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排序方式: 共有274条查询结果,搜索用时 15 毫秒
1.
3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1-14C]-3-Chloropropionyl-CoA binds covalently to enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [14C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue. 相似文献
2.
Amino acid sequence of an active site peptide of avian liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 总被引:3,自引:0,他引:3
Hydroxymethylglutaryl-CoA synthase is irreversibly inhibited by the active site-directed inhibitor 3-chloropropionyl-CoA. Enzyme modification has been postulated to involve alkylation of an active site cysteinyl sulfhydryl group. DEAE-Sephadex chromatography of tryptic digests prepared from enzyme inactivated using chloro[14C]propionyl-CoA suggested that bound radioactivity is localized on one peptide. Specificity of the modification was further demonstrated by reverse-phase high pressure liquid chromatography, which was used to isolate the radioactively labeled peptide in a chemically homogeneous form. Automated gas-phase Edman degradation techniques have been employed to confirm the assignment of cysteine as the inhibitor's target residue and to elucidate the sequence of amino acids which flank the 14C-carboxyethylated cysteine: Glu-Ser-Gly-Asn-Thr-Asp-Val-Glu-Gly-Ile-Asp-Thr-(Thr)- Asn-Ala-S-[14C]carboxyethylcysteine-Tyr-Gly-Gln-Thr-(Ala). These data represent the first assignment of active site structure for hydroxymethyl-glutaryl-CoA synthase. 相似文献
3.
Christian S. Lobsiger Josef P. Magyar Verdon Taylor Philip Wulf Andrew A. Welcher Amgen EST Program Ueli Suter 《Genomics》1996,36(3):379
The PMP22/EMP/MP20 gene family includes four closely related proteins, peripheral myelin protein-22 (PMP22), epithelial membrane protein-1 (EMP-1), epithelial membrane protein-2 (EMP-2), and epithelial membrane protein-3 (EMP-3), which share amino acid identities ranging from 33 to 43%. In addition, the lens-specific membrane protein MP20 represents a more distant relative. Functionally, this family of proteins is likely to play important roles in the control of cell proliferation, cell differentiation, and cell death. In particular, mutations affecting thePMP22gene are responsible for various hereditary peripheral neuropathies in humans and mice. We report the isolation and characterization of a mouse EMP-1 cDNA and the correspondingemp-1gene. Mouse EMP-1 displays 93% amino acid identity to rat EMP-1 and 39% identity to mouse PMP22. The cDNA-predicted EMP-1 protein contains four putative membrane-associated domains and can beN-linked glycosylatedin vitro.EMP-1 is encoded by a single-copy gene with the positions of introns exactly conserved betweenemp-1andPMP22,corroborating the hypothesis that both genes belong to the same family. Computer-predicted structural domains of EMP-1 are partially mirrored by the exon/intron structure ofemp-1.Most interestingly, exon 4, which covers the potential second transmembrane domain, a small intracellular loop, and half of the third transmembrane domain, encodes the most highly conserved regions between the EMP-1 and PMP22 proteins and is also remarkably conserved in the MP20 gene, indicating some shared functional significance for this module in the PMP22/EMP/MP20 family. 相似文献
4.
Characterization of the ribulosebisphosphate carboxylase-carbon dioxide-divalent cation-carboxypentitol bisphosphate complex 总被引:5,自引:0,他引:5
Ribulosebisphosphate carboxylase forms a stable quaternary complex with CO2, divalent cation, and carboxypentitol bisphosphate. Incorporation of nonexchangeable CO2 into the complex requires the presence of a divalent cation. MG2+, Mn2+, or Co2+ supports stoichiometric binding of CO2 activator. When the quaternary complex is formed in the presence of saturating CO2, stoichiometric amounts of cation are bound in a nonexchangeable fashion. Incorporation of Mn2+ into an enzyme-CO2-Mn2+-carboxypentitol bisphosphate complex permitted investigation of cation environment by electron spin resonance (ESR) techniques. Measurements at 9 and 35 GHz suggest rhombic distortion of the coordination sphere of bound Mn2+. A complex inner sphere liganding of the cation bound in the quaternary complex would account for both the ESR spectra and the marked stability of the complex with respect to cation exchange. 相似文献
5.
We have directly tested the ability of acetoacetate, upon activation to the CoA thioester, to channel into the cholesterogenic pathway prior to scrambling of its carbon skeleton with the acetate pool. The approach relies upon trapping [3-13C]acetoacetate-derived hydroxymethylglutaryl-CoA, hydrolyzing this metabolite, and esterifying the resulting hydroxymethylglutaric acid to allow gas chromatography/mass spectrometry analysis of the dimethyl esters for the 13C enrichment and labeling pattern. 99% enriched [3-13C] and [1,3,5-13C]hydroxymethylglutaric acid samples were synthesized, providing standards against which physiological samples could be compared. Cytosolic extracts from brain and liver of cholestyramine-fed rats were incubated with [3-13C]acetoacetate (2 mM) or with [1-13C]acetate (5 mM). In contrast to [13C]acetate-derived hydroxymethylglutarate, which shows the expected triple labeling pattern, [13C]acetoacetate-derived hydroxymethylglutarate from both liver and brain extracts is predominantly monolabeled. These data suggest that, after acetoacetate is activated to the CoA thioester, cytosolic hydroxymethylglutaryl-CoA synthase effectively commits much of this acetoacetyl-CoA to cholesterogenesis before thiolase can scramble the carbon skeleton of the acetoacetyl moiety into the acetate pool. This chemical approach represents an alternative method for testing the channeling of metabolites through sequential steps in a metabolic pathway. Such a method may be useful when physical or kinetic techniques prove to be unsuitable. 相似文献
6.
Spinach leaf phosphoribulokinase is sensitive to modification by ATP analogs that react with lysine residues. The 2',3'-dialdehyde derivative of ATP (oATP) inactivates enzyme in a slow, time-dependent fashion. The process follows first-order kinetics (kinact = 0.07 min-1), and the concentration dependence of inactivation indicates tight inhibitor binding (Ki = 106 microM). ATP offers good protection against inactivation (Kd = 67 microM), suggesting that oATP is directed toward the catalytic site. This conclusion is supported by the fact that oATP functions as an alternate substrate (Km = 0.55 mM). Inactivation of phosphoribulokinase by [14C]oATP results in a modification stoichiometry of 0.7/site. The 14C-labeled enzyme is stable to dialysis, suggesting that the covalent adduct formed between protein and oATP is not a simple Schiff's base. Adenosine di- and triphosphopyridoxals (Ado-P2-Pl and Ado-P3-Pl, respectively) also inhibit spinach phosphoribulokinase in a time-dependent fashion. In this case, activity loss is reversible unless the inhibited species is borohydride-reduced, suggesting that Ado-P2-Pl and Ado-P3-Pl form Schiff's bases with an amino group on the enzyme. Protection is afforded by the substrate ATP, suggesting that modification is active site-directed. Prolonged incubation of enzyme with these inhibitors does not result in complete inactivation of phosphoribulokinase. Residual activity is dependent on inhibitor concentration, as would be expected if equilibrium is established between the noncovalent E.I complex and the covalent (Schiff's base) E-I species. Kinetic data analysis indicates Ki values of 175 and 11 microM for Ado-P2-Pl and Ado-P3-Pl, respectively. Thus, the ATP-binding domain can easily accommodate the pyridoxal moiety which is tethered to the polyphosphate chain. The phosphorylated ATP analogs employed in this study exhibit substantially tighter binding to phosphoribulokinase than does fluorosulfonyl-benzoyladenosine (Ki = 4.8 mM), which we have previously demonstrated to be useful in selectively modifying the ATP-binding domain (Krieger, T. J., and Miziorko, H. M. (1986) Biochemistry 25, 3496-3501; Krieger, T. J., Mende-Mueller, L. M., and Miziorko, H. M. (1987) Biochim. Biophys. Acta 915, 112-119). Although the adduct formed between oATP and enzyme was unsuitable for structural analysis, borohydride reduction of the Schiff's base formed between enzyme and Ado-P3-[3H]Pl produced a species useful for investigation by protein chemistry techniques. A radiolabeled tryptic peptide was prepared, isolated, and sequenced; the data indicate that lysine 68 is the residue modified by Ado-P3-[3H]Pl. 相似文献
7.
L A Menahan W T Hron D G Hinkelman H M Miziorko 《European journal of biochemistry》1981,119(2):287-294
Kinetic and physical approaches have been employed to investigate the binding of acetoacetyl-CoA to hydroxymethylglutaryl-CoA synthase. The enzyme has an apparent Km for acetoacetyl-CoA (0.35 microM) which is more than an order of magnitude lower than the Ki (6--10 microM) measured for substrate inhibition by this metabolite. Hepatic acetoacetyl-CoA concentration, as measured by a sensitive and highly specific radioactive assay appears to be in the 1--10 microM range; the concentration decreases during diabetic ketoacidosis. Total hepatic activity of hydroxymethylglutaryl-CoA synthase and levels of mitochondrial enzyme protein, determined by radioimmunoassay, are not appreciably different in livers from control or ketoacidotic animals. In contrast to the decrease in hepatic acetoacetyl-CoA concentration observed during ketoacidosis, myocardial acetoacetyl-CoA levels are increased by at least tenfold when compared to controls. Elevated acetoacetyl-CoA levels may serve to inhibit fatty acid utilization by the heart. Thus, a consideration of the multiple interactions of acetoacetyl-CoA with the enzymes involved in ketone body production and utilization may be useful in evaluating the metabolic significance of this intermediate. 相似文献
8.
H M Miziorko D Shortle M D Lane 《Biochemical and biophysical research communications》1976,69(1):92-98
Carboxyamidomethylated J chain was shown to be an excellent substrate for the enzyme, pyrollidone carboxylyl peptidase, which specifically removed the cyclized amino terminal glutamyl residue. J chain lacking the “blocked” PCA group was subjected to automated Edman degradation and the amino terminal amino acid sequence determined as: PCA-Glu-Asp-Glu-Arg-Ile-Val-Leu-Val-Asp-Asn-Lys-CMCys-Lys-CMCys-Ala-Arg. Previous studies by others have identified a disulfide bridge between the heavy chain of immunoglobulins and a tripeptide identical in composition with the sequence at positions 15–17 in the J chain. These two sets of data locate the linkage of immunoglobulin heavy chain with Cys 15 of the J chain. 相似文献
9.
Nicky HGM Peters Inne HM Borel Rinkes Willem PTM Mali Maurice AAJ van den Bosch Remmert K Storm Peter W Plaisier Erwin de Boer Adriaan J van Overbeeke Petra HM Peeters 《Trials》2007,8(1):1-7
Background
In orthodontic treatment, anchorage control is a fundamental aspect. Usually conventional mechanism for orthodontic anchorage control can be either extraoral or intraoral that is headgear or intermaxillary elastics. Their use are combined with various side effects such as tipping of occlusal plane or undesirable movements of teeth. Especially in cases, where key-teeth are missing, conventional anchorage defined as tooth-borne anchorage will meet limitations. Therefore, the use of endosseous implants for anchorage purposes are increasingly used to achieve positional stability and maximum anchorage.Methods/Design
The intended study is designed as a prospective, multicenter randomized controlled trial (RCT), comparing and contrasting the effect of early loading of palatal implant therapy versus implant loading after 12 weeks post implantation using the new ortho-implant type II anchor system device (Orthosystem Straumann, Basel, Switzerland). 124 participants, mainly adult males or females, whose diagnoses require temporary stationary implant-based anchorage treatment will be randomized 1:1 to one of two treatment groups: group 1 will receive a loading of implant standard therapy after a healing period of 12 week (gold standard), whereas group 2 will receive an early loading of orthodontic implants within 1 week after implant insertion. Participants will be at least followed for 12 months after implant placement. The primary endpoint is to investigate the behavior of early loaded palatal implants in order to find out if shorter healing periods might be justified to accelerate active orthodontic treatment. Secondary outcomes will focus e.g. on achievement of orthodontic treatment goals and quantity of direct implant-bone interface of removed bone specimens. As tertiary objective, a histologic and microtomography evaluation of all retrieved implants will be performed to obtain data on the performance of the SLA surface in human bone evaluation of all retrieved implants. Additionally, resonance frequency analysis (RFA, Osstell? mentor) will be used at different times for clinically monitoring the implant stability and for histological comparison in order to measure the reliability of the resonance frequency measuring device.Trial registration
Current Controlled Trials ISRCTN97142521. 相似文献10.
Nikolaev S Montoya-Burgos JI Margulies EH;NISC Comparative Sequencing Program Rougemont J Nyffeler B Antonarakis SE 《PLoS genetics》2007,3(1):e2
Understanding the early evolution of placental mammals is one of the most challenging issues in mammalian phylogeny. Here, we addressed this question by using the sequence data of the ENCODE consortium, which include 1% of mammalian genomes in 18 species belonging to all main mammalian lineages. Phylogenetic reconstructions based on an unprecedented amount of coding sequences taken from 218 genes resulted in a highly supported tree placing the root of Placentalia between Afrotheria and Exafroplacentalia (Afrotheria hypothesis). This topology was validated by the phylogenetic analysis of a new class of genomic phylogenetic markers, the conserved noncoding sequences. Applying the tests of alternative topologies on the coding sequence dataset resulted in the rejection of the Atlantogenata hypothesis (Xenarthra grouping with Afrotheria), while this test rejected the second alternative scenario, the Epitheria hypothesis (Xenarthra at the base), when using the noncoding sequence dataset. Thus, the two datasets support the Afrotheria hypothesis; however, none can reject both of the remaining topological alternatives. 相似文献