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51.
gamma-Glutamyltranspeptidase purified from human kidneys contains 4-5 asparagine-linked sugar chains in each molecule. The sugar chains were released from the polypeptide portion of the enzyme by hydrazinolysis as oligosaccharides and separated by paper electrophoresis into one neutral and two acidic fractions. By sequential exoglycosidase digestion and methylation analysis, the neutral fraction, which comprised 69% of total oligosaccharides, was shown to be a mixture of bisected bi- and triantennary complex-type sugar chains with and without a fucose on the proximal N-acetylglucosamine residue and with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups in their outer chain moieties. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of bisected triantennary complex-type oligosaccharides with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc group in their outer chain moieties. Some of the outer chains of the acidic oligosaccharides were considered to be sialylated X-antigenic structures. 相似文献
52.
Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus. 总被引:44,自引:10,他引:34 下载免费PDF全文
M Matsuhashi M D Song F Ishino M Wachi M Doi M Inoue K Ubukata N Yamashita M Konno 《Journal of bacteriology》1986,167(3):975-980
A novel penicillin-binding protein, PBP-2' (Mr about 75,000), is known to be induced in excessively large amount by most beta-lactam compounds in cells of a clinically isolated strain of Staphylococcus aureus, TK784, that is highly resistant to beta-lactams and also most other antibiotics. This protein has very low affinities to most beta-lactam compounds and has been supposed to be the cause of the resistance of the cells to beta-lactams. A 14-kilobase DNA fragment was isolated from the cells that carried the gene encoding this penicillin-binding protein and also a genetically linked marker that is responsible for the resistance to tobramycin. This DNA was cloned on plasmid pACYC184 and was shown to cause both production of PBP-2' and resistance to tobramycin in Escherichia coli cells. However, the formation of PBP-2' in E. coli was only moderate and was independent of normal inducer beta-lactams. The PBP-2' formed in the E. coli cells showed slow kinetics of binding to beta-lactams similar to that of PBP-2' formed in the original S. aureus cells and gave a similar pattern of peptides to the latter when digested with the proteolytic V8 enzyme of S. aureus. 相似文献
53.
W Huang T Kimura K Mashima K Miyazaki H Masaki J Yamashita T Horio 《Journal of biochemistry》1986,100(3):687-696
We previously reported that sera from various kinds of animals contain a protein(s) capable of inhibiting the growth of the non-malignant epithelial cell line derived from Buffalo rat liver (BRL). In the present study, a similar epithelial cell-specific growth inhibitor (EGI) was purified to homogeneity from an acid-ethanol extract of human platelets. During purification, EGI was separated from the major component of type beta transforming growth factor (TGF-beta), which can stimulate the colony formation of the non-malignant fibroblastic cell line derived from rat kidney (NRK) in soft agar in the presence of epidermal growth factor (EGF). The purified EGI had an Mr of 27,000, and was composed of two subunits identical in Mr. It significantly inhibited the growth in monolayer cultures of three non-malignant epithelial cell lines, BRL, MDCK (from Madin-Darby canine kidney) and BSC-1 (from African green monkey kidney), at doses lower than 40 pg/ml in medium containing 10% fetal calf serum. Its inhibitory activity was stable against heating at 90 degrees C for 3 min, but not against treatment with 50 mM dithiothreitol. In addition, TGF-beta was also partially purified from the same extract. The purified TGF-beta did not show any inhibitory activity toward the growth of BRL, MDCK, BSC-1, or NRK. 相似文献
54.
Reactivity of a sulfhydryl group of the ras oncogene product p21 modulated by GTP binding 总被引:4,自引:0,他引:4
S Hattori T Yamashita T D Copeland S Oroszlan T Y Shih 《The Journal of biological chemistry》1986,261(31):14582-14586
We have studied the sensitivity of sulfhydryl groups of a highly purified p21 protein of the v-rasH oncogene to a thiol-specific reagent, N-ethylmaleimide (NEM). Approximately 70% of GTP binding and autokinase activities of p21 were inactivated by NEM, and excessive amounts of GTP or GDP protected p21 activities. Thiol titration revealed the presence of one fast reactive cysteine residue, the susceptibility of which is modulated by GTP binding. A total of 4 and 6 residues, respectively, became titratable upon denaturation and reduction, suggesting the presence of a disulfide bond. This GTP-modulated sulfhydryl group was identified as Cys-80 in the following tryptic peptide sequence: NH2-Thr-Gly-Glu-Gly-Phe-Leu-Cys-Val-Phe-Ala-Ile-Asn-Asn-Thr-Lys-COOH. This is based on the comparative tryptic peptide mapping of [14C]NEM-modified p21 in the presence and absence of GTP. The GTP-modulated peptide co-chromatographed with a synthetic peptide of the predicted sequence. Amino acid analysis of the purified [14C]NEM-modified peptide from tryptic digests of p21 also confirmed its identity. This region of p21 shares an extensive sequence homology with various G-proteins and appears to be in the vicinity of the GTP-binding domain of these proteins. 相似文献
55.
Possible involvement of aminopeptidase, an ecto-enzyme, in the inactivation of bradykinin by intact neutrophils 总被引:1,自引:0,他引:1
The subcellular localization of the bradykinin-inactivating activity was studied using guinea-pig neutrophils and the following results were obtained. The bradykinin-inactivating activities were found to be present in the cytosol and membrane fractions but not in the granular and nuclear fractions. The bradykinin-inactivating activity of the cytosol fraction was inhibited by N-carbobenzoxy-Gly-Pro, an inhibitor of prolyl endopeptidase, whereas that of the membrane fraction was inhibited by bestatin, an inhibitor of aminopeptidase. Prolyl endopeptidase and aminopeptidase activities were located predominantly in the cytosol and membrane fractions, respectively, and their activities were inhibited by their respective inhibitors. Prolyl endopeptidase and aminopeptidase activities measured with synthetic substrates were competitively inhibited by bradykinin, suggesting that bradykinin is a possible substrate for prolyl endopeptidase and aminopeptidase. Intact neutrophils inactivated bradykinin rapidly. However, when neutrophils were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent which inactivates ecto-enzymes selectively, both the bradykinin-inactivating activity and aminopeptidase activity of neutrophils decreased significantly without any inhibition of cytosol prolyl endopeptidase. The possibility that aminopeptidase, an ecto-enzyme, would be responsible for the inactivation of bradykinin by intact neutrophils was deduced from the results above, although both cytosol prolyl endopeptidase and membrane aminopeptidase could inactivate bradykinin. 相似文献
56.
K Yamashita A Hitoi N Tateishi T Higashi Y Sakamoto A Kobata 《Archives of biochemistry and biophysics》1985,240(2):573-582
Paper electrophoresis and Bio-Gel P-4 column chromatography of the oligosaccharides released from mouse kidney gamma-glutamyltranspeptidase by hydrazinolysis gave fractionation patterns quite distinct from those of the bovine and rat kidney enzymes. Structural studies of the fractionated oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis showed that mouse kidney gamma-glutamyltranspeptidase contains a series of bisected complex-type asparagine-linked sugar chains with the following oligosaccharides as their outer chain moieties: GlcNAc beta 1----, Sia alpha 2----Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, and sialylated N-acetyllactosamine repeating sugar chains. Some of these sugar chains were found for the first time in glycoproteins. 相似文献
57.
Internodal cells of Chara australis were subjected to two consecutiveintracellular perfusions with a Ca2+-free EGTA medium whichdisintegrated the tonoplast within about 10 minutes and thenwith a Ca2+-buffered medium. All perfusion media usually contained1 mM ATP. To stop the electrogenic pump, the internode was depletedof intracellular ATP. The excitability of the plasmalemma wasnot significantly influenced by intracellular free Ca2+ concentrationsup to 104 M. To trigger action potentials, minimum currentdensities of 1 to 2 µA cm2 had to be applied atall tested Ca2+ concentrations. In the absence of cytoplasmicATP, excitability was completely lost at all Ca2+ concentrations.
1 Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, D-5300 Bonn, FRG. (Received September 22, 1984; Accepted March 6, 1985) 相似文献
58.
Ionic Activity Gradients across the Surface Membrane of Cytoplasmic Droplets Prepared from Chara australis 总被引:1,自引:0,他引:1
The membrane potential and the ionic activity gradients of K+and Cl across the surface membrane of cytoplasmic dropletsprepared from Chara australis internodal cells, were measuredin high and low ionic strength bathing solutions using liquidion exchange microelectrodes selective for K+ and Cl.Our results indicate that K+ is close to electrochemical equilibriumwhereas Cl is not.
1 Present address: ICI Japan, Palace Hotel Annex Building, Marunouchi,Tokyo, Japan. 相似文献
59.
Polymorphic extracellular glucoamylase genes and their evolutionary origin in the yeast Saccharomyces diastaticus. 总被引:13,自引:6,他引:7 下载免费PDF全文
DNA coding for extracellular glucoamylase genes STA1 and STA3 was isolated from DNA libraries of two Saccharomyces diastaticus strains, each carrying STA1 or STA3. Cells transformed with a plasmid carrying either the STA1 or STA3 gene secreted glucoamylases having the same enzymatic and immunological properties and the same electrophoretic mobilities in acrylamide gel electrophoresis as those of authentic glucoamylases. Southern blot analysis of genomic DNA from S. diastaticus and a glucoamylase-non-secreting yeast, Saccharomyces cerevisiae, revealed that the STA1 and STA3 loci of S. diastaticus showed a high degree of homology, and that both yeast species (S. diastaticus and S. cerevisiae) contained DNA segments highly homologous to those of the extracellular glucoamylase genes. Restriction maps of the homologous DNA segments suggested that the extracellular glucoamylase genes of S. diastaticus may have arisen from recombination among the resident DNA segments in S. cerevisiae. 相似文献
60.
Nucleotide sequence of the extracellular glucoamylase gene STA1 in the yeast Saccharomyces diastaticus. 总被引:13,自引:6,他引:7 下载免费PDF全文
The complete nucleotide sequence of the extracellular glucoamylase gene STA1 from the yeast Saccharomyces diastaticus has been determined. A single open reading frame codes for a 778-amino-acid protein which contains 13 potential N-glycosylation sites. In the 5'- and 3'-flanking regions of the gene, there are striking sequence homologies to the corresponding regions of ADH1 for alcohol dehydrogenase and MAT alpha 2 for mating type control in the yeast Saccharomyces cerevisiae. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. The presumptive signal sequence showed a significant homology to that of Bacillus subtilis alpha-amylase precursor. The next segment, of ca. 320 amino acids, contains a threonine-rich tract in which direct repeat sequences of 35 amino acids exist, and is bordered by a pair of basic amino acid residues (Lys-Lys) which may be a proteolytic processing signal. The carboxy-terminal half of the precursor is a presumptive glucoamylase which contains several peptide segments showing a high degree of homology with alpha-amylases from widely diverse organisms including a procaryote (B. subtilis) and eucaryotes (Aspergillus oryzae and mouse). Analysis of both the nucleotide sequence of the STA1 gene and the amino acid composition of the purified glucoamylase suggested that the putative precursor is processed to yield subunits H and Y of mature enzyme by both trypsin-like and chymotrypsin-like cleavages. 相似文献