scholarly journals Subunit interactions in tyrosinase from frog epidermis in immobilized enzyme systems

1981 ◽  
Vol 197 (3) ◽  
pp. 581-589 ◽  
Author(s):  
J L Iborra ◽  
J A Ferragut ◽  
J A Lozano
Keyword(s):  
Fluorescence Spectra ◽  
Specific Activity ◽  
Guanidinium Chloride ◽  
Subunit Interactions ◽  
Low Concentrations ◽  
Tryptophan Residues ◽  
Unfolded State ◽  
The Matrix ◽  
Dopa Oxidase ◽  
Tetrameric Form

1. Frog epidermis tyrosinase was coupled to Sepharose activated with low concentrations of CNBr. The tetrameric form of the enzyme was linked to the matrix via its subunits. Dissociation of the bound active enzyme with guanidinium chloride yielded an active immobilized dimeric derivative. 2. Immobilized dimeric derivative was able to interact with soluble subunits formed transiently during renaturation. An 85% recovery of the native dopa oxidase specific activity was achieved after hybridization. 3. Fluorescence spectra of different immobilized derivatives suggested that tryptophan residues were involved in the interactions between tyrosinase subunits. 4. It is suggested that the activation of the subunits of tyrosinase involves a conformational change towards a more unfolded state, which favours a reassociation to the dimeric active state.

scholarly journals The mechanism of folding of globular proteins. Suitability of a penicillinase from Staphylococcus Aureus as a model for refolding studies

1976 ◽  
Vol 155 (2) ◽  
pp. 325-330 ◽  
Author(s):  
B Robson ◽  
R H. Pain
Keyword(s):  
Staphylococcus Aureus ◽  
Enzymic Activity ◽  
Optical Rotatory Dispersion ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Tyrosine Residues ◽  
Low Concentrations ◽  
Unfolded State ◽  
Solvent Molecules ◽  
Homogeneous Preparation

1. A homogeneous preparation of penicillinase (penicillin amido-β-lactamhydrolase, EC 3.5.2.6) was isolated and purified from cultures of Staphylococcus aureus by a simple two-stage procedure. 2. The native protein contains 20-30% helix as determined by optical-rotatory-dispersion and circular-dichroism measurements. Some 54(+/-5)% of the 13 tyrosine residues are exposed to solvent molecules of diameter 0.44 and 0.94 nm. 3. Conditions that allow full recovery of enzymic activity and native conformation from the fully unfolded state in 4M-guanidinium chloride were defined. 4. Refolding of the protein was shown to be inhibited by intermolecular interaction, by small changes in ionization and by low concentrations (0.025 M) of phenol.

scholarly journals Kinetic studies on the regulation of rabbit liver pyruvate kinase

1973 ◽  
Vol 131 (2) ◽  
pp. 287-301 ◽  
Author(s):  
M. G. Irving ◽  
J. F. Williams
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Rabbit Liver ◽  
Hill Coefficient ◽  
Saturation Curve ◽  
Low Concentrations ◽  
Ammonium Sulphate Fractionation ◽  
The Hill

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K+ and optimum activity was recorded with 30mm-K+, 4mm-MgADP-, with a MgADP-/ADP2- ratio of 50:1, but inhibition occurred with K+ concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg2+ was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (nH+2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent Km for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis–Menten response was obtained when phosphoenolpyruvate was the variable substrate (Km+0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg2+.

Leach Behavior of SRL TDS-131 Defense Waste Glass in Water at High&Low Flow Rates

1983 ◽  
Vol 26 ◽  
Author(s):  
Aaron Barkatt ◽  
William Sousanpour ◽  
Alisa Barkatt ◽  
Morad A. Boroomand ◽  
Pedro B. Macedo
Keyword(s):  
Contact Time ◽  
Protective Layer ◽  
High Flow ◽  
Low Flow ◽  
Matrix Elements ◽  
Flow Rates ◽  
Low Concentrations ◽  
Controlling Mechanism ◽  
The Matrix ◽  
Leach Behavior

ABSTRACTLeach tests carried out on SRL TDS-131 Defense Waste Class indicate that at high flow rates the controlling mechanism is simple corrosion. The matrix elements (Si, Al) are leached out at rates similar to those of the leaching of the alkalis and of boron, and the leaching process is nearly linear with time. At slow flow rates (below 1 m/yr) leaching becomes controlled by the build-up of a protective layer. Al and most of the Si remain in the leached surface layer. The leach rates decrease in the course of the test before leveling off at constant values which are almost inversely proportional to the contact time, indicating that leachate concentrations have become solubility-limited. The low concentrations observed at this stage indicate the formation of alteration products.

scholarly journals Comparison of the activity and conformation changes of lactate dehydrogenase H4 during denaturation by guanidinium chloride

1991 ◽  
Vol 277 (1) ◽  
pp. 207-211 ◽  
Author(s):  
Y Z Ma ◽  
C L Tsou
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Cross Linking ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Original Activity ◽  
Limited Region ◽  
Low Concentrations ◽  
Two Stages ◽  
Inhibited Enzyme

The inactivation and unfolding of lactate dehydrogenase (LDH) during denaturation by guanidinium chloride (GuHCl) under diverse conditions have been compared. Unfolding of the native conformation, as monitored by fluorescence and c.d. measurements, occurs in two stages with increasing GuHCl concentrations, and the inactivation approximately coincides with, but slightly precedes, the first stage of unfolding. The enzyme is inhibited to about 60-70% of its original activity by cross-linking with glutaraldehyde or in the presence of 1 M-(NH4)2SO4, with its conformation stabilized as shown by the requirement for higher GuHCl concentrations to bring about both inactivation and unfolding. Low concentrations of GuHCl (0.2-0.4 M) activate the cross-linked and the (NH4)2SO4-inhibited enzyme back to the level of the native enzyme. For the enzyme stabilized by (NH4)2SO4 or by cross-linking with glutaraldehyde, inactivation occurs at a markedly lower GuHCl concentration than that required to bring about its first stage of unfolding. It is concluded that the active site of LDH is situated in a limited region relatively fragile in conformation as compared with the molecule as a whole. The GuHCl activation of LDH stabilized in (NH4)2SO4 or by cross-linking with glutaraldehyde suggests that this fragility and consequently flexibility of the active site is required for its catalytic activity.

scholarly journals Potentiometric Determination of Chlorate Impurities in Hypochlorite Solutions

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Dmitry V. Girenko ◽  
Al’ona A. Gyrenko ◽  
Nikolai V. Nikolenko
Keyword(s):  
Sodium Hypochlorite ◽  
Sodium Sulfite ◽  
Systematic Errors ◽  
Expanded Uncertainty ◽  
Iodide Ions ◽  
Low Concentrations ◽  
Sophisticated Equipment ◽  
The Matrix ◽  
Fold Excess

The method of iodometric determination of chlorates impurities in sodium hypochlorite solutions for medical and veterinary purposes was developed. This method does not require sophisticated equipment and can be implemented directly where the solutions are used. The method is based on the different rates of interaction of ClO- and ClO3- with iodide ions depending on the acidity of the medium. We have shown that blank titration is advisable to improve the accuracy of the determination of low concentrations of chlorates in the matrix of hypochlorite which is present in excess since in this case possible systematic errors due to the presence of oxidizing impurities in the reagents are prevented. To quantify the low concentrations of chlorates, we proposed to remove 85-95% of hypochlorite ions by means of reducing their excess with sodium sulfite at pH 10.5. The solution of sodium sulfite does not require standardization before each analysis in the proposed procedure. The possibility of quantitative determination of chlorate impurities in the range of 2-50 mg/L in the presence of 50-500–fold excess of sodium hypochlorite with an error of 5% has been proved. The expanded uncertainty of chlorate determination did not exceed 0.6 mg/L.

ACC-N-Malonyltransferase activity during zygotic embryogenesis and germination of chick-pea seeds

2000 ◽  
Vol 10 (1) ◽  
pp. 71-76 ◽  
Author(s):  
Clemente Martin-Remesal ◽  
Maria del Carmen Gomez-Jimenez ◽  
Angel J. Matilla
Keyword(s):  
Enzymatic Activity ◽  
Specific Activity ◽  
Embryonic Axis ◽  
Zygotic Embryogenesis ◽  
Chick Pea ◽  
Low Concentrations ◽  
Mtase Activity ◽  
Storage Organs ◽  
Radicle Emergence

AbstractSome physiological characteristics of ACC-Nmalonyltransferase (ACC-N-MTase) have been studied in the seeds of chick-pea (Cicer arietinum L.). This enzymatic activity was detectable during all periods of zygotic embryogenesis; however, the highest values were found in the dry seed. In dry seeds, the enzymatic activity was greater in the embryonic axis than in the cotyledons. During the onset of germination, activity increased more strongly in the cotyledons than in the embryonic axis, reaching its highest values in the storage organs coinciding with radicle emergence (18 to 24 h). Removal of the cotyledons strongly diminished enzymatic activity in the embryonic axis. Low concentrations of ethrel (50 µM) stimulated the axis ACC-N-MTase. The highest specific activity was found in the apical meristem of the embryonic axis, declining over the length of the organ. The role of ACC-N-MTase activity in the germinative process is discussed, together with the regulatory effect of ethylene on this ACCconjugating enzyme.

Analytical validation of eight methods of thyroglobulin measurement in fine-needle aspiration washouts

2020 ◽  
pp. 000456322096836
Author(s):  
Florence Boux de Casson ◽  
Rémi Beloeil ◽  
Anne-Sophie Gauchez ◽  
Charlotte Oris ◽  
Monique Leban ◽  
...  
Keyword(s):  
Matrix Effect ◽  
Limit Of Detection ◽  
Needle Aspiration ◽  
Decision Threshold ◽  
Serum Samples ◽  
Cervical Lymph Nodes ◽  
Analytical Error ◽  
Low Concentrations ◽  
The Matrix ◽  
Assay Methods

Background Thyroglobulin (Tg) assay in washout fluids of fine needles, after cervical lymph nodes aspiration, is used for detecting metastases from differentiated thyroid carcinomas. Assay methods are the same as for Tg in serum. However, with non-serum samples, methods require extensive validation to notably check for the absence of matrix effect. This study fits this context. Our objectives were to assess analytic performances, in washout fluid, of eight different Tg assay methods and to compare them to validated data in serum. Methods Eleven medical laboratories participated in this study. The matrix tested was phosphate-buffer saline containing 1% bovine serum albumin (PBS-1% BSA). Samples used were dilutions, in this buffer, of Certified Reference Material (CRM 457). We verified, for all methods, the limit of detection, precision, linearity, trueness and accuracy. Results In PBS-1% BSA, the functional sensitivities (FS) were comparable to those expected for serum. All the methods were linear. The relative biases of trueness were between –24.5 and 10.2% around 1  µg/L. Total analytical error was ≤40% near the functional sensitivity values. Conclusion No quantitatively important matrix effect was observed. All the methods showed their ability to measure Tg in PBS-1% BSA, over the concentration range of interest, with acceptable total analytical error. We validated the functional sensitivity value as a decision threshold in thyroidectomized patients after treatment and with low concentrations of serum Tg.

scholarly journals Conformational changes at the active site of creatine kinase at low concentrations of guanidinium chloride

1993 ◽  
Vol 291 (1) ◽  
pp. 103-107 ◽  
Author(s):  
H M Zhou ◽  
X H Zhang ◽  
Y Yin ◽  
C L Tsou
Keyword(s):  
Creatine Kinase ◽  
Fluorescent Probe ◽  
Conformational Change ◽  
Conformational Changes ◽  
Active Site ◽  
Emission Intensity ◽  
Enzyme Inactivation ◽  
Amino Groups ◽  
Guanidinium Chloride ◽  
Low Concentrations

It has been previously reported that, during denaturation of creatine kinase by guanidinium chloride (GdmCl) or urea [Tsou (1986), Trends Biochem. Sci. 11, 427-429], inactivation occurs before noticeable conformational change can be detected, and it is suggested that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole. In this study, the thiol and amino groups at or near the active site of creatine kinase are labelled with o-phthalaldehyde to form a fluorescent probe. Both the emission intensity and anisotropy decrease during denaturation indicating exposure of this probe and increased mobility of the active site. The above conformational changes take place together with enzyme inactivation at lower GdmCl concentrations than required to bring about intrinsic fluorescence changes of the enzyme. At the same GdmCl concentration, the rate of exposure of the probe is comparable with that of inactivation and is several orders of magnitude faster than that for the unfolding of the molecule as a whole.

scholarly journals Conformational changes in gastric mucoproteins induced by caesium chloride and guanidinium chloride

1974 ◽  
Vol 141 (3) ◽  
pp. 641-646 ◽  
Author(s):  
David Snary ◽  
Adrian Allen ◽  
Roger H. Pain
Keyword(s):  
Molecular Weight ◽  
Conformational Changes ◽  
Sedimentation Coefficient ◽  
Water Structure ◽  
Water Soluble ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Gastric Mucus ◽  
Caesium Chloride ◽  
Low Concentrations

1. Caesium chloride and guanidinium chloride were shown to cause conformational changes in the high-molecular-weight mucoprotein A of water-soluble gastric mucus with no change in molecular weight. 2. Increasing concentrations of CsCl decrease the viscosity of the mucoprotein bringing about a transition which is essentially complete in 0.1m-CsCl. The shear-dependence of viscosity of the mucoprotein is abolished by low concentrations of CsCl. The normally highly expanded molecule becomes contracted in CsCl to a molecule having the same symmetry but a smaller volume and decreased solvation, in keeping with an increased sedimentation coefficient (18.7S→33S). 3. This contracted form does not revert to the native conformation on removal of the CsCl. 4. A mechanism is discussed in terms of the effect of the Cs+and Cl−ions on water structure and the water–mucoprotein interaction. 5. Guanidinium chloride causes the CsCl-treated material to expand, in keeping with a decrease in s025,w (33S→26S). This is analogous to the known unfolding effect of guanidinium chloride on proteins and suggests that guanidinium chloride solubilizes groups involved in stabilizing the contracted structure. Removal of the guanidinium chloride results in a limited aggregation of four mucoprotein molecules. 6. These results show that caution must be exercised before interpreting the physical properties of mucoproteins which have been treated with CsCl and/or guanidinium chloride.

Activation of detergent-solubilized rat hepatic 5′-iodothyronine deiodinase by NADPH and nonglutathione cytosolic components

1988 ◽  
Vol 66 (1) ◽  
pp. 71-76
Author(s):  
Kenzo Sawada ◽  
Brian C. W. Hummel ◽  
Paul G. Walfish
Keyword(s):  
Specific Activity ◽  
Microsomal Membrane ◽  
Relative Mass ◽  
Fractional Precipitation ◽  
Iodothyronine Deiodinase ◽  
Low Concentrations ◽  
Similar Dependence ◽  
Maximal Activation ◽  
Cytosolic Factors

An investigation was made of the possible role of the hepatic microsomal membrane in the activation of 5′-iodothyronine deiodinase (5′-DI) by a cytosolic activating system consisting of fraction A (relative mass (Mr) > 60000), fraction B (Mr, approximately 13 000), and NADPH. Activation of 5′-DI in washed microsomes was compared with that of a microsome extract prepared by solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate and further purification by fractional precipitation with polyethylene glycol and by DEAE-Sephacel chromatography. All 5′-DI preparations exhibited qualitatively similar dependence upon NADPH and cytosolic factors in fractions A and B for 5′-DI activation and were relatively unresponsive to NADH. Activation of solubilized preparations, unlike that of intact microsomes, was more readily inhibited by low concentrations of detergent and not inhibited by NADPH concentrations above 0.25 mM. Attempted purification of 5′-DI failed to produce a substantial increase in specific activity of the enzyme. It is concluded that, while glutathione-independent cytosolic factors and NADPH can activate 5′-DI in the absence of an intact microsomal membrane, some membrane constituents removed during solubilization and purification of the enzyme are required for maximal activation.

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