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51.
Summary The pepN gene of Escherichia coli K-12 has been cloned onto a multi-copy plasmid and shown to encode a polypeptide which co-migrates with purified peptidase N. Transformed strains have been shown to contain up to a one hundred fold increase in the amount of peptidase N. We isolated the peptidase N protein and determined the sequence of its first 15 amino acids. By restriction mapping, we identified and subcloned the 5 region of the pepN gene and then determined its nucleotide sequence. Comparison of the actual amino acid sequence with that predicted from the extended open reading frame found in the DNA sequence indicated that peptidase N is not synthesized as a pre-protein precursor. The presumed region preceding the open reading frame contained nucleotide sequence having homology to the procaryotic promoter consensus sequences for the -35 and the -10 regions and the ribosome binding site. 相似文献
52.
It has recently been reported that insulin-degrading enzyme (IDE) contains an allosteric site which binds polyanions such as ATP and PPPi. This site is distinct from the catalytic site where homotrophic allosteric effects are produced. In this study, we have characterized the binding of ATP to this anion binding site using the fluorescent ATP analog 2',3'-O-(2,4,6-trinitrophenyl)-adenosine triphosphate (TNP-ATP), which exhibits a higher affinity to the enzyme than ATP itself. TNP-ATP binding to IDE was accompanied by a more than 4-fold increase in fluorescence. The dissociation constant (K(D)) of TNP-ATP was determined as 1.15 microM, while the activation constant (K(A)) was determined to be 1.6 microM. Competition experiments were used to show that ATP (Ki = 1.3 mM) and PPPi (Ki = 0.9mM) bind with a higher affinity than ADP (2.2 mM) and AMP (4.0 mM). Adenosine did not bind to the anion binding site. 相似文献
53.
Esther Pilla Ulrike M?ller Guido Sauer Francesca Mattiroli Frauke Melchior Ruth Geiss-Friedlander 《The Journal of biological chemistry》2012,287(53):44320-44329
Sumoylation affects many cellular processes by regulating the interactions of modified targets with downstream effectors. Here we identified the cytosolic dipeptidyl peptidase 9 (DPP9) as a SUMO1 interacting protein. Surprisingly, DPP9 binds to SUMO1 independent of the well known SUMO interacting motif, but instead interacts with a loop involving Glu67 of SUMO1. Intriguingly, DPP9 selectively associates with SUMO1 and not SUMO2, due to a more positive charge in the SUMO1-loop. We mapped the SUMO-binding site of DPP9 to an extended arm structure, predicted to directly flank the substrate entry site. Importantly, whereas mutants in the SUMO1-binding arm are less active compared with wild-type DPP9, SUMO1 stimulates DPP9 activity. Consistent with this, silencing of SUMO1 leads to a reduced cytosolic prolyl-peptidase activity. Taken together, these results suggest that SUMO1, or more likely, a sumoylated protein, acts as an allosteric regulator of DPP9. 相似文献
54.
Guillon-Munos A Oikonomopoulou K Michel N Smith CR Petit-Courty A Canepa S Reverdiau P Heuzé-Vourc'h N Diamandis EP Courty Y 《The Journal of biological chemistry》2011,286(29):25505-25518
Kallikrein-related peptidases (KLKs) are an emerging group of secreted serine proteases involved in several physiological and pathological processes. We used a degradomic approach to identify potential substrates of KLK12. MDA-MB-231 cells were treated either with KLK12 or vehicle control, and the proteome of the overlying medium was analyzed by mass spectrometry. CCN1 (cyr61, ctgf, nov) was among the proteins released by the KLK12-treated cells, suggesting that KLK12 might be responsible for the shedding of this protein from the cell surface. Fragmentation of CCN1 by KLK12 was further confirmed in vitro, and the main cleavage site was localized in the hinge region between the first and second half of the recombinant protein. KLK12 can target all six members of the CCN family at different proteolytic sites. Limited proteolysis of CCNs (cyr61, ctgf, nov) was also observed in the presence of other members of the KLK family, such as KLK1, KLK5, and KLK14, whereas KLK6, KLK11, and KLK13 were unable to fragment CCNs. Because KLK12 seems to have a role in angiogenesis, we investigated the relations between KLK12, CCNs, and several factors known to be involved in angiogenesis. Solid phase binding assays showed that fragmentation of CCN1 or CCN5 by KLK12 prevents VEGF(165) binding, whereas it also triggers the release of intact VEGF and BMP2 from the CCN complexes. The KLK12-mediated release of TGF-β1 and FGF-2, either as intact or truncated forms, was found to be concentration-dependent. These findings suggest that KLK12 may indirectly regulate the bioavailability and activity of several growth factors through processing of their CCN binding partners. 相似文献
55.
The pyrokinins (PK) are multifunctional neuropeptides found in a variety of arthropod species, including the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae). A series of biostable pyrokinin analogs based on the shared C-terminal pentapeptide core region were fed in solutions of artificial diet to the pea aphid over a period of three days and evaluated for antifeedant and aphicidal activity. The analogs contained either modified Pro residues Oic or Hyp and or a d-amino acid in key positions to enhance resistance to tissue-bound peptidases and retain activity in a number of PK bioassays. A series of PK analogs conjugated with two lengths of polyethyleneglycol (PEG) polymers were also evaluated in the aphid feeding assay. Three of the biostable PK analogs demonstrated potent antifeedant activity, with a marked reduction in honeydew formation and very high mortality after 1 day. In contrast, a number of unmodified, natural pyrokinins and several other analogs containing some of the same structural components that promote biostability were inactive. Two of the most active analogs, Oic analog PK-Oic-1 (FT[Oic]RL-NH(2)) and PEGylated analog PK-dF-PEG(8) [(P(8))-YF[dF]PRL-NH(2)], featured aphicidal activity calculated at LC(50)'s of 0.042nmol/μl [0.029μg/μl] (LT(50) of 1.0 day) and 0.126nmol/μl (LT(50) of 1.3 days), respectively, matching the potency of some commercially available aphicides. Notably, a PEGylated analog of a PK antagonist can block over 55% of the aphicidal effects of the potent PK agonist PK-Oic-1, suggesting that the aphicidal effects are mediated by a PK receptor. The mechanism of this activity has yet to be established, though the aphicidal activity of the biostable analogs may result from disruption of digestive processes by interfering with gut motility patterns, a process shown to be regulated by the PKs in other insects. The active PK analogs represent potential leads in the development of selective, environmentally friendly aphid pest control agents. 相似文献
56.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(3):151-169
AbstractPeptide boronic acid dipeptide compounds were analyzedfor their ability to inhibit recombinant human dipeptidylpeptidase IV (CD26, DPPIV). Rate constants for the peptide boronates are difficult to obtain because the active boronic acid dipeptide exists in equilibrium with a cyclic inactive species in aqueous solution. Rate constants were determined for the inhibition of DPPIV using several peptide boronates at different pH values. Val-boropro forms the most tightly bound complex with DPPIV; the first order half life for dissociation of the inactive enzyme/inhibitor complex at 23°C is approximately 27 days. 相似文献
57.
Dipeptidyl peptidase-IV expression and activity in human glomerular endothelial cells 总被引:2,自引:0,他引:2
Pala L Mannucci E Pezzatini A Ciani S Sardi J Raimondi L Ognibene A Cappadona A Vannelli BG Rotella CM 《Biochemical and biophysical research communications》2003,310(1):28-31
Glucagon-like peptide-1 (GLP-1), a meal-stimulated gastrointestinal insulinotropic hormone inactivated by dipeptidyl peptidase-IV (DPP-IV), is reduced in type 2 diabetic patients. The present study shows that 2-week exposure of human glomerular endothelial cells to high glucose (22 mM) determines a highly significant increase in DPP-IV activity and mRNA expression, which cannot be entirely accounted for by hyperosmolarity. On the other hand, incubation of purified DPP-IV in a buffer solution added with high glucose does not affect enzyme activity. These results suggest that high glucose increases expression and activity of DPP-IV, possibly contributing to GLP-1 reduction in type 2 diabetic patients. 相似文献
58.
Thirion J Wattiaux R Jadot M 《Biology of the cell / under the auspices of the European Cell Biology Organization》2003,95(2):99-105
We found a dipeptidyl aminopeptidase activity in the parasitic protozoan Giardia lamblia with properties similar to the lysosomal cathepsin C of rat-liver lysosomes. Subcellular fractionation of this parasite indicated that the cathepsin C activity is located in organelles not distinguishable from the ones containing acid phosphatase, a known marker enzyme of Giardia lysosome-like peripheral vesicles. Contrary to the rat lysosomal enzyme, Giardia cathepsin C behaved like a membrane protein. Moreover, the enzyme was not solubilized by Triton X-100 or Triton X-100/SDS at 0 degrees C but could be substantially solubilized by octylglucoside, Triton X-100 at 37 degrees C or by a pretreatment with the cholesterol complexing agent beta-cyclodextrin before the Triton/SDS treatment carried out at 0 degrees C. These observations suggest that binding/anchorage of this enzyme to membranes occurs in cholesterol-rich microdomains. 相似文献
59.
Singleton MR Taylor SJ Parrat JS Littlechild JA 《Extremophiles : life under extreme conditions》2000,4(5):297-303
The gene encoding pyrrolidone carboxyl peptidase (Pcp) has been cloned from the hyperthermophilic archaeon Thermococcus litoralis. The recombinant enzyme has been expressed in Escherichia coli, purified, and char-acterized. The T. litoralis Pcp demonstrates strong sequence homology to previously characterized bacterial Pcps. Some investigations have been carried
out on enzyme substrate specificity and stability.
Received: July 4, 2000 / Accepted: July 21, 2000 相似文献
60.
Besteiro S Williams RA Coombs GH Mottram JC 《International journal for parasitology》2007,37(10):1063-1075
Leishmania occurs in several developmental forms and thus undergoes complex cell differentiation events during its life-cycle. Those are required to allow the parasite to adapt to the different environmental conditions. The sequencing of the genome of L. major has facilitated the identification of the parasite's vast arsenal of proteolytic enzymes, a few of which have already been carefully studied and found to be important for the development and virulence of the parasite. This review focuses on these peptidases and their role in the cellular differentiation of Leishmania through their key involvement in a variety of degradative pathways in the lysosomal and autophagy networks. 相似文献