共查询到20条相似文献,搜索用时 0 毫秒
1.
V M Hughes 《Stain technology》1986,61(4):201-214
A new lead-precipitation technique for demonstrating magnesium-activated actomyosin adenosine triphosphatase (ATPase) at physiological pH and electrolyte levels in fixed skeletal muscle sections is reported. This method is compared with standard acid- and alkali-denatured muscle stained for calcium myosin ATPase as well as calcium-formalin denatured and pyrophosphate-formalin denatured muscle also stained for calcium myosin ATPase. The technique was developed using hamster skeletal muscle; however, it has also been applied to human, rat, and cat muscle. The fiber-type staining intensities of the formalin-denatured magnesium actomyosin ATPase closely resemble those of the formalin-denatured calcium myosin ATPase in rodents, but intensities in Type 1 fibers are reversed relative to calcium myosin ATPase in human muscle. Cat muscle shows intermediate characteristics. 相似文献
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Price JA 《Journal of biochemical and biophysical methods》2007,70(3):441-444
Phospholipase A(2) is an important enzyme in various pathologies. Although fluorescent substrate assays for it have been recently developed, there is a need for an assay with inexpensive commercially available substrates, useful when samples interfered with fluorescent assays, that is nonisotopic, continuous, conducted at physiological pH, and in a 96 well format. A reaction using bromothymol blue was developed that meets all these requirements. 相似文献
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《Journal of biochemical and biophysical methods》2008,70(3):441-444
Phospholipase A2 is an important enzyme in various pathologies. Although fluorescent substrate assays for it have been recently developed, there is a need for an assay with inexpensive commercially available substrates, useful when samples interfered with fluorescent assays, that is nonisotopic, continuous, conducted at physiological pH, and in a 96 well format. A reaction using bromothymol blue was developed that meets all these requirements. 相似文献
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Ke Tang Jing Liu Zhuoshun Yang Huafeng Zhang Jingwei Ma Duyun Ye 《Biochemical and biophysical research communications》2010,400(3):432-436
The inflammation-resolving lipid mediator lipoxin A4 (LXA4), which is derived from arachidonic acid in the context of inflammation, can be generated physiologically in vivo. However, the mechanism of physiologic formation of LXA4 remains elusive. In this report, we provide evidence that platelet-derived microparticles contain lipoxygenase 12 (12-LO) protein and act as a mediator in transferring 12-LO to mast cells, leading to the production of LXA4 by mast cells. Absence of either leukotriene, the precursor for LXA4, in mast cells or 12-LO in microparticles abolished LXA4 production. Using a mouse model, we demonstrated that platelet-derived microparticles were taken up by peritoneal mast cells in vivo and triggered LXA4 production. We also found that similar to LXA4, platelet-derived microparticles attenuated LPS- or dextran sulfate sodium-induced inflammation by regulating inflammatory cytokines. Together, these data suggest a critical role of platetlet-derived microparticles as a signal mediator, at least in LXA4 production, resulting in significant immunoregulatory consequences. 相似文献
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The cellular pathway of sucrose transfer from the endosperm cavity to the starchy endosperm of developing grains of wheat (Triticum turgidum) has been elucidated. The modified aleurone and sub-aleurone cells exhibit a dense cytoplasm enriched in mitochondria and endoplasmic relicilium. Significantly, the sub-aleurone cells are characterized by secondary wall ingrowths. Numerous plasmodesmata interconnect all cells between the modified aleurone and starchy endosperm. The pro-tonophore carbonylcyanide-m-chlorophenyl hydrazone (CCCP) slowed [14C]sucrose uptake by grain tissue slices enriched in modified aleurone and sub-aleurone cells but had no effect on uptake by the starchy endosperm. The fluorescent weak acid sulphorhodamine G (SRG) was preferentially accumulated by the modified aleurone and sub-aleurone cells, and this uptake was sensitive to CCCP. The combined plasma membrane surface areas of the modified aleurone and sub-aleurone cells appeared to be sufficient to support the in vivo rates of sucrose transfer to the starchy endosperm. Plasmolysis of intact excised grain inhibited [14C]sucrose transfer from the endosperm cavity to the starchy endosperm. The sulphydryl group modifier p-chloromercuribenzenesulphonie acid (PCMBS) decreased [14C]sucrose uptake by the modified aleurone and sub-aleurone cells but had little effect on uptake by the starchy endosperm. In contrast, when PCMBS and [14C]sucrose were supplied to the endosperm cavity of intact excised grain, PCMBS slowed accumulation by all tissues equally. Estimates of potential sucrose fluxes through the interconnecting plasmodesmata were found to be within the published range. It is concluded that the bulk of sucrose is accumulated from the endosperm cavity by the modified aleurone and sub-aleurone cells and subsequently transferred through the symplast to the starchy endosperm. 相似文献
6.
A new xylanase from thermoacidophilic Alicyclobacillus sp. A4 with broad-range pH activity and pH stability 总被引:1,自引:0,他引:1
Yingguo Bai Jianshe Wang Zhifang Zhang Peilong Yang Pengjun Shi Huiying Luo Kun Meng Huoqing Huang Bin Yao 《Journal of industrial microbiology & biotechnology》2010,37(2):187-194
We have identified a highly pH-adaptable and stable xylanase (XynA4) from the thermoacidophilic Alicyclobacillus sp. A4, a strain that was isolated from a hot spring in Yunnan Province, China. The gene (xynA4) that encodes this xylanase was cloned, sequenced, and expressed in Escherichia coli. It encodes a 338-residue polypeptide with a calculated molecular mass of 42.5 kDa. The deduced amino acid sequence is most
similar to (53% identity) an endo-1,4-β-xylanase from Geobacillus stearothermophilus that belongs to family 10 of the glycoside hydrolases. Purified recombinant XynA4 exhibited maximum activity at 55°C and
pH 7.0, had broad pH adaptability (>40% activity at pH 3.8–9.4) and stability (retaining >80% activity after incubation at
pH 2.6–12.0 for 1 h at 37°C), and was highly thermostable (retaining >90% activity after incubation at 60°C for 1 h at pH
7.0). These properties make XynA4 promising for application in the paper industry. This is the first report that describes
cloning and expression of a xylanase gene from the genus Alicyclobacillus. 相似文献
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We have developed a new type of 2'-hydroxyl protecting group for the automated machine synthesis of RNA oligomers: a 2-hydroxyisophthalate formaldehyde acetal (HIFA). The unique feature of this protecting group is that, as the bis ester, it is relatively stable to the acidic conditions that are used for repeated removal of dimethoxytrityl groups during chain elongation, but the final deprotection step in alkali, which cleaves the chain from the support and removes the base and phosphate protecting groups, converts it to the bis carboxylate and this can be removed relatively rapidly by treatment with mild acid. Conversion of the bis ester to the bis carboxylic acid increases the rate of acid-catalyzed hydrolysis of the acetal by 42-fold at pH 1, and, possibly, by 1320-fold at pH 3. The bis ester is 112 times more stable than the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl group (Fpmp) towards hydrolysis at pH 1, while the bis acid is only 2.35 times more stable than Fpmp at pH 3. In synthesis of the dimers UpU and UpG, with a coupling time of 5 min, the dimethoxytrityl cation assay indicated coupling yields of > 98%. 相似文献
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K H R?hm 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1975,356(6):903-914
The hydrolysis of dipeptides by purified yeast dipeptidase (EC 3.4.13.?) shows marked deviations from Michaelis-Menten kinetics over a wide range of pH. Quite anomalous kinetics is observed between pH 6 and 7, indicating a drastic change in the enzyme's properties. A reasonable explanation is provided by the assumption of a conformational transition brought about either by pH shifts or, at a constant pH, by changes in the substrate concentration. The transition, which may have a half-life on the order of minutes under appropriate conditions, is a distinctly cooperative process, with a dependence on ligand concentration higher than first order. The two forms of the enzyme differ clearly from each other with respect to various properties. The magnitudes and pH dependence of the kinetic parameters as well as the type of inhibition (or activation) exerted by amino acids and other ligands are different, as are their heat stabilities and the rates of inactivation by photooxidation of proteolytic degradation. Neither the molecular weight nor the gross conformation of the enzyme changes during the transition, so it seems to be due to a local isomerization affecting mainly the geometry of the active site. The sensitivity of dipeptidase to changes in the concentrations of substrates and other ligands is most pronounced exactly at the values of pH known to prevail in the living yeast cell. Thus the observed effects, which modulate dipeptidase activities within wide and limits, according to the amounts of dipeptides and amino acids present, are likely to play a role in the regulation of the enzyme in vivo. 相似文献
11.
C Cerini V Peyrot C Garnier L Duplan S Veesler J P Le Caer J P Bernard H Bouteille R Michel A Vazi P Dupuy B Michel Y Berland J M Verdier 《The Journal of biological chemistry》1999,274(32):22266-22274
Lithostathine is a calcium carbonate crystal habit modifier. It is found precipitated under the form of fibrils in chronic calcifying pancreatitis or Alzheimer's disease. In order to gain better insight into the nature and the formation of fibrils, we have expressed and purified recombinant lithostathine. Analytical ultracentrifugation and quasi-elastic light scattering techniques were used to demonstrate that lithostathine remains essentially monomeric at acidic pH while it aggregates at physiological pH. Analysis of these aggregates by electron microscopy showed an apparently unorganized structure of numerous monomers which tend to precipitate forming regular unbranched fibrils. Aggregated forms seem to occur prior to the apparition of fibrils. In addition, we have demonstrated that these fibrils resulted from a proteolysis mechanism due to a specific cleavage of the Arg(11)-Ile(12) peptide bond. It is deduced that the NH(2)-terminal undecapeptide of lithostathine normally impedes fiber formation but not aggregation. A theoretical model explaining the formation of amyloid plaques in neurodegenerative diseases or stones in lithiasis starting from lithostathine is described. Therefore we propose that lithostathine, whose major function is unknown, defines a new class of molecules which is activated by proteolysis and is not involved in cytoskeleton nor intermediate filament functions. 相似文献
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Purification of a high-molecular-mass form of phospholipase A2 from rat kidney activated at physiological calcium concentrations. 总被引:10,自引:0,他引:10
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Rat kidney contains a soluble phospholipase A2 (PLA2), which is chromatographically identical with a previously identified hormonally regulated form of the enzyme in rat renal mesangial cells. This kidney enzyme has been purified by sequential column fractionation. The purified enzyme is a 110 kDa polypeptide which can hydrolyse arachidonoyl phosphatidylcholine and arachidonoyl phosphatidylethanolamine, but has low activity towards arachidonoyl phosphatidylinositol. The enzyme is considerably larger than most previously isolated forms of secretory or intracellular PLA2, and is stimulated by physiological concentrations of Ca2+, with half-maximal activation occurring at 500 nM-Ca2+. The hormonal regulation and Ca2(+)-dependency of this enzyme strongly suggest that it plays a role in hormonally regulated arachidonic acid release and prostaglandin production in the kidney. 相似文献
14.
《Comptes rendus biologies》2014,337(11):609-624
The biological information coming from electrophysiologic sensors like ECG, pulse sensor or from molecular signal devices like NMR spectrometry has to be visualized and manipulated in a compressed way for an efficient medical use by clinicians, if stored in scientific data bases or in personalized patient records repositories. Here, we define a new transform called Dynalet based on Liénard ordinary differential equations susceptible to model the mechanism at the source of the studied signal, and we propose to apply this new technique first to the modelling and compression of real biological periodic signals like ECG and pulse rhythm. We consider that the cardiovascular activity results from the summation of cellular oscillators located in the cardiac sinus node and we show that, as a result, the van der Pol oscillator (a particular Liénard system) fits well the ECG signal and the pulse signal. The reconstruction of the original signal (pulse or ECG) using Dynalet transform is then compared with that of Fourier, counting the number of parameters to be set for obtaining an expected signal-to-noise ratio. Then, we apply the Dynalet transform to the modelling and compression of molecular spectra obtained by protein NMR spectroscopy. The reconstruction of the original signal (peak) using Dynalet transform is again compared with that of Fourier. After reconstructing visually the peak, we propose to periodize the signal and give it to hear, the whole process being called the protein “stethoscope”. 相似文献
15.
Characterization of single and double inactivation strains reveals new physiological roles for group 2 sigma factors in the cyanobacterium Synechocystis sp. PCC 6803
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Pollari M Gunnelius L Tuominen I Ruotsalainen V Tyystjärvi E Salminen T Tyystjärvi T 《Plant physiology》2008,147(4):1994-2005
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M G Mainwaring S D Lugo R A Fingal O H Kapp S N Vinogradov 《The Journal of biological chemistry》1986,261(23):10899-10908
Lumbricus terrestris HbO2 and HbCO dissociated below pH 5.0; a time-dependent alteration to the met form occurred at pH less than 5 and pH less than 4.5, respectively. The extent of dissociation was unaffected by alkaline earth cations but was decreased by an increase in ionic strength. HbO2 and HbCO exposed to pH 4.0-4.8 were centrifuged to obtain the undissociated pellet (P1) and dissociated supernatant (S1) fractions. S1 was reassociated at pH 7.0 by dialysis against various buffers and then centrifuged to obtain the reassociated pellet (P2) and unreassociated supernatant (S2) fractions. Reassociation was possible only if S1 was dialyzed against water prior to return to neutral pH; otherwise precipitation occurred starting at about pH 5.3. The extent of reassociation varied from about 40 to 80%, was usually higher for HbCO than HbO2, and was unaffected by an increase in ionic strength or by Ca(II). Gel filtration of P2 on Sephacryl S-300 at neutral pH gave one peak IaR, eluting at a slightly greater volume than the native Hb; S1 and S2 gave in addition, three peaks, Ib (200 kDa), II (65 kDa), and III (18 kDa). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that P2 was slightly deficient in subunit M relative to the Hb, that Ib was deficient in subunits D1 and D2 and that II and III consisted of subunits D1 + D2 + T and subunit M, respectively. Scanning transmission electron microscopy of P2 showed that it was smaller than the native hemoglobin: 25 nm in diameter and 16 nm in height, instead of 30 X 20 nm. Comparison of the results of the dissociations of Lumbricus Hb at alkaline pH (Kapp, O. H., Polidori, G., Mainwaring, M., Crewe, A. V., Vinogradov, S. N. (1984) J. Biol. Chem. 259, 628-639) with those obtained in this study suggested that the Hb quaternary structure was not multimeric and that an alternative model had to be considered. In the proposed model it is assumed that subunits D1 and D2 form a scaffolding or bracelet, decorated with 12 complexes of M and T subunits. 相似文献
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A new set of Arabidopsis expressed sequence tags from developing seeds. The metabolic pathway from carbohydrates to seed oil 总被引:24,自引:0,他引:24
White JA Todd J Newman T Focks N Girke T de Ilárduya OM Jaworski JG Ohlrogge JB Benning C 《Plant physiology》2000,124(4):1582-1594
Large-scale single-pass sequencing of cDNAs from different plants has provided an extensive reservoir for the cloning of genes, the evaluation of tissue-specific gene expression, markers for map-based cloning, and the annotation of genomic sequences. Although as of January 2000 GenBank contained over 220,000 entries of expressed sequence tags (ESTs) from plants, most publicly available plant ESTs are derived from vegetative tissues and relatively few ESTs are specifically derived from developing seeds. However, important morphogenetic processes are exclusively associated with seed and embryo development and the metabolism of seeds is tailored toward the accumulation of economically valuable storage compounds such as oil. Here we describe a new set of ESTs from Arabidopsis, which has been derived from 5- to 13-d-old immature seeds. Close to 28,000 cDNAs have been screened by DNA/DNA hybridization and approximately 10,500 new Arabidopsis ESTs have been generated and analyzed using different bioinformatics tools. Approximately 40% of the ESTs currently have no match in dbEST, suggesting many represent mRNAs derived from genes that are specifically expressed in seeds. Although these data can be mined with many different biological questions in mind, this study emphasizes the import of photosynthate into developing embryos, its conversion into seed oil, and the regulation of this pathway. 相似文献
20.
Reaction of AMP with formaldehyde and 3-mercaptopropionic acid at pH 11.7 gave a new AMP derivative, N6-[(2-carboxyethyl)thiomethyl]-AMP (I) in 91% yield and reaction at pH 3.1 gave another new derivative, N6,N6-bis[(2-carboxyethyl)thiomethyl]-AMP (II) in 57% yield. The structures were determined by their 13C and 1H nuclear magnetic resonance spectra coupled with those of the simple analogues, N6-[(2-carboxyethyl)thiomethyl]-9-methyladenine (III) and N6,N6-bis[(2-carboxyethyl)thiomethyl]-9-methyladenine (IV) which were synthesized from 9-methyladenine in the same way as for derivatives I and II. ADP and ATP were treated in the same way as AMP to afford the corresponding carboxyl derivatives, N6-[(2-carboxyethyl)thiomethyl]-ADP (V), N6-[(2-carboxyethyl)thiomethyl]-ATP (VI), N6,N6-bis[(2-carboxyethyl)thiomethyl]-ADP (X) and N6,N6-bis[(2-carboxyethyl)thiomethyl]-ATP (XI) in 71%, 75%, 53% and 40% yield, respectively. These compounds were coupled to 1,3-diaminopropane with a water-soluble carbodiimide to give the corresponding amino derivatives, N6-([N-3-aminopropyl)carbamoylethyl]thiomethyl)-ADP (VIII), N6-(N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ATP (IX), N6,N6-bis([N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ADP (XIII), and N6,N6-bis([N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ATP (XIV), which were further bound to CNBr-activated dextran to give new polymer-bound derivatives of ADP and ATP. These free and bo-nd derivatives were tested for their coenzymic activities against several kinases. The activities of the ADP derivatives, V, VIII, X, XIII, dextran-bound VIII, and dextran-bound XIII against acetate kinase were 82%, 81%, 68%, 55%, 35%, and 15%, respectively, relative to ADP and those of the ATP derivatives, VI, IX, XI, XIV, dextran-bound IX, and dextran-bound XIV against hexokinase were 88%, 94%, 60%, 81%, 58%, and 49%, respectively, relative to ATP. 相似文献