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141.
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Human plasma platelet-activating factor acetylhydrolase. Purification and properties 总被引:14,自引:0,他引:14
Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid synthesized by a variety of cell types upon appropriate stimulation. PAF is a potent hypotensive factor and it activates platelets and inflammatory cells at concentrations as low as 10(-10) M. Removal of the acetyl moiety at the sn-2 position abolishes the biological activity and this reaction is catalyzed by a specific acetylhydrolase present in plasma and animal tissues. Ultracentrifugation in density gradients showed that 30% of the activity is associated with high density lipoproteins and 70% with low density lipoproteins. We have purified the plasma low density lipoprotein-associated activity to near homogeneity using a rapid assay based on the separation of [3H]acetate from 1-O-alkyl-2-[3H]acetyl-sn-glycerol-3-phosphocholine on disposable reversed-phase columns. The enzyme was purified by 25,000-fold and approximately 10% of the starting activity was recovered. Plasma PAF-acetylhydrolase has an apparent molecular weight of 43,000, does not require calcium, has preference for micellar versus monomeric substrate, and exhibits surface dilution kinetics. The purified protein has an apparent Km of 13.7 microM and a Vmax of 568 mumol/h/mg with micellar PAF. It can act both on 1-O-alkyl and 1-acyl substrates and on ethanolamine analogs of PAF. However, the enzyme has a marked preference for the sn-2 acetyl residue and therefore can be considered as a specific PAF-acetylhydrolase. 相似文献
144.
Characterization of three group A klebicin plasmids: localization of their E colicin immunity genes 总被引:3,自引:0,他引:3
We have investigated the immunity to E colicins conferred by three group A klebicin plasmids. pP5a, which encodes klebicin A1-P5, like pClo-DF13, confers immunity to colicin E6 on Escherichia coli K12, whilst pP5b and pP3, which encode klebicins A2-P5 and A3-P3 respectively, both confer immunity to colicin E3. We have determined the restriction endonuclease and functional maps of the three group A klebicin plasmids. By sub-cloning and transposon mutagenesis we have investigated the relationship between the klebicin immunity and the E colicin immunity conferred by these plasmids. The colicin E6 and the klebicin A1 immunity are encoded by a single gene present on pP5a. The colicin E3 and the klebicin A2 immunity are encoded by a single gene present on pP5b. The colicin E3 and the klebicin A3 immunity are encoded by separate genes present on pP3. Recombinant pML8412, which is derived from the ColE6-CT14 plasmid and encodes colicin E6 immunity, confers klebicin A1-P5 immunity upon Klebsiella pneumoniae UNF5023. Recombinant pKC23, which is derived from the ColE3-CA38 plasmid and confers colicin E3 immunity, confers immunity to klebicin A2-P5, but not to klebicin A3-P3. 相似文献
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Boudreau James C.; Do Ly Thi; Sivakumar Latha; Oravec Joseph; Rodriguez Carlos A. 《Chemical senses》1987,12(3):437-458
Single unit recordings were taken from sensory ganglion cellsin the petrosal ganglion (PG) of the glossopharyngeal nerveof the rat. These taste units were examined with respect tospontaneous and evoked discharge patterns and responsivenessto a wide variety of chemical compounds, most of natural occurrence.Spontaneous activity patterns, with few exceptions, tended tobe extremely irregular with both bursting (clusters of 23spikes) and grouping (large groups of spikes as in evoked discharges).Most interspike interval histograms of spontaneous activitywere multimodal, similar to rat geniculate ganglion (GG) units.Evoked discharges usually displayed grouping of spikes, andlong latencies of onset and persistence of discharge after rinsewere sometimes seen. Little response was shown to nucleotidesor salts. Units responsive to amino acids tended to show largedischarge to only one or two amino acids; and the most responsiveamino acid usually varied from cell to cell. Units responsiveto alkaloids only responded to a few alkaloids with atropineand quinine being the most stimulatory. Units responsive toacids only discharged to a few of the acids tested and oftenacids of low pH elicited no discharge. Saccharin activated unitsresponsive to both sugar and alkaloids. A few units highly responsiveto both sugar and alkaloids were seen. The units were placedinto four clusters on the basis of chemicals activating themand certain neurophysiological characteristics: PG salt units,PG acid units and, tentatively, amino acid (sugar) units andX (alkaloid and alkaloid plus) units. The PG salt units didnot show the exclusive sensitivity to sodium and lithium compoundsas did the GG salt units. The PG acid units could also be differentiatedfrom the GG acid units. The petrosal amino acid and X units,on the other hand, could not be differentiated from similarunits in the rat GG. 相似文献
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James Howie 《BMJ (Clinical research ed.)》1987,294(6578):1017-1018
150.
Complexes (2:1) of chymotrypsin with human alpha 2-macroglobulin have been prepared in the presence of 200 mM methylamine such that 90% of the chymotrypsin remains noncovalently bound to the alpha 2-macroglobulin. Reaction of this complex with the active-site-directed spin-labeling reagent 4-[(ethoxyfluorophosphinyl)oxy]-2,2,6,6-tetramethylpiperidinyl+ ++-1-oxy results in nitroxide labeling of the active-site serine residue of the complexed chymotrypsin. Electron spin resonance (ESR) spectra of this complex were recorded at 275 K in buffer and at 263 K in 50% glycerol. At 263 K in 50% glycerol the spectrum is that expected for a rigid glass, whereas at room temperature the ESR spectrum shows that the chymotrypsin is only slightly immobilized compared with free spin-labeled chymotrypsin. These findings are discussed in relation to possible models of inhibition of protease activity by alpha 2-macroglobulin. It is concluded that the trap mechanism of Barrett and Starkey [Barrett, A. J., & Starkey, P. M. (1973) Biochem. J. 133, 709-724] is the only model currently considered that can account for the present findings. 相似文献