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).

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.

Characterization of Tamm-Horsfall Urinary Glycoprotein

1973 ◽  
Vol 31 ◽  
pp. 420-421
Author(s):  
John P. Robinson ◽  
J. David Puett
Keyword(s):  
Electron Microscopy ◽  
Circular Dichroism ◽  
Secondary Structure ◽  
Quaternary Structure ◽  
Normal Adult ◽  
Morphological Properties ◽  
Adult Males ◽  
Circular Dichroïsm ◽  
Urinary Glycoprotein

Much work has been reported on the chemical, physical and morphological properties of urinary Tamm-Horsfall glycoprotein (THG). Although it was once reported that cystic fibrotic (CF) individuals had a defective THG, more recent data indicate that THG and CF-THG are similar if not identical.No studies on the conformational aspects have been reported on this glycoprotein using circular dichroism (CD). We examined the secondary structure of THG and derivatives under various conditions and have correlated these results with quaternary structure using electron microscopy.THG was prepared from normal adult males and CF-THG from a 16-year old CF female by the method of Tamm and Horsfall. CF female by the method of Tamm and Horsfall.

Germin: physicochemical properties of the glycoprotein which signals onset of growth in the germinating wheat embryo

1987 ◽  
Vol 65 (12) ◽  
pp. 1039-1048 ◽  
Author(s):  
William C. McCubbin ◽  
Cyril M. Kay ◽  
Theresa D. Kennedy ◽  
Byron G. Lane
Keyword(s):  
Circular Dichroism ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Helical Structure ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Cross Linking ◽  
Wheat Embryo ◽  
Circular Dichroïsm

The size and structure of germin, the homooligomeric glycoprotein which marks the onset of growth in germinating wheat embryos, has been examined by gel filtration, ultracentrifugation, electron microscopy, chemical cross-linking, and optical techniques (circular dichroism). Germin has a sedimentation coefficient (S20,w) of 7.3S, and a Stokes' radius (RS) of 4.5 nm, the latter value being compatible with the dimensions of the particle observed by negative staining in the electron microscope. By three methods (sedimentation equilibrium, sodium dodecyl sulphate (SDS) – polyacrylamide electrophoresis, S20,w/RS), the mean particle mass of the two closely related forms of germin (G and G′) is ca. 130 kilodaltons (kDa). Cross-linking with dimethyl suberimidate indicates that the oligomer is homopentameric, compatible with the molecular mass of the protomer (ca. 26 kDa) as determined by SDS–polyacrylamide gel electrophoresis. Using the Provencher and Glockner analysis to interpret circular dichroism measurements (in the far ultraviolet), both forms of germin contain about 10–20% α-helical structure, 50–60% β-sheet/turn structure, and 20–30% random coil. In a structure-inducing environment (45% trifluoroethanol), the α-helical structure increases to a value (35–40%) similar to that predicted by Chou–Fasman analysis of the protein sequence deduced by cDNA sequencing.

Physical studies on the ribosomal "A" protein from two archaebacteria, Halobacterium cutirubrum and Methanobacterium thermoautotrophicum

1984 ◽  
Vol 62 (1) ◽  
pp. 44-48 ◽  
Author(s):  
A. T. Gudkov ◽  
S. Yu Venyaminov ◽  
A. T. Matheson
Keyword(s):  
Escherichia Coli ◽  
Circular Dichroism ◽  
Secondary Structure ◽  
Quaternary Structure ◽  
Methanobacterium Thermoautotrophicum ◽  
Sedimentation Equilibrium ◽  
Effect Of Temperature ◽  
Extreme Halophile ◽  
Moderate Halophile ◽  
Circular Dichroïsm

Physical studies on the effect of temperature and ionic conditions on the secondary, tertiary, and quaternary structure of the ribosomal "A" protein, equivalent to L7/L12 in Escherichia coli, from two archaebacteria were performed using circular dichroism and sedimentation equilibrium measurements. The two archaebacteria investigated were Halobacterium cutirubrum, an extreme halophile, and Methanobacterium thermoautotrophicum, a thermophile which also showed properties of a moderate halophile. The changes in the secondary structure and the thermostability of these proteins were directly related to the internal salt concentrations of the two archaebacteria. At the higher salt concentrations the changes in the secondary structure resulted in changes in the tertiary and quaternary structure of these proteins.

scholarly journals Conformational studies of biliproteins from the insects pieris brassicae and cerura vinula

1988 ◽  
Vol 43 (1-2) ◽  
pp. 84-90 ◽  
Author(s):  
Hugo Scheer ◽  
Hartmut Kayser
Keyword(s):  
Circular Dichroism ◽  
Optical Activity ◽  
Fluorescence Spectra ◽  
Protein A ◽  
Protein Secondary Structure ◽  
Helical Structure ◽  
Pieris Brassicae ◽  
Bile Pigments ◽  
Urea Denaturation ◽  
Circular Dichroïsm

Chromophore conformation and protein secondary structure of biliproteins from the butterfly, Pieris brassicae, and the moth, Cerura vinula, have been investigated by absorption, circular dichroism and fluorescence spectroscopy. The chromophore of the P. brassicae protein, biliverdin IXy, has probably a cyclic-helical structure similar to that of free bile pigments of the biliverdin type. Though achiral by structure the chromophore displays strong optical activity in the native protein-bound state, but becomes inactive after urea denaturation of the protein. A minor biliprotein from P. brassicae shows absorption, circular dichroism and fluorescence spectra identical to the main biliprotein. In the biliprotein from Cerura vinula the structure of the pigment is still unknown. It has a semi-open conformation intermediate between that of the Pieris proteins and that of the phycobiliprotein, C-phycocyanin, and it retains optical activity after urea denaturation. The band widths and the size of the Stokes shifts of the fluorescence spectra indicate a high degree of conformational flexibility of the chromophores in the two Pieris pigments, and a decreased flexibility in the one from Cerura. In the biliproteins from both insects the polypeptides are low in a-helix content compared to that of phycobiliproteins. From these and earlier data, insect and algal biliproteins seem to be related only distantly if at all, but there exist also considerable differences among insect biliproteins from different species

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.

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