Spelling suggestions: "subject:"pure sciences"" "subject:"cure sciences""
1 |
Human Lung Tryptase Purification and CharacterizationSmith, Timothy J. 01 May 1985 (has links)
Human lung tryptase (HLT), a mast cell derived trypsin-like enzyme, was isolated from whole human lung tissue obtained at autopsy. Increased yields from this purification process allowed extensive characterization of the enzyme. One of the critical steps in the purification scheme was the use of a linear heparin gradient to elute active material from cellulose phosphate. Gel filtration studies in 1.0 M NaCl yielded an apparent M(,r) of 135,000, and subsequent electrophoresis on sodium dodecyl sulfate-polyacrylamide gels demonstrated the presence of two active species with apparent M(,r) = 30,900 and 31,600. Enzymatic activity was sensitive to NaCl concentrations above 0.05 M and was only 50% in 0.15 M NaCl, decreasing to 18% in 0.6 M NaCl. The effects of synthetic and natural inhibitors were studied, confirming the enzyme's trypsin-like characteristics and demonstrating that naturally occurring serum inhibitors are incapable of diminishing its activity. A complete amino acid analysis showed a high tryptophan content. Antisera to human lung tryptase was generated, and the immunological identity of active fractions was investigated. The stability of HLT in various buffer systems was extensively studied, and 10 mM MES buffer, pH 6.1 appeared to provide the best conditions during extended storage and purification. The effect of heparin on the enzyme's activity using the synthetic substrates Z-Lys-SBzl was studied, and heparin concentrations of 10 micromolar stabilized HLT and allowed full expression of activity even at low ionic strengths. In the presence of heparin the enzyme retained full activity after 24 hours at 37(DEGREES)C, whereas in the absence of heparin, activity was lost after 30 min at this temperature. Heparin had a similar effect on HLT's ability to cleave natural substrates such as fibronectin. Assays comparing the activity of HLT on the substrates Z-Lys-SBzl and Z-Arg-SBzl were performed. The K(,m) and V(,max) of HLT for the above substrates were determined. The substrate 4-methylumbelliferyl-p-guanidinobenzoate (MUG-B) has been used to perform an active site titration on HLT, and a k(,cat) of 610/sec was calculated for the substrate Z-Arg-SBzl.
|
2 |
Purification and Characterization of Enoyl-acp Reductase From Euglena GracilisTucker, Margie M. 01 May 1990 (has links)
Enoyl-(acyl-carrier-protein) reductase was purified from the phytoflagellate Euglena gracilis. Its purification employed DEAE-Sephacel chromatography, Matrex Orange chromatography, and affinity chromatography using acyl carrier protein (ACP) covalently bound to Sepharose as the affinity ligand. Matrex Orange chromatography resolved two different enoyl-ACP reductases having different characteristics. Euglena gracilis appears to resemble higher plants in the possession of two isoforms of this enzyme. Antibodies specific for the cofactor binding site of NADP (H)-requiring dehydrogenases were obtained. They were isolated from a polyclonal population of antibodies directed against yeast glucose-6-phosphate dehydrogenase by affinity chromatography using chicken liver malic enzyme as the affinity ligand. The affinity purified antibodies were covalently bound to Sepharose. Glucose-6-phosphate dehydrogenase and malic enzyme were both bound by the antibody column and were eluted by their cofactor, NADP$\sp+$, identifying the site of recognition of the enzymes by the antibodies as the cofactor binding site. The utility of this antibody affinity column was demonstrated by its ability to bind enoyl-ACP reductase, which was eluted by its cofactor, NADPH. Preliminary studies of the E. gracilis fatty acid synthase (FAS) genes were undertaken using the plasmid pFAS4 (Witkowski et al., 1987), which contains a cDNA insert to part of the rat liver FAS mRNA and was a gift of Dr. Stuart Smith. The insert was cleaved with KpnI and PstI to generate probes specific for the ketoreductase, ACP, and thioesterase domains of the FAS. DNA from wild type E. gracilis and from a mutant, W$\sb{10}$BSML, which lacks chloroplast DNA, was subjected to field inversion gel electrophoresis and the DNA alkaline-blotted onto Nylon membranes. Hybridization of the three probes to the DNA was performed; all three probes hybridized to nuclear DNA, but none of the three hybridized to chloroplast DNA. The three probes also hybridized to a band which was neither nuclear nor cholorplast DNA. This DNA, which was larger than the chloroplast genome, may represent E. gracilis mitochondrial DNA sequences.
|
3 |
Purification and Characterization of an Inhibitor of Thymidine Uptake From Culture Supernatants of Human Tonsil LymphocytesZarnegar, Abdolreza 01 May 1987 (has links)
Lymphocytes from human tonsils were cultured in the absence of serum for 3 days. In the presence of the concentrated culture supernatant the proliferative response of PBL, to con A, as measured by the uptake of ('3)H-tdr, was significantly reduced. The suppressor substance was referred to as SMAL (suppressor of mitogen activated lymphocytes). The estimated molecular weight of SMAL under nondenaturing conditions was 100,000-300,000. SMAL also suppressed the incorporation of ('3)H-tdr by a variety of mouse and human tumor cell lines. The activity of SMAL was sensitive to pronase and heating at 100(DEGREES)C for 30 minutes but insensitive to RNase. Treatment with DNase, however, enhanced the activity of SMAL. SMAL activity was also destroyed by treatment with 5% TCA, 0.4 M HCl or 60% acetonitrile, but resistant to 6 M urea or dialysis against pH 2 buffer for 24 hours. SMAL activity was precipitated in 40-80% ammonium sulfate saturation. When applied to a phenyl-sepharose column no activity was recovered. SMAL was not produced by heat-killed tonsil lymphocytes or lymphocytes-treated with cycloheximide. Maximal production occurred in the first 24 hours of culture, and progressively less was produced in subsequent 24-hour intervals. Both T- and B lymphocyte-enriched culture supernatants contained SMAL. SMAL adhered strongly to DEAE-cellulose, but less than two-fold purification was achieved. Using QMA-Accell anion exchange medium, a 5-fold purification of SMAL with higher specific activity was obtained with HPLC. Activity of SMAL was recovered after native polyacrylamide gel electrophoresis by electroblotting to DEAE-cellulose paper followed by eluting the bound materials with salt. Two active components, one corresponding to a large and/or less negatively charged molecule and another corresponding to a small and/or highly acidic molecule, were recovered. HPLC-purified SMAL at relatively low doses inhibited the uptake and phosphorylation of ('3)H-tdr, without significant effect on cell proliferation. The inhibition of ('3)H-tdr uptake was favored over that of ('3)H-udr or ('3)H-adr, and this effect was reversible. At relatively high doses of HPLC-purified SMAL, the growth of mouse thymoma EL-4 and human T cell leukemia CEM-CM(,3) cell lines was inhibited.
|
4 |
The continuum hypothesis in algebraic set theoryKusalik, Timothy January 2009 (has links)
No description available.
|
5 |
Periodic orbits in systems of two non-rigid particles on the torusMaye, Steven January 2009 (has links)
No description available.
|
6 |
Derived categories of coherent sheaves on smooth projective curvesTomberg, Artour January 2011 (has links)
No description available.
|
7 |
Nearly rigid analytic modular forms and their values at CM pointsFranc, Cameron January 2011 (has links)
No description available.
|
8 |
The Einstein constraint equations on compact 3-dimensional manifoldsTcheng, Alexandra January 2011 (has links)
No description available.
|
9 |
Jacobi formsde Quehen, Victoria January 2011 (has links)
No description available.
|
10 |
Analysis of three stochastic models for discrete populationsCottrell, David Daniel January 2010 (has links)
No description available.
|
Page generated in 0.0574 seconds