scholarly journals Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein

1978 ◽  
Vol 169 (2) ◽  
pp. 371-380 ◽  
Author(s):  
J A Ayala ◽  
M Nieto
Keyword(s):  
Circular Dichroism ◽  
Adenosine Triphosphatase ◽  
Thermal Denaturation ◽  
Enzymic Activity ◽  
Intrinsic Fluorescence ◽  
Guanidinium Chloride ◽  
Subunit Dissociation ◽  
Micrococcus Lysodeikticus ◽  
Maximum Stability ◽  
Circular Dichroïsm

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.

scholarly journals Optical properties and denaturation by guanidinium chloride and urea of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of four molecular forms of the enzyme

1977 ◽  
Vol 161 (2) ◽  
pp. 321-331 ◽  
Author(s):  
M Nieto ◽  
J A Ayala
Keyword(s):  
Circular Dichroism ◽  
Adenosine Triphosphatase ◽  
Quaternary Structure ◽  
Molecular Form ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Molecular Forms ◽  
Guanidinium Chloride ◽  
Micrococcus Lysodeikticus ◽  
Circular Dichroïsm

1. The fluorescence and circular dichroism of four homogeneous preparations of ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus differing in molecular structure and enzymic properties were examined at pH 7.5 and 25 degrees. Emission was maximum at 325 and 335 nm and the relative intensities at these wavelengths may be used to characterize the different ATPase preparations. The circular-dichroism spectra exhibited negative extrema at 208 and 220 nm, and the relative value of the molar ellipticity at these wavelengths was also different for each molecular form of the enzyme. 2. The four preparations undergo two consecutive major unfolding transitions in guanidinium chloride (midpoints at 0.94 and 1.5 M denaturant), with concomitant destruction of the quaternary structure of the protein. A comparatively minor alteration in the ATPase structure also occurred in 0.05-0.2M-guanidine and led to complete inactivation of the enzyme. The inactivation and the first unfolding transition were reversible by dilution of the denaturant; the transition with midpoint at 1.5M-guanidine was irreversible. 3. Similar results were obtained in urea, except that the successive transitions had midpoints at concentrations of denaturant of 0.4, 2.0 and 4.5M. Low concentrations of urea caused a noticeable activation of the enzyme activity and alterations of the electrophoretic mobility of the ATPase. 4. A model is proposed in which one of the major subunits, alpha, is first dissociated and unfolded reversibly by the denaturants, followed by the irreversible unfolding and dissociation of the other major subunit, beta, from subunit delta and/or the components of relative mobility 1.0 in dodecyl sulphate/polyacrylamide-gel electrophoresis (rho).

The thermal denaturation of bovine cardiac G-actin

1977 ◽  
Vol 55 (4) ◽  
pp. 325-331 ◽  
Author(s):  
C. C. Contaxis ◽  
C. C. Bigelow ◽  
C. G. Zarkadas
Keyword(s):  
Circular Dichroism ◽  
Conformational Change ◽  
Thermodynamic Analysis ◽  
Thermal Denaturation ◽  
State Transition ◽  
Thermal Unfolding ◽  
Difference Spectroscopy ◽  
Maximum Stability ◽  
Ph Range ◽  
Circular Dichroïsm

The thermal denaturation of bovine cardiac G-actin has been studied by ultraviolet difference spectroscopy and circular dichroism between pH 7.5 and 10.5. As with proteins previously studied, thermal unfolding is incomplete compared with unfolding by urea or GuHCl. However, the same conformational change is observed over the pH range studied, and the available evidence indicates it is a two-state transition. Thermodynamic analysis of the data shows that ΔH0 and ΔS0 are strongly dependent on the temperature, that ΔCp is 1300 cal deg−1 mol−1, and that G-actin has a temperature of maximum stability near −5 °C.

scholarly journals Fluorescence and circular-dichroism studies on the Streptomyces R61 dd-carboxypeptidase–transpeptidase. Penicillin binding by the enzyme

1973 ◽  
Vol 135 (3) ◽  
pp. 493-505 ◽  
Author(s):  
Manuel Nieto ◽  
Harold R. Perkins ◽  
Jean-Marie Frère ◽  
Jean-Marie Ghuysen
Keyword(s):  
Circular Dichroism ◽  
Thermal Denaturation ◽  
Penicillin G ◽  
Affinity Binding ◽  
Guanidinium Chloride ◽  
Positive Maximum ◽  
Peptide Substrates ◽  
High Affinity ◽  
Circular Dichroïsm ◽  
New Hypothesis

The circular dichroism of the dd-carboxypeptidase–transpeptidase from Streptomyces R61 shows in the near u.v. a set of weak extrema at 289nm (positive) and at 282, 275 and 268nm (all negative). In the far u.v. it shows negative extrema at 217–218 and 208nm, crossover at 202nm and a positive maximum at about 194nm. The u.v. absorption of the enzyme shows it to contain tyrosine and tryptophan in approx. 3.4:1 ratio. The enzyme is fluorescent with a maximum emission at 318–320nm. The near-u.v. circular dichroism of the protein is extensively affected by binding of penicillin G, but the far u.v. is unaffected. Binding of the antibiotic also causes quenching of the fluorescence of the enzyme. The latter effect has been used to study the binding of penicillin G to the enzyme and the influence exerted upon it by salts, denaturants and peptide substrates and inhibitors. High-affinity binding of penicillin appears to be comparatively slow and reversible, and can occur under conditions in which the protein is enzymically inactive. The thermal denaturation of the enzyme in guanidinium chloride at pH7 is affected by binding of the antibiotic. The presence of even large concentrations of β-mercaptoethanol neither impaired the activity of the enzyme nor prevented its inhibition by penicillin G or cephalosporin C. A new hypothesis for the molecular mechanism of the interaction of the enzyme with penicillin is proposed.

scholarly journals Conformational changes in the extracellular β-lactamase I from Bacillus cereus 569/H/9

1974 ◽  
Vol 143 (1) ◽  
pp. 137-141 ◽  
Author(s):  
Richard B. Davies ◽  
E. P. Abraham ◽  
D. G. Dalgleish
Keyword(s):  
Bacillus Cereus ◽  
Conformational Change ◽  
Conformational Changes ◽  
Thermal Denaturation ◽  
Complete Recovery ◽  
Enzymic Activity ◽  
Active Enzyme ◽  
Guanidinium Chloride ◽  
Subsequent Removal ◽  
Circular Dichroïsm

1. The thermal denaturation and precipitation of β-lactamase I from Bacillus cereus 569/H/9 at 60°C are reversible, a soluble and almost fully active enzyme being obtained after solution of the precipitate in 5m-guanidinium chloride or 8m-urea and subsequent removal of the denaturing agent. 2. Inactivation of β-lactamase I occurs rapidly between 50° and 55°C and is shown by circular-dichroism spectra to be accompanied by an extensive conformational change. 3. A change to a different conformation occurs in 6m-urea. This change is also reversible; refolding with almost complete recovery of enzymic activity occurs within 5min of dilution of the denaturing agent. 4. Inactivation of β-lactamase I at pH3.0 and 11.0 is also associated with conformational changes, since a proportion of the lost activity is recovered within 5min of adjustment of the pH to 7.0.

Effect of Temperature and pH on the Secondary Structure and Denaturation Process of Jumbo Squid Hepatopancreas Cathepsin D.

2019 ◽  
Vol 26 (7) ◽  
pp. 532-541 ◽  
Author(s):  
Cadena-Cadena Francisco ◽  
Cárdenas-López José Luis ◽  
Ezquerra-Brauer Josafat Marina ◽  
Cinco-Moroyoqui Francisco Javier ◽  
López-Zavala Alonso Alexis ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Secondary Structure ◽  
Cathepsin D ◽  
Thermal Denaturation ◽  
Protein Denaturation ◽  
Marine Organisms ◽  
Effect Of Temperature ◽  
Jumbo Squid ◽  
Α Helix ◽  
Circular Dichroïsm

Background: Cathepsin D is a lysosomal enzyme that is found in all organisms acting in protein turnover, in humans it is present in some types of carcinomas, and it has a high activity in Parkinson's disease and a low activity in Alzheimer disease. In marine organisms, most of the research has been limited to corroborate the presence of this enzyme. It is known that cathepsin D of some marine organisms has a low thermostability and that it has the ability to have activity at very acidic pH. Cathepsin D of the Jumbo squid (Dosidicus gigas) hepatopancreas was purified and partially characterized. The secondary structure of these enzymes is highly conserved so the role of temperature and pH in the secondary structure and in protein denaturation is of great importance in the study of enzymes. The secondary structure of cathepsin D from jumbo squid hepatopancreas was determined by means of circular dichroism spectroscopy. Objective: In this article, our purpose was to determine the secondary structure of the enzyme and how it is affected by subjecting it to different temperature and pH conditions. Methods: Circular dichroism technique was used to measure the modifications of the secondary structure of cathepsin D when subjected to different treatments. The methodology consisted in dissecting the hepatopancreas of squid and freeze drying it. Then a crude extract was prepared by mixing 1: 1 hepatopancreas with assay buffer, the purification was in two steps; the first step consisted of using an ultrafiltration membrane with a molecular cut of 50 kDa, and the second step, a pepstatin agarose resin was used to purification the enzyme. Once the enzyme was purified, the purity was corroborated with SDS PAGE electrophoresis, isoelectric point and zymogram. Circular dichroism is carried out by placing the sample with a concentration of 0.125 mg / mL in a 3 mL quartz cell. The results were obtained in mdeg (millidegrees) and transformed to mean ellipticity per residue, using 111 g/mol molecular weight/residue as average. Secondary-structure estimation from the far-UV CD spectra was calculated using K2D Dichroweb software. Results: It was found that α helix decreases at temperatures above 50 °C and above pH 4. Heating the enzyme above 70°C maintains a low percentage of α helix and increases β sheet. Far-UV CD measurements of cathepsin D showed irreversible thermal denaturation. The process was strongly dependent on the heating rate, accompanied by a process of oligomerization of the protein that appears when the sample is heated, and maintained a certain time at this temperature. An amount typically between 3 and 4% α helix of their secondary structure remains unchanged. It is consistent with an unfolding process kinetically controlled due to the presence of an irreversible reaction. The secondary structure depends on pH, and a pH above 4 causes α helix structures to be modified. Conclusion: In conclusion, cathepsin D from jumbo squid hepatopancreas showed retaining up to 4% α helix at 80°C. The thermal denaturation of cathepsin D at pH 3.5 is under kinetic control and follows an irreversible model.

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