ISSN:0173-0835    

DOI:10.1002/elps.1150180702

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractA set of eleven biallelic and three multiallelic molecular markers have been developed to analyze populations ofHistoplasma capsulatum. All markers are amplified by polymerase chain reaction (PCR) and can be readily scored using minimal amounts of template DNA. The 11 biallelic loci have polymorphic restriction endonuclease sites or small insertions or deletions which may be assessed by agarose gel electrophoresis. These markers are inherited in an unambiguous manner and are ideal for assessing structure and gene flow within US populations ofH. capsulatum, but are monomorphic in non‐US populations. Both length and sequence variation are present in the multiallelic loci, which can be scored by direct sequencing, polyacrylamide gel electrophoresis, or single‐strand conformation polymorphism (SSCP): As they are hyper‐variable, the multiallelic loci can be used to type isolates and to assess the level of genetic variation within populations. Preliminary results indicate that the three multiallelic markers presented are sufficient to distinguish isolates at the individual level and are polymorphic in both US and non‐US populations. This collection of molecular markers will be a useful tool in population and epidemiology studies ofH. cap

ISSN:0173-0835    

DOI:10.1002/elps.1150180703

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractDeoxyribonuclease I (DNase I) is an actin monomer‐sequestering actin binding protein (ABP) that inhibits the rate and extent of actin polymerisationin vitroby forming a high affinity, stoichiometric 1:1 complex. Using capillary zone electrophoresis (CZE), we have studied the interaction between G‐actin and DNase I to evaluate the capability of CZE to determine the dissociation constant (Kdvalue) for this interaction. We used (i) an uncoated fused‐silica capillary and ultraviolet (UV) detection at 214 nm; (ii) a hydrophilic‐coated capillary with UV detection at 214 nm; and (iii) a hydrophilic‐coated capillary with laser‐induced fluorescence (LIF) detection. Using procedure (ii), aKdvalue of approximately 0.03 μMwas obtained by simulation of binding data. We conclude that CZE combined with a LIF detector has the capacity to extend the determination ofKdvalues from the micromolar range to the nanomolar range. Subsequent determination ofKdvalues for other actin‐binding proteins should provide information on interactions between the binding sites on actin for these proteins and their spatia

ISSN:0173-0835    

DOI:10.1002/elps.1150180704

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractTrypsin inhibitory proteins of low buoyant density (ρ ≤ 1.35 g/mL) fractions were prepared by CsCl density gradient ultracentrifugation of 4Mguanidinium hydrochloride extracts of lumbar beagle and greyhound annulus fibrosus and nucleus pulposus from animals aged 1 to 6 years. Affinity blotting with biotinylated trypsin was used to identify active trypsin inhibitory protein species; these species were also identified immunologically by Western blotting using antibodies against bovine pancreatic trypsin inhibitor (BPTI), and human inter‐α‐trypsin inhibitor (ITI). None of the trypsin inhibitory species evident in Western blots were reactive with anti‐human α1‐proteinase inhibitor (α‐1‐PI), α2‐macroglobulin or secretory leucocyte proteinase inhibitor. The greyhound intervertebral disc samples generally had higher levels of active trypsin inhibitor species per unit weight of tissue extracted, and a more extensive range of inhibitor species. Inhibitor species of 30, 32, 34 kDa were identified in both beagle and greyhound intervertebral disc samples; these species were generally most prominent in the annulus fibrosus samples. In contrast, the nucleus pulposus samples contained relatively large trypsin inhibitor species; the anti‐BPTI detected an inhibitor species of ∼ 85–90 kDa; anti‐ITI detected species of 120–250 kDa; biotinylated trypsin detected species of 60–110 kDa. A small molecular mass trypsin inhibitor species of 6 kDa, which was of similar mobility to BPTI, was also detected in annulus fibrosus samples; however, this spec

ISSN:0173-0835    

DOI:10.1002/elps.1150180705

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe electrophoretic analysis by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE; reduced and unreduced) of fractions, collected from a size exclusion‐high performance liquid chromatography (SE‐HPLC) separation of gluten proteins using a column with pore size of around 400Å, showed clear resolution for the seven elution ranges studied in two Australian bread wheat lines. Polymeric proteins — high molecular weight (HMW) glutenin subunits, low molecular weight (LMW) glutenin subunits, HMW albumins and some modified ω‐gliadins — appeared exclusively in the region within the first peak of the chromatogram (fractions 1 to 5), the limit being a region that resolved as a small peak before the large peak of gliadins and where some ω‐gliadins eluted. A larger proportion of HMW glutenin subunits and B subunits contributed to polymer formation of higher molecular weight. The polymer size decreased as the proportion of the other protein co

ISSN:0173-0835    

DOI:10.1002/elps.1150180706

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractAdvances in protein database design and the software used to access the sequence data has led to progress in using protein attributes such as amino acid composition and peptide masses to identify proteins separated by two‐dimensional electrophoresis. However, Edman degradation remains the principal technique for protein identification and it presents a significant bottle‐neck in the progress towards rapid protein identification. Simple modifications to the sequencing hardware, which automate the delivery of protein spots into the sequencer, and parallel sequencing of the protein spots represent a significant advance in the use of Edman degradation to rapidly generate the powerful protein attribute, anN‐terminal sequenc

ISSN:0173-0835    

DOI:10.1002/elps.1150180707

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractMicropreparative two‐dimensional polyacrylamide gel electrophoresis has been used to separate milk whey proteins from the Tammar wallaby (Macropus eugenii). We have used a combination of amino acid analysis andN‐terminal sequence tagging as a rapid and sensitive method to identify the major whey proteins. Using these techniques, we confidently identified α‐lactalbumin and late lactation protein. While these are the only twoM. eugeniiwhey proteins with a corresponding SWISS‐PROT entry, we demonstrate that by using amino acid analysis and matching across species boundaries, we can identify previously unsequenced conserved wallaby whey proteins including β‐lactoglobulin and se

ISSN:0173-0835    

DOI:10.1002/elps.1150180708

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractIn muscle cells actin exists as a mixture of monomeric (G‐actin) and filamentous actin (F‐actin) and ionic conditions strongly favor the formation of F‐actin. The existence of unpolymerized actin depends, among other factors, on proteins that bind to G‐actin, the so‐called G‐actin‐binding proteins (G‐ABPs). We have coupled monomeric actin to divinylsulphone‐activated agarose (Mini‐Leak) to isolate G‐ABPs in human skeletal muscle. Eluted proteins were analyzed by two‐dimensional gel electrophoresis (2‐DE), which shows that some proteins are selectively retained. Deoxyribonuclease I (DNase I) is known to bind residues at the “pointed end” of actin (subdomains 2 and 4) with a high affinity. When DNase I is bound to the actin Mini‐Leak before applying the skeletal muscle extract, the 2‐DE gels of the eluted proteins reveals differences when compared to gels of proteins eluted from actin‐Mini‐Leak and DNase I‐Mini‐Leak affinity columns. This strategy should detect ABPs which bind to sites other than the DNase I‐

ISSN:0173-0835    

DOI:10.1002/elps.1150180709

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe mouse macrophage cell‐line RAW264.7, stimulated with lipopolysaccharide, was used as a model for the study of the production of tumor necrosis factor (TNF) isoforms. TNF is synthesised initially as a 26 kDa transmembrane precursor, which is then processed enzymatically by a protease to release a mature molecule of 17 kDa. Dose‐dependent production of transmembrane TNF was assessed by fractionation of cell membranes and Western blot analysis followed by autoradiography and densitometry. Isoforms of both the precursor and mature molecules were separated using two‐dimensional (2‐D) electrophoresis with immobilised pH gradient 3–10 linear gels as the first dimension. After radiolabelling of cells with35S, both cell‐associated and supernate‐associated TNF isoforms were immunoprecipitated. A large number of protein spots were visualised on the 2‐D gel map, for both the transmembrane and mature TNF species, more than have been detected previously using one‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The likelihood that these putative isoforms were the result of differential glycosylation was tested by preincubating the cells with tunicamycin. This had the effect of reducing the number of protein spots, notably the higher molecular weight species. There were a number of precursor TNF isoforms that were unchanged upon tunicamycin treatment and these presumably reflect protein modifications oth

ISSN:0173-0835    

DOI:10.1002/elps.1150180710

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe theory of mass transport coupled to macromolecular interactions under chemical kinetic control forms the basis of four different models of the electrophoretic mobility‐shift assay of complexes formed between dimerizing proteins and DNA. The theory of mass action was applied to the set of simultaneous dimerization (either simple or ligand‐induced) and DNA‐binding reactions in order to fix the initial equilibrium composition of mixtures to be assayed. Theoretical mobility‐shift patterns were obtained for a range of protein concentrations at constant DNA concentration by numerical solution of the set of simultaneous transport‐reaction equations appropriate for each model. In those cases in which dimerization in solution is modeled (including heterodimerization), analysis of the peaks in the patterns provides apparent binding constants, which, when extrapolated to infinite dilution of protein, yield acceptable estimates of equilibrium constants. Those for binding of dimer are products of two or three equilibrium constants, from which the equilibrium binding constant can be extracted, privided that dimerization and, where required, ligand‐binding constants are determined by independent physicochemical methods. Dimerization of protein when bound to DNA is distinctive in that extrapolation to infinite dilution of protein is n

ISSN:0173-0835    

DOI:10.1002/elps.1150180711

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY