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21.
Ouabain-binding site of (Na+ + K+)-ATPase in right-side-out vesicles has not an externally accessible SH group 总被引:1,自引:0,他引:1
G Scheiner-Bobis M Zimmermann U Kirch W Schoner 《European journal of biochemistry》1987,165(3):653-656
The fluorescing sulfhydryl reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) inactivates purified (Na+ + K+)-ATPase at 20 microM. This inactivation results in a decrease of the ouabain-binding capacity of the enzyme. Treatment of (Na+ + K+)-ATPase, embedded in right-side-out-oriented vesicles, by DACM does not affect ouabain binding to the enzyme. Incorporation of DACM into the alpha subunit of (Na+ + K+)-ATPase embedded in right-side-out vesicles is also not affected by the presence or absence of 100 microM ouabain. It is therefore concluded that a sulfhydryl group does not reside within the ouabain-binding site of (Na+ + K+)-ATPase. 相似文献
22.
Rudolf Geyer Silvia Diabaté Hildegard Geyer Hans-Dieter Klenk Heiner Niemann Stephan Stirm 《Glycoconjugate journal》1987,4(1):17-32
Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-3H]mannose, or withd-[6-3H]glucosamine, and the small subunit (HA2; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA
bovine serum albumin
- endo D (F,H)
endo--N-acetyl-d-glucosaminidase D (F,H)
- HA
hemagglutinin (HA1, large subunit of HA
- HA2
small subunit
- FPV
fowl plague virus
- PNGase F
peptide:N-glycosidase F
- SDS
sodium dodecylsulfate 相似文献
23.
S G Whittaker F K Zimmermann B Dicus W W Piegorsch S Fogel M A Resnick 《Mutation research》1989,224(1):31-78
The diploid yeast strain D61.M was used to study induction of mitotic chromosome loss. The test relies upon the uncovering and expression of multiple recessive markers reflecting the presumptive loss of the chromosome VII homologue carrying the corresponding wild-type alleles. The underlying 'loss event' is probably complex since the predicted centromere-linked lethal tetrad segregations for chromosome VII are not recovered. Instead, the homologue bearing the multiple recessive markers is patently homozygous. An interlaboratory study was performed in which 16 chemicals were tested under code in 2 laboratories. The results generated by the Berkeley and Darmstadt laboratories were in close agreement. Acetonitrile, ethyl acetate, 4-acetylpyridine, propionitrile and nocodazole were identified as potent inducers of mitotic chromosome loss. Acetone, dimethyl sulfoxide and 2-methoxyethyl acetate either elicited weak responses or yielded ambiguous results. Water, carbon tetrachloride, 4-fluoro-D,L-phenylalanine, amphotericin B, griseofulvin, cadmium chloride, ethyl methanesulfonate and methylmercury(II) chloride failed to induce chromosome loss. These data suggest that the system described herein represents a reliable assay for chemically induced chromosome loss in yeast. 相似文献
24.
Differences in water binding were measured in the leaf cells ofMesembryanthemum crystallinum L. plants grown under high-salinity conditions by using nuclear-magnetic-resonance (NMR) imaging. The 7-Tesla proton NMR imaging system yielded a spatial resolution of 20·20·100 m3. Images recorded with different spin-echo times (4.4 ms to 18 ms) showed that the water concentrations in the bladder cells (located on the upper and lower leaf surface), in the mesophyll cells and in the water-conducting vessels were nearly identical. All of the water in the bladder cells and in the water-conducting vessels was found to be mobile, whilst part of the water in the mesophyll cells was bound. Patches of mesophyll cells could be identified which bound water more strongly than the surrounding mesophyll cells. Optical investigations of leaf cross-sections revealed two types of mesophyll cells of different sizes and chloroplast contents. It is therefore likely that in the small-sized mesophyll cells water is strongly bound. A long-term asymmetric water exchange between the mesophyll cells and the bladder cells during Crassulacean acid metabolism has been described in the literature. The high density of these mesophyll cells in the lower epidermis is a possible cause of this asymmetry.Abbreviations CAM
Crassulacean acid metabolism
- NMR
nuclear magnetic resonance
- TE
spin-echo time 相似文献
25.
Role of phospholipase A2 in the stimulation of sponge cell proliferation by homologous lectin 总被引:2,自引:0,他引:2
M Gramzow H C Schr?der U Fritsche B Kurelec A Robitzki H Zimmermann K Friese M H Kreuter W E Müller 《Cell》1989,59(5):939-948
Using the Geodia cydonium system, we showed that after incubation of competent sponge cells in the presence of lectin, phospholipase A2 was released from the cells. The substrates for this enzyme, phosphatidylethanolamine and phosphatidylcholine, were identified in the extracellular material of sponge tissue. In addition, the phospholipase A2 inhibitor calelectrin was identified by immunobiochemical techniques; this molecule was associated with the aggregation factor. Reconstitution experiments strongly suggested that phospholipase A2 catalyzed the release of arachidonic acid, which is then taken up by the cells. Intracellularly, arachidonic acid was metabolized primarily to prostaglandin E2. Inhibition studies revealed that prostaglandin E2 is involved in the ultimate increase of DNA synthesis. These findings suggest that the phospholipase A2-arachidonic acid system is involved in the matrix-initiated signal transduction pathway in sponges. 相似文献
26.
Regulated expression of nuclear protein(s) in myogenic cells that binds to a conserved 3'' untranslated region in pro alpha 1 (I) collagen cDNA. 总被引:5,自引:1,他引:4
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T Herget M Burba M Schmoll K Zimmermann A Starzinski-Powitz 《Molecular and cellular biology》1989,9(7):2828-2836
We describe the identification and DNA-binding properties of nuclear proteins from rat L6 myoblasts which recognize an interspecies conserved 3' untranslated segment of pro alpha 1 (I) collagen cDNA. Levels of the two pro alpha 1 (I) collagen RNAs, present in L6 myoblasts, decreased drastically between 54 and 75 h after induction of myotube formation in serum-free medium. Both mRNAs contained a conserved sequence segment of 135 nucleotides (termed tame sequence) in the 3' untranslated region that had 96% homology to the human and murine pro alpha 1 (I) collagen genes. The cDNA of this tame sequence was specifically recognized by nuclear protein(s) from L6 myoblasts, as judged by gel retardation assays and DNase I footprints. The tame-binding protein(s) was able to recognize its target sequence on double-stranded DNA but bound also to the appropriate single-stranded oligonucleotide. Protein that bound to the tame sequence was undetectable in nuclear extracts of L6 myotubes that did not accumulate the two collagen mRNAs. Therefore, the activity of this nuclear protein seems to be linked to accumulation of the sequences that it recognizes in vitro. The collagen RNAs and the nuclear tame-binding proteins reappeared after a change of medium, which further suggests that the RNAs and the protein(s) are coordinately regulated. 相似文献
27.
28.
R Friolet J P Colombo F Lazeyras W P Aue R Kretschmer A Zimmermann C Bachmann 《Biochemical and biophysical research communications》1989,159(2):815-820
Hyperammonemia is a major contributing factor to the neurological abnormalities observed in hepatic encephalopathy and in congenital defects of ammonia detoxication. In rats variable changes in labile energy rich phosphates in the brain have been observed in hyperammonemia using biochemical methods. Using 31P-NMR spectroscopy however no significant changes of the relative concentrations of the energy rich phosphates alpha, beta and gamma-ATP, phosphocreatine, inorganic phosphate and the pH were found in the fronto parietal cortex of the urease treated hyperammonemic rat. Alterations in the metabolites of these compounds do not appear to be a major pathomechanism of ammonia toxicity in this brain area. 相似文献
29.
Photochemical labeling of bovine pancreatic ribonuclease A with 8-azidoadenosine 3',5'-bisphosphate 总被引:1,自引:0,他引:1
A simple method has been developed for the preparation of 5'-32P-labeled 8-azidoadenosine 3',5'-bisphosphate (p8N3Ap) for use in photoaffinity labeling studies. Irradiation of a complex between p8N3Ap and bovine pancreatic ribonuclease A (RNase A) with light of 300-350 nm led to the covalent attachment of the nucleotide to the enzyme. RNase A could also be labeled in the dark with prephotolyzed p8N3Ap. In either case, the nucleotide reacted with the same tryptic peptide, encompassing amino acids 67-85 of the protein. The site of labeling was determined to be either Thr-78 or Thr-82, both of which are close to or at the pyrimidine binding site of the enzyme. This result is consistent with recent nuclear magnetic resonance and X-ray studies which indicate that 8-substituted adenine nucleotides interact with the pyrimidine binding site of RNase A. 相似文献
30.
The mannitol-specific enzyme II (mannitol permease) of the Escherichia coli phosphotransferase system (PTS) catalyzes the concomitant transport and phosphorylation of D-mannitol. Previous studies have shown that the mannitol permease (637 amino acid residues) consists of 2 structural domains of roughly equal size: an N-terminal, hydrophobic, membrane-bound domain and a C-terminal, hydrophilic, cytoplasmic domain. The C-terminal domain can be released from the membrane by mild proteolysis of everted membrane vesicles [Stephan, M.M., & Jacobson, G.R. (1986) Biochemistry 25, 8230-8234]. In this report, we show that phosphorylation of the intact permease by [32P]HPr (a general phosphocarrier protein of the PTS) followed by tryptic separation of the two domains resulted in labeling of only the C-terminal domain. Phosphorylation of the C-terminal domain occurred even in the complete absence of the N-terminal domain, showing that the former contains most, if not all, of the critical residues comprising the interaction site for phospho-HPr. The phosphorylated C-terminal domain, however, could not transfer its phospho group to mannitol, suggesting that the N-terminal domain is necessary for mannitol binding and/or phosphotransfer from the enzyme to the sugar. The elution profile of the C-terminal domain after molecular sieve chromatography showed that the isolated domain is monomeric, unlike the native permease which is likely a dimer in the membrane. Experiments employing a deletion mutation of the mtlA gene, which encodes a protein lacking the first phosphorylation site in the C-terminal domain (His-554) but retaining the second phosphorylation site (Cys-384), demonstrated that a phospho group could be transferred from phospho-HPr to Cys-384 of the deletion protein, and then to mannitol, only in the presence of the full-length permease.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献