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11.
Plasminogen activator secreted by lymphosarcoma (ascites) of mice was purified up to 163-fold by ammonium sulphate fractionation
at 35% saturation and chromatography on p-aminobenzamidine-Sepharose 4B. The purified activator contained specific activity
of 9980 IU/mg. The plasminogen activator displayed homogeneity by polyacrylamide slab gel electrophoresis and high performance
liquid chromatography. The activator consisted of a single polypeptide chain with an apparent molecular weight of 66,000 daltons
as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions as well as gel filtration
on Sephadex G-100. Distinct differences between this activator and urokinase were discernible in respect of specific activities,
fibrin affinity and immunochemical properties. The lymphosarcoma activator appears to be of tissue-type origin since it showed
gross similarity to standard tissue plasminogen activator in terms of modes of binding to fibrin and immunological attributes. 相似文献
12.
Sreedhara Sangadala Subramanian Sivakami Joseph Mendicino 《Molecular and cellular biochemistry》1991,101(2):125-143
Summary Two specific -N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3GalNAc-Mucin to yield Gal3(GlcNAc6)GalNAc-Mucin and a 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3(GlcNAC6)GalNAc-mucin to yield GlcNAc3Gal3 (GlcNAc6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal1,3GalNAc chains was 0.53 µM; for UDP-N-acetylglucosamine, 12 µM; and for Gal 1,3GalNAc NO2ø, 4 mM. The activity of the 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal3GalNAc chains in Cowper's gland mucin glycoprotein.The best substrate for the partially purified 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal1,3(GlcNAc6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal1,3GalNAc side chains.The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the 6- and 3-glucosaminyltransferases were shown to be Gal3(GlcNAC6) GalNAc and GlcNAc3 Gal3(GlcNAC6)GalNAc respectively.Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a 6-glucosaminyltransferase converts Gal3GalNAc chains in mucin glycoproteins to Gal3(GlcNAc6)GalNAc chains. This product is the substrate for a second 3-glucosaminyltransferase which converts the Gal3(GlcNAc6)GalNAc chains to GlcNAc3Gal(GlcNAc6)GalNAc chains in the glycoprotein. The 3-glucosaminyltransferase did not utilize Gal3GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.Abbreviations NeuNAc
N-acetylneuraminic acid
- GalNAcol
N-acetylgalactosaminitol
- CGMG
Cowper's gland mucin glycoprotein
- GalNAc-CGMG
Cowper's gland mucin glycoprotein containing GalNAc side chains O-glycosidically linked to serine or threonine
- Gal3GalNAc-CGMC
Cowper's gland mucin glycoprotein containing Gal3GalNAc side chains
- MES
2-(N-morpholino) Ethane Sulfonic acid
- PBS
Phosphate Buffered Saline 相似文献
13.
V. Subramanian 《Journal of industrial microbiology & biotechnology》1986,1(2):119-127
Summary Methane-grown cells ofMethylococcus capsulatus andMethylosinus trichosporium readily oxidized propene and various isomers of butene to their respective epoxides. When examined in a proton NMR spectrum using tris([3-trifluoromethylhydroxymethylene]-d-camphorato), europium III derivative as an optically active chemical shift reagent, the products propylene oxide and 1,2-epoxybutane were found to contain equal amounts of both isomers. Methane-grown cells of both bacteria had considerable levels of reducing equivalents to catalyze the epoxidation of gaseous olefins. Cells depleted of reductants catalyzed the oxidation in the presence of low levels of methanol or formaldehyde with a stoichiometry of about 2:1. The rates of epoxidation of propene and 1-butene in a continuous reactor were 2–3-times that of a batch-wise reaction; the epoxidation activity, however, was lost within 3 h. The inactivation was attributed to the reactivity of the accumulated epoxides in the reactor. Propene and 1-butene oxidation by both bacteria were drastically inhibited by the respective products. Thus, the major problem in the application of microorganisms for production of epoxides from gaseous olefins is the rapid separation of the reactive products. 相似文献
14.
The binding of lysozyme with bromophenol blue (BPB) at various dye concentrations and pH was carried out at 25 degrees C by equilibrium dialysis, ultraviolet (UV) difference and circular dichroism (CD) spectral techniques. Binding isotherms at pH 5.0 show non-cooperative binding at low dye concentrations, which change over to cooperative binding at higher concentrations indicating biphasic nature. However, binding isotherms at pH 7.0 and 9.0 show cooperative binding only, at all concentrations of the dye. The number of available binding sites decreases with the increase of pH. Gibbs free energy change, calculated on the basis of Wyman's binding potential concept, decreases with the increase of pH. Binding isotherms at pH 5.0 obtained at a lower temperature of 8 degrees C, also indicate the biphasic nature similar to those observed at 25 degrees C, but with a slight decreased strength of binding. The UV difference spectra of the complex do not show any distinct peaks in the 285 to 297 nm region eliminating any possible interaction of BPB with tryptophan and tyrosine residues of the lysozyme molecule. The CD spectra of lysozyme-BPB complex show a decrease in ellipticities with reference to native lysozyme in the near UV and far UV regions. This indicates that the lysozyme-BPB complex has a lower helical content probably due to the conformational changes induced into the native enzyme. The appearance of new positive peaks at 315 nm in the near UV region and at 592 nm in the visible region of the CD spectra may be due to the induced asymmetry into the BPB molecule as a result of its binding to a cationic residue (probably a lysine residue) of lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
We have examined the accessibility to diethylpyrocarbonate of spinach chloroplast 4.5S ribosomal RNA when free and when it is part of the ribosomal structure. The modifications in free 4.5S RNA were found mostly in single-stranded regions of the secondary structure model proposed in our previous paper (Kumagai, I. et al. (1982) J.B.C. 257, 12924-28): adenines at positions 17, 19, 33, 36, 54, 55, 60, 64, 68, 72, 77, 86 and 87 were identified as the reactive residues. On the other hand, in 4.5S RNA in 70S ribosomes or 50S subunits, adenine 33 was exclusively modified, and its reactivity was much higher than in free 4.5S RNA. This highly accessible A33 of spinach 4.5S RNA is located within a characteristic seven nucleotide sequence, which is found in the 4.5S rRNAs from spinach, tobacco and a fern but deleted in 4.5S RNAs from maize and wheat. 相似文献
16.
Maize chloroplast DNA encodes a protein sequence homologous to the bacterial ribosome assembly protein S4. 总被引:11,自引:6,他引:5
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A cloned restriction fragment of maize chloroplast DNA (Bam H1 fragment 5) is shown to contain an open reading frame which encodes a basic protein of 201 amino acid residues with 40-50% sequence homology to E. coli ribosomal protein S4. Based on the experimentally determined sequence homology between the highly conserved bacterial ribosomal protein L12 and its chloroplast homologue (Bartsch M., Kimura, M. and Subramanian, A.R. (1982) Proc. Natl. Acad. Sci. USA 79, 6871), we conclude that this reading frame represents the maize chloroplast S4 gene. The nucleotide sequence of a 1100 base pair DNA segment containing the putative gene is presented. 相似文献
17.
18.
The potential contribution of maternal age to tetrahydrocannabinol's (THC) in utero effects in rats was studied. Pregnant animals were intubated with 25, 10 or 0 mg/kg of THC from gestation day six to parturition. Animals in the 10 and 0 mg/kg groups were pair fed to those given the 25 mg/kg dose. Each series of doses was administered to females three, four or six months of age. THC lowered maternal weight gain and weights of offspring at birth and at 21 days of age, but did not affect litter size, spontaneous alternation or passive avoidance learning in offspring. Increased maternal age was associated with smaller litter size and lower birth weight and weight at 21 days, but did not interact significantly with THC. 相似文献
19.
We have isolated a gene, AAT1, encoding an aspartate aminotransferase (AspAT) from a Saccharomyces cerevisiae genomic library. AAT1 encodes a 451 amino acid protein with a predicted molecular weight of 51,687, which is likely to be the yeast mitochondrial AspAT. Sequence comparison of this yeast AspAT with AspATs from other organisms shows a high degree of homology in regions previously shown to be important for catalysis. However, the yeast mitochondrial AspAT contains four obvious insertions with respect to all other known AspATs, suggesting that the AAT1-encoded protein represents a distinct AspAT. 相似文献
20.
The N-1-Naphthylphthalamic Acid-Binding Protein Is an Integral Membrane Protein 总被引:6,自引:3,他引:3
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N-1-Naphthylphthalmic acid (NPA)-binding protein is a plasmalemma (PM) protein involved in the control of cellular auxin efflux. We re-evaluated the spatial relationship of this protein with the PM of zucchini (Cucurbita pepo L.) hypocotyls. First, Triton X-114 partitioning indicated that the NPA-binding protein was more hydrophobic than most PM proteins. Second, the NPA-binding activity was found to be resistant to proteolytic digestion in membranes. Maximum concentrations of binding sites for NPA were virtually identical in untreated and proteinase K-treated PMs: 19.2 and 20.6 pmol [3H]NPA bound/mg protein, respectively. The insensitivity of the NPA-binding protein was not due to its presence inside tightly sealed vesicles or due to lack of protease activity in the conditions tested. This protein could be made sensitive to proteolytic degradation upon solubilization by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in the presence of sodium molybdate. Proteinase K treatment decreased the concentration of binding sites to 0.84 pmol [3H]NPA bound/mg protein from 9.2 for untreated, solubilized PM. Third, this activity could not be solubilized by chaotropic agents or sodium carbonate treatment of intact PM. This study indicates that the NPA-binding protein may be an integral membrane protein and contradicts previously reported findings that suggested that this protein was peripheral to the PM. 相似文献