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31.
A eukaryotic fumarase is for the first time unequivocally shown to contain two distinct substrate-binding sites. Pig heart fumarase is a tetrameric enzyme consisting of four identical subunits of 50 kDa each. Besides the true substrates L-malate and fumarate, the active sites (sites A) also bind their analogs D-malate and oxaloacetate, as well as the competitive inhibitor glycine. The additional binding sites (sites B) on the other hand also bind the substrates and their analogs D-malate and oxaloacetate, as well as L-aspartate which is not an inhibitor. Depending on the pH, the affinity of sites B for ligands (Kd being in the millimolar range) is 1-2 orders of magnitude lower than the affinity of sites A (of which Kd is in the micromolar range). However, saturating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays very special properties. One molecule of this ligand is indeed able to bind into a site A and a site B at the same time. Four molecules of pyromellitic acid were found to bind per molecule fumarase, and the affinity of the enzyme for this ligand is very high (Kd = 0.6 to 2.2 microM, depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosine residue is found to be located in site A, whereas an essential methionine residue resides in or near site B. Upon limited proteolysis, a peptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small local conformational changes in the enzyme are picked up by circular dichroism measurements in the near-UV region. This spectrum is built up of two tryptophanyl triplets, the first one of which is modified upon saturating the active sites (A), and the second one upon saturating the low affinity binding sites (B). 相似文献
32.
Robben J.; der Schueren J.Van; Verhasselt P.; Aert R.; Volckaert G. 《Protein engineering, design & selection : PEDS》1995,8(2):159-165
The deletion of nine residues from the C-terminus of the bacterialchloramphenicol acetyltransferase (CAT) results in depositionof the mutant protein in cytoplasmic inclusion bodies and lossof chloramphenicol resistance in Escherichia coli. This foldingdefect is relieved by C-terminal fusion of the polypeptide withas few as two residues. Based on these observations, efficientpositive selection for the cloning of DNA fragments has beendemonstrated. The cloning vector encodes a C-terminally truncatedCAT protein. Restriction sites in front of the stop codon allowthe insertion of target DNA, resulting in the production ofproperly folded CAT fusion proteins and regained chloramphenicolresistance. The positive selection of recombinants is accomplishedby growth of transformants on chloramphenicol-containing agarplates. The method appears particularly convenient for the cloningof DNA fragments amplified by the PCR because minimal informationto restore CAT folding can be included in the primers. The cloningof random sequences shows that the folding defect can be relievedby fusion to a wide variety of peptides, providing great flexibilityto the positive selection system. This vector may also contributeto the determination of the role of the C-terminus in CAT folding. 相似文献
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AE Speckens AM Van Hemert JH Bolk HG Rooijmans MW Hengeveld 《Canadian Metallurgical Quarterly》1996,26(4):745-752
BACKGROUND: Measurement of intracardiac hemodynamic parameters has been limited to brief periods in the acute care setting. We developed and evaluated an implantable hemodynamic monitor that is capable of measuring chronic right ventricular oxygen saturation and pulmonary artery pressure. METHODS AND RESULTS: The device consists of an electronic controller placed subcutaneously and two transvenous leads placed in the right ventricle (reflectance oximeter) and pulmonary artery (variable capacitance pressure sensor). Implantation was performed in 10 patients with severe left ventricular dysfunction. Average implant pulmonary artery pressures were systolic, 52 +/- 16 mm Hg; diastolic, 29 +/- 11 mm Hg; and mean, 40 +/- 12 mm Hg. The mean right ventricular oxygen saturation at implant was 51%. Provocative maneuvers, including postural changes, sublingual nitroglycerin, and bicycle exercise, demonstrated expected changes in measured oxygen saturation and pulmonary artery pressures over time. At follow-up of 0.5 to 15.5 months, there were no significant differences between pulmonary artery pressures or oxygen saturation values transmitted from the device and simultaneous measurement with balloon flotation catheters. Four of the pulmonary artery leads dislodged and three demonstrated sensor drift, whereas two of the oxygen saturation sensors failed. Four patients died and four received transplants. Pathological study did not demonstrate injury to the right ventricular outflow tract or pulmonic valve. CONCLUSIONS: Chronic measurement of hemodynamic parameters in the outpatient setting with implantable sensor technology appears to be feasible. The devices are well tolerated without significant untoward effects, and the sensors generally function well over time, providing reliable information. Clinical usefulness remains to be established. 相似文献
35.
R Herruzo-Cabrera L Malo-González ME Calle Purón MJ Vizcaíno-Alcaide J Del Rey-Calero 《Canadian Metallurgical Quarterly》1993,9(4):442-446
A cross-sectional study was performed to obtain risk factors for hepatitis B disease, HBsAg carriers and immunised personnel, among 2470 workers in a general hospital in Madrid, Spain. The data obtained were analyzed with multiple logistic regression to obtain beta coefficients for variables. The results of the analysis show that being a nurse or being regularly exposed to blood are the most important risk factors for hepatitis B acquisition. The length of time working at the same job activity was also a risk factor. The resulting beta coefficients allow the construction for a hepatitis non-immunised, HBsAg carrier and immunised HBV status, which can select subjects for a hepatitis B vaccination program. 相似文献
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39.
ABSTRACT: The thermal stability of Bacillus licheniformis α-amylase at low moisture content was studied, based on isothermal experiments performed in a temperature range 113 to 125 °C. The thermal inactivation was monitored by measuring the decrease in thermal denaturation enthalpy and/or the decrease in enzymic activity on pnitrophenyl-α-D-maltoheptaoside, or on starch as a substrate. Based on enthalpy readings, an enzymic system with a z-value of 10.4 °C was observed when using a relative humidity of 81% at 4 °C. A theoretical study showed that this system could be used as a Time Temperature Integrator (TTI) to monitor the safety of sterilization processes of numerous food products. 相似文献
40.
Low molecular weight aldehydes were formed during surface water preozonization, their levels showing a positive correlation with increasing ozone dose applied and with increasing water temperature. A strong negative correlation was observed between aldehyde levels and U.V. absorbance at 254 nm.Coagulation had no influence on the aldehydes present and the influence of rapid double layer filtration varied strongly with temperature : significant removals were only observed above 10°C.Mutagenic activity generated by preozonization in Salmonella typhimurium TA98 shows an ozone dose depending relationship different from the formation of linear aldehydes. Its removal by coagulation is not effective but rapid double layer filtration reduces mutagenic activity to marginal levels.In this respect too no clear parallel can be drawn between the presence of low molecular weight aldehydes and mutagenic activity. 相似文献