Dextran-gel filtration of enzymes in the presence of their high-molecular-weight substrates.
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ABSTRACT:
SUBMITTER: Auricchio F
PROVIDER: S-EPMC1264828 | biostudies-other | 1966 Jan
REPOSITORIES: biostudies-other
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Project description:1. The behaviour of rat liver alpha-glucosidases on dextran gel (Sephadex G-100) columns was studied. A ;retardation' of an acid alpha-glucosidase activity was observed. This activity was identified as lysosome alpha-(1-->4)-glucosidase. A single gel-filtration step resulted in a 700-fold purification of the enzyme. The same technique was also used to purify the acid alpha-glucosidase of human kidney. 2. The acid alpha-glucosidases of both tissues show very similar pH optima when tested with maltose or glycogen as substrate.
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Project description:Here, we present a high-throughput virtual top-down proteomics approach that restores the molecular weight (MW) information in shotgun proteomics and demonstrates its utility in studying proteolytic events in programmed cell death. With gel-assisted proteome position integral shift (GAPPIS), we quantified over 7000 proteins in staurosporine-induced apoptotic HeLa cells and identified 84 proteins exhibiting in a statistically significant manner at least two of the following features: (i) a negative MW shift; (ii) an elevated ratio in a pair of a semitryptic and tryptic peptide, (iii) a negative shift in the standard deviation of MW estimated for different peptides, and (iv) a negative shift in skewness of the same data. Of these proteins, 58 molecules were previously unreported caspase 3 substrates. Further analysis identified the preferred cleavage sites consistent with the known caspase cleavages after the DXXD motif. As a powerful tool for high-throughput MW analysis simultaneously with the conventional expression analysis, the GAPPIS assay can prove useful in studying a broad range of biological processes involving proteolytic events.
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Project description:1. Gels were prepared with recrystallized acrylamide and bisacrylamide. Electrophoresis was in tris-sodium acetate-EDTA buffer for 0.5 to 3hr. Gels were scanned at 280 or 265mmu. Techniques are described for slicing and radioactive counting. 2. The mobility of RNA was inversely related to the sedimentation coefficient and varied with gel concentration. Electrophoresis in 2.2-2.6% gels gives a fractionation similar to density-gradient centrifugation. It shows the two ribosomal RNA components, the 45s precursor, transfer RNA and minor components. In 5% and 7.5% gels, 4s and 5s RNA separated and ribosomal RNA was excluded. 3. The resolution is greater and more detailed than by centrifugation, and many samples can be analysed simultaneously and rapidly.
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Project description:Unlike general peroxidases, Pleurotus ostreatus MnP2 was reported to have a unique property of direct oxidization of high-molecular-weight compounds, such as Poly R-478 and RNase A. To elucidate the mechanism for oxidation of polymeric substrates by MnP2, a series of mutant enzymes were produced by using a homologous gene expression system, and their reactivities were characterized. A mutant enzyme with an Ala substituting for an exposing Trp (W170A) drastically lost oxidation activity for veratryl alcohol (VA), Poly R-478, and RNase A, whereas the kinetic properties for Mn(2+) and H(2)O(2) were substantially unchanged. These results demonstrated that, in addition to VA, the high-molecular-weight substrates are directly oxidized by MnP2 at W170. Moreover, in the mutants Q266F and V166/168L, amino acid substitution(s) around W170 resulted in a decreased activity only for the high-molecular-weight substrates. These results, along with the three-dimensional modeling of the mutants, suggested that the mutations caused a steric hindrance to access of the polymeric substrates to W170. Another mutant, R263N, contained a newly generated N glycosylation site and showed a higher molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Interestingly, the R263N mutant exhibited an increased reactivity with VA and high-molecular-weight substrates. The existence of an additional carbohydrate modification and the catalytic properties in this mutant are discussed. This is the first study of a direct mechanism for oxidation of high-molecular-weight substrates by a fungal peroxidase using a homologous gene expression system.
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Project description:Optimizing high molecular weight extractions
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Project description:The human serum proteome has been extensively screened for biomarkers. However, the large dynamic range of protein concentrations in serum and the presence of highly abundant and large molecular weight proteins, make identification and detection changes in the amount of low-molecular weight proteins (LMW, molecular weight ? 30kDa) difficult. Here, we developed a gel-filter method including four layers of different concentration of tricine SDS-PAGE-based gels to block high-molecular weight proteins and enrich LMW proteins. By utilizing this method, we identified 1,576 proteins (n = 2) from 10 ?L serum. Among them, 559 (n = 2) proteins belonged to LMW proteins. Furthermore, this gel-filter method could identify 67.4% and 39.8% more LMW proteins than that in representative methods of glycine SDS-PAGE and optimized-DS, respectively. By utilizing SILAC-AQUA approach with labeled recombinant protein as internal standard, the recovery rate for GST spiked in serum during the treatment of gel-filter, optimized-DS, and ProteoMiner was 33.1 ± 0.01%, 18.7 ± 0.01% and 9.6 ± 0.03%, respectively. These results demonstrate that the gel-filter method offers a rapid, highly reproducible and efficient approach for screening biomarkers from serum through proteomic analyses.
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Project description:Dextran has been frequently used during intracoronary imaging, such as in optical coherence tomography, optical frequent domain imaging, and coronary angioscopy. We report a case of dextran-induced anaphylaxis in a 70-year-old male with chronic coronary disease. Upon admission, we performed coronary angiography and coronary angioscopy on the patient. After the intracoronary imaging, the patient's blood pressure suddenly fell to 50 mmHg and a rash appeared on his chest. The patient was diagnosed as having dextran-induced anaphylactic shock. Epinephrine was administered repeatedly, and his blood pressure gradually recovered after administering a total of 6 mg epinephrine. There was no recurrence of the anaphylactic shock, and the patient was discharged 12 days later. The incidence of dextran-induced anaphylactic reactions is extremely low; however, they can be fatal. The possibility of anaphylactic shock induced by dextran should be kept in mind by all cardiovascular interventionalists performing intracoronary imaging.Learning objectiveDextran has been frequently used during intracoronary imaging. We report on a case of dextran-induced anaphylaxis in a 70-year-old male with chronic coronary disease. While the incidence of dextran-induced anaphylactic reactions is extremely low, it can lead to fatal events. The possibility of anaphylactic shock induced by dextran should be kept in mind by all cardiovascular interventionalists while performing intracoronary imaging.
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Project description:Ultracentrifugation studies of purified mouse hepatic catalase revealed that 5-7% of the total material consists of a form with a higher molecular weight than the bulk of the catalase. The two components were separated by sucrose-gradient centrifugation. Polyacrylamide-gel electrophoresis (in borate buffer) demonstrated that high-molecular-weight catalase is enriched in a more slowly migrating component, and sodium dodecyl sulphate/polyacrylamide gel-electrophoresis demonstrated that the molecular weight of the subunits of the high-molecular-weight material is identical with that of the subunits of the major form. These results suggest that high-molecular-weight catalase consists of subunits that are not markedly distinct from those present in the normal catalase tetramer.
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