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51.
Rickettsia prowazekii accumulated radioactivity from [adenine-2,8-3H]NAD but not from [nicotinamide-4-3H]NAD, which demonstrated that NAD was not taken up intact. Extracellular NAD was hydrolyzed by rickettsiae with the products of hydrolysis, nicotinamide mononucleotide and AMP, appearing in the incubation medium in a time- and temperature-dependent manner. The particulate (membrane) fraction contained 90% of this NAD pyrophosphatase activity. Rickettsiae which had accumulated radiolabel after incubation with [adenine-2,8-3H]NAD were extracted, and the intracellular composition was analyzed by chromatography. The cells contained labeled AMP, ADP, ATP, and NAD. The NAD-derived intracellular AMP was transported via a pathway distinct from and in addition to the previously described AMP translocase. Exogenous AMP (1 mM) inhibited uptake of radioactivity from [adenine-2,8-3H]NAD and hydrolysis of extracellular NAD. AMP increased the percentage of intracellular radiolabel present as NAD. Nicotinamide mononucleotide was not taken up by the rickettsiae, did not inhibit hydrolysis of extracellular NAD, and was not a good inhibitor of the uptake of radiolabel from [adenine-2,8-3H]NAD. Neither AMP nor ATP (both of which are transported) could support the synthesis of intracellular NAD. The presence of intracellular [adenine-2,8-3H]NAD within an organism in which intact NAD could not be transported suggested the resynthesis from AMP of [adenine-2,8-3H]NAD at the locus of NAD hydrolysis and translocation. 相似文献
52.
53.
The sea urchin multicatalytic protease: purification, biochemical analysis, subcellular distribution, and relationship to snRNPs 总被引:4,自引:0,他引:4
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We have purified and extensively characterized a 19-S particle from sea urchin eggs. This particle is the sea urchin homologue of the "prosome", a particle originally identified in duck erythroblasts. We now show that these sea urchin prosomes contain multiple proteolytic activities. As shown for analogous particles from other cells, these particles hydrolyze synthetic substrates containing neutral hydrophobic or basic amino acids at the carboxy terminus of the synthetic peptides. They contain 16-20 small proteins ranging in molecular weight from 20,000 to 32,000. Peptide mapping shows that most of the polypeptides are unique, however, three exist in two isoelectric forms. We have investigated the possible function of the sea urchin multicatalytic proteases (MCPs) by determining their subcellular distribution, their relationship to egg snRNPs, and their possible role in translational repression. There are almost as many MCPs (2 x 10(8] as ribosomes (6.6 x 10(8] or mRNPs (1.8 x 10(7] per egg. This suggests that like ribosomes, the MCPs are stored in the egg for use during later development. We find that a substantial proportion of egg MCPs move into nuclei by the late blastula stage. Using a specific antibody against one of the sea urchin MCP proteins and antibodies against U1-U6, La, and Ro RNPs, we show that the sea urchin particle is distinct from these RNPs, although the anti-U1-U6 RNP antibody cross-reacts with a single MCP protein. In addition, the sea urchin MCP appears to be associated with a large structure in the cytoplasm of unfertilized eggs and is released under the same conditions that activate egg mRNPs in vitro. 相似文献
54.
K E Jaeger F J Adrian H E Meyer R E Hancock U K Winkler 《Biochimica et biophysica acta》1992,1120(3):315-321
Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) secreted by Pseudomonas aeruginosa PAC1R was purified from cell-free growth medium by preparative isoelectric focusing. After blotting the N-terminal amino acid sequence and the amino acid composition were determined and compared to P. fragi and P. cepacia lipases yielding significant homology between all three species. Additionally, a consensus sequence K-Y-P-i-v-l-V-H-G was identified residing at the N-terminus of Pseudomonas lipases and in the central part of Staphylococcus lipases. Treatment of lipase with the serine-specific inhibitor diethyl p-nitrophenyl phosphate caused a rapid and complete inhibition of enzyme activity indicating the presence of a serine at the catalytic site as expected from lipase consensus sequences. Upon charge-shift electrophoresis the electrophoretic mobility of purified lipase was shifted either anodally or cathodally in the presence of sodium deoxycholate and cetyltrimethylammoniumbromide, respectively. This result demonstrates that extracellular lipase of P. aeruginosa exhibits an amphiphilic character like intrinsic membrane proteins. 相似文献
55.
Purification, assay and kinetic features of HIV-1 proteinase 总被引:6,自引:0,他引:6
1) The aspartic proteinase of the human immunodeficiency virus type 1 (HIV-1) was purified from cultures of recombinant E. coli. The enzyme preparation is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. 2) A rapid assay procedure for the proteinase was established which makes use of the cleavage of a radiolabeled decapeptide and the separation of substrate and labeled product by ion-exchange resin. 3) Activity of the enzyme is optimal at an ionic strength of 2.5-3.5M; also, the inhibitor pepstatin is a more potent inhibitor at higher ionic strength. This can be attributed to a tighter binding of both substrate and inhibitor in high-salt buffer. 4) The Km value of the decapeptide substrate is independent of the pH in the range of 3.5-7.5, while kcat shows a bell-shaped curve with a maximum at pH 5.2. The shape of the curve can be attributed to pKa values of 4.2 and 6.2 of groups on the enzyme. Pepstatin inhibition is optimal below pH 5.5, but becomes weak above pH 6. 相似文献
56.
Giovanni Fontana Roberto De Bernardi Federico Ferro Anita Gemignani Maurizio Raiteri 《Journal of neurochemistry》1996,66(1):161-168
Abstract: l -Glutamate, NMDA, dl -α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin-like immunoreactivity (SRIF-LI) from primary cultures of rat hippocampal neurons. In Mg2+ -containing medium, the maximal effects (reached at ∼100 µ M ) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µ M (AMPA), 39 µ M (glutamate), 41 µ M (KA), and 70 µ M (NMDA). The metabotropic receptor agonist trans -1-aminocyclopentane-1,3-dicarboxylate did not affect SRIF-LI release. The release evoked by glutamate (100 µ M ) was abolished by 10 µ M dizocilpine (MK-801) plus 30 µ M 1-aminophenyl-4-methyl-7,8-methylenedioxy-5 H -2,3-benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK-801 but insensitive to GYKI 52466. The AMPA- and KA-evoked releases were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) or by GYKI 52466 but were insensitive to MK-801. The release of SRIF-LI elicited by all four agonists was Ca2+ dependent, whereas only the NMDA-evoked release was prevented by tetrodotoxin. Removal of Mg2+ caused increase of basal SRIF-LI release, an effect abolished by MK-801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved. 相似文献
57.
Secretogranin II: Relative Amounts and Processing to Secretoneurin in Various Rat Tissues 总被引:2,自引:0,他引:2
Bernd Leitner Reiner Fischer-Colbrie Gerhard Scherzer Hans Winkler 《Journal of neurochemistry》1996,66(3):1312-1317
Abstract: Secretoneurin is a 33-amino-acid peptide produced in vivo from secretogranin II. An antiserum raised against this peptide recognizes both the free peptide and its precursors. By HPLC and radioimmunoassay we characterized the immunoreactive molecules and determined the levels of immunoreactivity in various rat organs. In adrenal medulla and to a lesser degree in the anterior pituitary processing of secretogranin II to secretoneurin was very limited, whereas in all other organs studied (brain, intestine, endocrine pancreas, thyroid gland, and posterior pituitary) a high degree of processing was apparent. Thus, practically all of the immunoreactivity was present as free secretoneurin. This was also true for serum. When the total amount of secretoneurin immunoreactivity was calculated for the various organs, the largest pools in descending order were in the intestine, CNS, anterior pituitary, pancreas, and adrenal gland. This makes it likely that secretoneurin in serum is mainly derived from the intestine. The high degree of processing of secretogranin II in most organs is consistent with the concept that this protein acts as a precursor of a functional peptide, i.e., secretoneurin. 相似文献
58.
A specific subform of the human cytomegalovirus transactivator protein pUL69 is contained within the tegument of virus particles. 总被引:10,自引:10,他引:0
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The polypeptide encoded by the open reading frame UL69 of human cytomegalovirus (HCMV), which is homologous to the immediate-early regulator ICP27 of herpes simplex virus, has recently been identified as a transactivator protein that exerts a broad stimulatory effect on gene expression (M. Winkler, S. A. Rice, and T. Stamminger, J. Virol. 68:3943-3954, 1994). Here, we provide evidence that pUL69 is a phosphorylated tegument protein of HCMV. This finding could be demonstrated by Western blot (immunoblot) analyses with purified virions and a specific antiserum against pUL69. These experiments revealed that one phosphorylated subform of the three pUL69 polypeptides that are synthesized in infected fibroblast cells is contained within the HCMV virion. After the treatment of purified virions with detergents, pUL69 could not be detected within the membrane fraction, suggesting that it is either a capsid or a tegument protein. Its presence within dense bodies, however, shows that pUL69 is a constituent of the viral tegument. 相似文献
59.
Thomas Schnyder Peter Tittmann Hanspeter Winkler Heinz Gross Theo Wallimann 《Journal of structural biology》1995,114(3)
Octamers of mitochondrial creatine kinase (Mi-CK) wore modified with the thiol-specific reagents N-ethylmaleimide or the gold-coupled derivative, maleidoyl undecagold. The kinetics of inhibition of the Mi-CK catalysis was shown to be comparable for both reagents, suggesting that the large gold cluster complex is accessible to the reactive cysteines. SDS-PAGE analysis revealed that two of eight cysteines per Mi-CK monomer were labeled with maleidoyl undecagold with a similar affinity for the functional maleimide group. Gel exclusion chromatography of labeled molecules showed that the octameric structure of Mi-CK was preserved after thiol modification. Freeze-dried gold-labeled octamers visualized by electron microscopy under cryoconditions were enhanced in contrast and showed a well-preserved fourfold symmetry of the end-on view, Image analysis of gold-labeled Mi-CK exhibited an averaged end-on view with four strong contrast signals located at the periphery of the notamer, whereas the center of the molecule remained electron translucent. We conclude that the two cysteine residues per monomer labeled with maleidoyl undecagold are located at the octamer's perimeter and we discuss the possible role of these reactive cysteines in enzyme catalysis. 相似文献
60.
Dr. Federico Carbone Prof. Dr. Ruggero Matteucci Dr. Brian R. Rosen Prof. Dr. Antonio Russo 《Facies》1994,30(1):1-13
Summary From a study of two areas, Jesira and the Bajuni Archipelago, about 400 km apart, a general pattern can be established for
the Recent facies, together with the morphological and taxonomic features of the corals. Present day coral development is
characterized by true fringing reefs in the Bajuni Archipelago and by scattered patches and knolls in the Jesira area. The
coral fauna, consisting of 27 genera and 63 species so far (including all uncertainties, but not sight records), is rather
poor, though coral communities are locally well developed. These figures probably reflect incomplete study and sampling. Although
comparison with other areas may therefore be premature, a preliminary biogeographical analysis suggests that this fauna is
more closely related to that of the Red Sea than to East Africa and the Seychelles. This differs from other published biogeographical
work on Indian Ocean coral faunas, but further study of the corals in this and neighbouring areas of the Indian Ocean is needed
in order to resolve this apparent anomaly. 相似文献