in proteolytic processing or degrees of phosphorylation or glycosylation, but rather to allelic variations in Dspp. Characterization of the DPP coding region identified 4 allelic variants. Among the 4 alleles, 27 sequence variations were identified, including 16 length polymorphisms ranging from 3 to 63 nucleotides. None of the length variations shifted the reading frame, and all localized to the highly redundant region of the DPP code. The 4 alleles encode DPP domains having 551, 575, 589, or 594 amino acids and completely explain the DPP size variations. DPP length variations are polymorphic and are not associated with dentin defects.  相似文献   
5.
The assignment of 1H and 13C NMR signals of a complex type triantennary asialooligosaccharide was examined using 2D selective-TOCSY–DQFCOSY and HSQC–TOCSY experiments. The 2D selective-TOCSY–DQFCOSY experiment exhibits a 2D DQFCOSY spectrum of an individual monosaccharide in the undecasaccharide, although the NMR signals of several monosaccharides in the triantennary undecasaccharide are heavily overlapped. Selective excitation of each anomeric proton signal and subsequent TOCSY experiment afforded transverse magnetization corresponding to all of the proton signals of the monosaccharide. This magnetization was then developed with the corresponding DQFCOSY pulse sequence to afford the DQFCOSY spectrum of the individual monosugars. In this case, four GlcNAc-b, -e, -j, and -h residues were excited as a mixture. In order to assign 13C signals, a conventional 2D HSQC–TOCSY spectrum was examined and compared with an unambiguous assignment of 2D selective-TOCSY–DQFCOSY thus obtained. This systematic analysis made it possible to obtain an assignment of the 1H and 13C NMR signals of the triantennary undecasaccharide. In addition, these experiments also revealed all of the glycosyl positions in the triantennary undecasaccharide.  相似文献   
6.
7.
Methionine γ‐lyse (MGL) catalyzes the α, γ‐elimination of l ‐methionine and its derivatives as well as the α, β‐elimination of l ‐cysteine and its derivatives to produce α‐keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site‐directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51–Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5′‐phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate‐assisted catalysis.  相似文献   
8.
Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.  相似文献   
9.
Mutations in parkin gene are responsible for autosomal recessive Parkinson’s disease (ARPD) and its loss-of-function is assumed to affect parkin ubiquitin ligase activity. Accumulation of its substrate may induce dopaminergic neurodegeneration in the substantia nigra (SN) of ARPD. Here, we show that parkin interacts with programmed cell death-2 isoform 1 (PDCD2-1) and promotes its ubiquitination. Furthermore, accumulation of PDCD2-1 was found in the SN of ARPD as well as in sporadic PD, suggesting that common failure of the ubiquitin-proteasome system is associated with neuronal death in both ARPD and sporadic PD.Structured summary:MINT-6805975, MINT-6806032, MINT-6806051, MINT-6806070:PDCD2 (uniprotkb:Q16342) physically interacts (MI:0218) with Parkin (uniprotkb:O60260) by anti tag coimmunoprecipitation (MI:0007)MINT-6805947:Parkin (uniprotkb:O60260) physically interacts (MI:0218) with PDCD2 (uniprotkb:Q16342) by two hybrid (MI:0018)MINT-6806000: PDCD2 (uniprotkb:Q16342) physically interacts (MI:0218) with ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007).  相似文献   
10.
NADH-dependent enzyme reducing acetophenone derivatives with high stereoselectivities and wide substrate specificities from Geotrichum candidum NBRC 4597 was isolated, purified, characterized, and used for asymmetric synthesis. Through five-step purification including ammonium sulfate fractionation and a series of chromatographies, the enzyme was purified about 150-fold with a yield of 5.6%. The active enzyme has a molecular mass of 73 kDa determined by gel filtration chromatography, and the SDS-PAGE result reveals that the molecular size of the subunit is 36 kDa. These results indicate that the enzyme consists of a homodimer of a 36 kDa subunit. The acetophenone reductase exhibited the highest activity at 50°C and optimal pH at 5.5. The enzyme was the most stable at 40°C. No metal ions considerably activated the enzyme, and such metal ions as Cu2+, Cd2+, and Zn2+ strongly inhibited the activity of the enzyme. The V max and the apparent K m value of the reductase were 77.0 μmol/min per milligram of protein and 0.296 mM for acetophenone, respectively. The N-terminal and internal amino acid sequences were determined by peptide sequencer. Furthermore, the purified enzyme was used for asymmetric reduction of acetophenone, resulting in the formation of corresponding (S)-alcohol with 99% ee.  相似文献   
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