scholarly journals Location of aromatic amino acids and helix content in Escherichia coli ribonucleic acid polymerase

1971 ◽  
Vol 123 (1) ◽  
pp. 117-122 ◽  
Author(s):  
B. H. Nicholson
Keyword(s):  
Escherichia Coli ◽  
Aromatic Amino Acids ◽  
Difference Spectrum ◽  
Optical Rotatory Dispersion ◽  
Intact Protein ◽  
Model Compounds ◽  
Tyrosine Residues ◽  
Tryptophan Residues ◽  
Direct Spectrum ◽  
The Difference

1. The perturbing effect of glycerol on the direct spectrum of Escherichia coli DNA-dependent RNA polymerase has been studied. 2. By comparison with model compounds and with the unfolded polymerase in 3.8m-urea it was possible to determine the ratio of tyrosine and tryptophan residues present. On reduction of the urea-treated enzyme with 2-mercaptoethanol, no further change in the difference spectrum occurred. 3. The amino acid composition of the enzyme is given. 4. In the intact protein approx. 30% of the tryptophan and 54% of the tyrosine residues were exposed. In conjunction with the extinction value and molecular weight this corresponded to 7 tryptophan residues and 57 tyrosine residues on the surface and 16 tryptophan residues and 48 tyrosine residues ‘buried’. 5. The optical rotatory dispersion of the enzyme was unaffected by 20% glycerol. 6. The helix content calculated from Moffit plots over 560–300nm was 13%, and from the 233nm trough 13%.

scholarly journals 19F-n.m.r. studies of ligand binding to 5-fluorotryptophan- and 3-fluorotyrosine-containing cyclic AMP receptor protein from Escherichia coli

1990 ◽  
Vol 266 (2) ◽  
pp. 545-552 ◽  
Author(s):  
F Sixl ◽  
R W King ◽  
M Bracken ◽  
J Feeney
Keyword(s):  
Escherichia Coli ◽  
Chemical Shift ◽  
Cyclic Amp ◽  
Conformational Changes ◽  
Cyclic Gmp ◽  
Chemical Shifts ◽  
Receptor Protein ◽  
Intact Protein ◽  
Tyrosine Residues ◽  
Tryptophan Residues

Two fluorine-containing analogues of the cyclic AMP receptor protein (CRP) from Escherichia coli were prepared by biosynthetic incorporation of 5-fluorotryptophan (5-F-Trp) and 3-fluorotyrosine (3-F-Tyr). The 19F-n.m.r. spectrum of the [5-F-Trp]CRP showed two signals corresponding to the two tryptophan residues, and that of the [3-F-Tyr]CRP showed six signals (two overlapping) corresponding to the six tyrosine residues: these results are as expected for a symmetrical dimer. A comparison of the 19F-n.m.r. spectra of the CRP analogues in the presence and in the absence of cyclic AMP reveals that the chemical shifts of both tryptophan residues and of two of the six tyrosine residues show differences. Since none of these residues is in direct contact with the bound nucleotide (although Trp-85 is fairly close), these shift changes must arise from induced conformational effects. The 19F-n.m.r. spectra of complexes with cyclic GMP showed chemical-shift perturbations different from those caused by cyclic AMP, indicating that different conformational changes are induced by the binding of cyclic GMP. The 19F-n.m.r. spectrum of the complex of [3-F-Tyr]CRP with tubercidin 3′,5′-(cyclic)monophosphate (which can activate transcription) showed essentially the same chemical-shift changes as seen for the cyclic AMP complex, indicating that similar conformational changes have been induced by the nucleotide binding. [3-F-Tyr]CRP in the presence of an equimolar amount of the 20 bp self-complementary DNA oligomer 5′-AATGTGAGTTAACTCACATT-3′ and excess cyclic AMP gave an 19F-n.m.r. spectrum that was almost identical with that for the [3-F-Tyr]CRP-cyclic AMP complex, indicating that the binding of DNA does not induce significant conformational changes involving the tyrosine residues. Proteolysis of [3-F-Tyr]CRP with chymotrypsin produced a 31 kDa fragment that is a dimer containing the cyclic AMP-binding domain. This fragment contains five of the six tyrosine residues, and its 19F-n.m.r. chemical shifts were essentially the same as those of the intact protein except for one missing signal (signal F): this signal could be assigned to Tyr-206 and shown to be unperturbed by the binding of cyclic nucleotide to the intact [3-F-Tyr]CRP. The similarity of the 19F-n.m.r. chemical shifts in the alpha-fragment and the intact CRP indicates that the alpha-fragment retains the same structure as found in the intact protein.(ABSTRACT TRUNCATED AT 400 WORDS)

The interaction of bovine milk caseins with the detergent sodium dodecyl sulphate. II. The effect of detergent binding on spectral properties of caseins

1970 ◽  
Vol 37 (2) ◽  
pp. 259-267 ◽  
Author(s):  
G. C. Cheeseman ◽  
Dorothy J. Knight
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Difference Spectrum ◽  
Temperature Dependent ◽  
Difference Spectra ◽  
Negative Change ◽  
Tryptophan Residues ◽  
Hydrophobic Binding ◽  
The Difference

SummaryThe dissociation of casein aggregates by the detergent sodium dodecyl sulphate (SDS) gave rise to difference spectra and these spectra were characteristic for each of the different types of casein. Increase in absorption by the chromophore groups, tyrosine and tryptophan, when αs1- and β-casein aggregates were dissociated indicated binding of the detergent at regions of the molecule containing these residues. A decrease in absorption when κ-casein was dissociated indicated that the tyrosine and tryptophan residues were not in the region of the molecule to which the detergent was bound and that in the κ-casein aggregate these residues were in a more hydrophobic environment. Peaks on the difference spectra were obtained at 280 and 288 nm for αs1-casein and 284 and 291 nm for β-casein and troughs at 278 and 286 nm for κ-casein. The difference spectrum reached a maximum value when the αsl- and β-casein aggregates were dissociated and the further binding of SDS did not alter this value. The large negative change in the difference spectrum of κ-casein did not occur until after most of the aggregates were dissociated and did not reach a maximum until binding with SDS was complete. The value obtained for ΔOD was found to be temperature-dependent for β-casein-SDS interaction, but not for αs1- and κ-casein. Changes in spectra were also observed when αs1- and κ-casein interacted to form aggregates. The data obtained confirmed the importance of hydrophobic binding in casein aggregate formation and indicated the possible involvement of tyrosine and tryptophan residues in this binding.

scholarly journals Effects of anions on a monomeric and a dimeric arginine kinase

1975 ◽  
Vol 149 (2) ◽  
pp. 387-395 ◽  
Author(s):  
E O Anosike ◽  
D C Watts
Keyword(s):  
Creatine Kinase ◽  
Mixed Type ◽  
Arginine Kinase ◽  
Difference Spectrum ◽  
Ionization State ◽  
Temperature Dependent ◽  
Tyrosine Residues ◽  
Dead End ◽  
Low Concentrations ◽  
The Difference

1. Some effects of anions on the rates of phosphoarginine synthesis by monomeric (lobster) and by dimeric (Holothuria forskali) arginine kinases are reported. 2. As with creatine kinase, acetate ions activate both enzymes: Cl- was also found to activate both although this was an inhibitor of creatine kinase. 3. NO3- inhibits the lobster enzyme. Inhibition is of the mixed type with respect to MgATP. Ki > Ki' and Ks > Ks' indicating that the presence of NO3- promotes the binding of substrate and vice versa. 4. NO3- alone has no effect on the difference spectrum of the lobster enzyme but in the presence of arginine, MgATP, MgADP, MgAMP or MgIDP the difference spectrum is greatly enhanced. A profound effect on the ionization state of tyrosine residues is inferred. 5. With the Holothuria enzyme low concentrations of NO3- activate in a manner that is competitive with arginine. Higher concentrations cause inhibition of the mixed type with respect to arginine in a similar manner to that found with MgATP for the lobster kinase. 6. Of a range of anions tested only NO3- and NO2- enhanced the inhibition of enzyme activity by MgADP, indicating the formation of a pseudo-transition-state dead-end complex, enzyme-arginine-NO3--MgADP. The effect was essentially independent of temperature with the Holothuria enzyme, but with the lobster enzyme was much less marked and temperature dependent. The difference may reflect the different stabilities of the monomer and dimer enzymes, although with neither arginine kinase is the stabilization of the dead-end complex as marked as is found with creatinine kinase.

Über die Einwirkung von freien Sauerstoffradikalen auf Rinderserumalbumin in Lösung

1963 ◽  
Vol 18 (8) ◽  
pp. 618-630
Author(s):  
J. Stauff ◽  
R. Jaenicke ◽  
H. Wolf
Keyword(s):  
Phosphate Buffer ◽  
Uv Light ◽  
Covalent Bond ◽  
Difference Spectrum ◽  
Optical Rotatory Dispersion ◽  
Fenton’S Reagent ◽  
Fenton's Reagent ◽  
Molecular Weights ◽  
Dissociation Equilibrium ◽  
The Difference

In order to determine the influence of OH and O2H-radicals on proteins, bovine serum albumin (BSA) in aqueous solution was treated with Fenton’s reagent [Fe(II)SO4+EDTA+H2O2] and with ultraviolet light (λ > 2800 Å) in the presence of H2O2. The action of free radicals produced in this way did not change the properties of the native protein with respect to the sedimentation in the ultracentrifuge or optical rotatory dispersion and electrophoresis under normal conditions. Ampèrometric titration indicated partial oxidation of SH-groups and of 3—5 SS-groups which are not reducible by NaBH4.Heat aggregation investigated by means of light-scattering was suppressed at pH 7.5 and strongly accelerated at pH 4.6 (range of coagulation), the latter being a result of increased entropy of activation of coagulation velocity.The difference spectrum against native BSA had positive values of Δε and two maxima at 2480 and 2950 Å.Ultracentrifugation at room temperature in phosphate buffer (pH 7.3, μ=0.18) furnishes a molecular weight of 63 300. In a solution of 8 M urea and borate buffer (pH 9, μ=0.05) fragments with molecular weights between 25 000 and 37 000 were observed while in phosphate buffer (pH 7.3, without urea) at temperatures higher than 46 °C an anomalous behaviour of the concentration gradient indicated an effect which possibly depends on a dissociation equilibrium.As a consequence oxygen radicals seem to attack not only SH- and SS-groups but at least one covalent bond of the peptide chain. Some experiments of heat aggregation with BSA treated with γ-rays (60Co) gave the same results as BSA treated with Fenton’s reagent or UV-light+H2O2.

Properties of P503 in Escherichia coli

1973 ◽  
Vol 19 (10) ◽  
pp. 1239-1242
Author(s):  
Joyce Kamitakahara-Pearlstone ◽  
Rozanne Poulson ◽  
W. J. Polglase
Keyword(s):  
Escherichia Coli ◽  
Protoporphyrin Ix ◽  
Difference Spectrum ◽  
Proliferating Cells ◽  
Intact Cells ◽  
E Coli ◽  
The Difference ◽  
Porphyrin Synthesis ◽  
Natural Amino Acids ◽  
K 12

The pigment (believed to be a tetrahydroporphyin) which appears, transiently, at 503 nm (P503) in the difference spectrum of intact cells of Escherichia coli B did not accumulate in cultures grown in medium supplemented with L-methionine. Accumulation of P503 was not prevented by structural analogues of L-methionine, nor by a mixture of natural amino acids excluding L-methionine. A methionine-requiring mutant of E. coli K-12 lacked P503 when grown in the presence of L-methionine, but P503 was present if D,L-ethionine was substituted for L-methionine in the growth medium. When L-methionine was added to air-oxidized cells which had been grown on minimal (glucose–salts) medium, the spectrum of P503 was produced specifically and rapidly. This effect of L-methionine in non-proliferating cells together with the inhibition by L-methionine of P503 accumulation in growing cultures may result from the stimulation of coproporphyrinogenase by methionine. The data indicate that P503 is an early intermediate in the conversion of coproporphyrinogen III to protoporphyrin IX rather than an intermediate in the porphyrin synthesis "by-pass' to coproporphyrin III.

scholarly journals Subunit interactions in horse spleen apoferritin. Dissociation by extremes of pH

1973 ◽  
Vol 133 (2) ◽  
pp. 289-299 ◽  
Author(s):  
R. R. Crichton ◽  
Charles F. A. Bryce
Keyword(s):  
Conformational Changes ◽  
Difference Spectrum ◽  
Low Ph ◽  
Tryptophan Residue ◽  
Tyrosine Residues ◽  
Ph Values ◽  
Lysine Residues ◽  
Arginine Residues ◽  
The Difference ◽  
Ph Range

1. The dissociation of horse spleen apoferritin as a function of pH was analysed by sedimentation-velocity techniques. The oligomer is stable in the range pH2.8–10.6. Between pH2.8 and 1.6 and 10.6 and 13.0 both oligomer and subunits can be detected. At pH values between 1.6 and 1.0 the subunit is the only species observed, although below pH1.0 aggregation of the subunits to a particle sedimenting much faster than the oligomer occurs. 2. When apoferritin is first dissociated into subunits at low pH values and then dialysed into buffers of pH1.5–5.0, the subunit reassociates to oligomer in the pH range 3.1–4.3. 3. U.v.-difference spectroscopy was used to study conformational changes occurring during the dissociation process. The difference spectrum in acid can be accounted for by the transfer of four to five tyrosine residues/subunit from the interior of the protein into the solvent. This process is reversed on reassociation, but shows the same hysteresis as found by sedimentation techniques. The difference spectrum in alkali is more complex, but is consistent with the deprotonation of tyrosine residues, which appear to have rather high pK values. 4. In addition to the involvement of tyrosine residues in the conformational change at low pH values, spectral evidence is presented that one tryptophan residue/subunit also changes its environment before dissociation and subsequent to reassociation. 5. Analysis of the dissociation and reassociation of apoferritin at low pH values suggests that this is a co-operative process involving protonation and deprotonation of at least two carboxyl functions of rather low intrinsic pK. The dissociation at alkaline pH values does not appear to be co-operative. 6. Of the five tyrosine residues/subunit only one can be nitrated with tetranitromethane. Guanidination of lysine residues results in the modification of seven out of a total of nine residues/subunit. Nine out of the ten arginine residues/subunit react with cyclohexanedione.

scholarly journals The distribution of plasmids among a representative collection of Scottish strains of salmonellae

1986 ◽  
Vol 97 (2) ◽  
pp. 199-204 ◽  
Author(s):  
D. J. Platt ◽  
D. J. Brown ◽  
D. S. Munro
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Antibiotic Resistant ◽  
R Plasmids ◽  
The Difference

SummaryThe distribution of plasmids was studied in a representative collection of salmonella strains which comprised 98Salmonella typhimuriumand 96 other serotypes. Plasmids were detected in 72% of strains (mean 1·3 plasmids/strain) and individual strains harboured between 0 and 7 plasmids. They were more common amongS. typhimuriumthan other serotypes (incidence 92 and 53%; mean 1·9 and 0·8 plasmids/strain respectively). Although a higher proportion ofS. typhimurium(33%) were antibiotic-resistant compared to other serotypes (14%) the evidence presented indicated that R-plasmids were not responsible for the difference observed in the number and distribution of plasmids in these strains. These results were discussed in comparison with similar studies ofEscherichia coliand other enteric genera.

Difference Spectrophotometric Estimation of Santonin

1984 ◽  
Vol 67 (5) ◽  
pp. 939-941
Author(s):  
Abdel-Azim M Habib ◽  
Abdalla A Omar ◽  
Taha M Sarg
Keyword(s):  
Difference Spectrum ◽  
Crude Drug ◽  
The Difference

Abstract Santonin gives a characteristic alkaline vs acidic difference spectrum. This was used for its estimation in pharmaceuticals and in the crude drug. Santonin was first extracted and purified through a specific partition procedure; then the difference absorbance was measured either at the maximum, 285 nm, or the minimum, 242 nm. The percentage of santonin can be calculated either by reference to the difference absorbance of a reference santonin sample, treated similarly, or by making use of the determined absorptivity. Measurement at the maximum is advisable, especially when the crude drug is assayed, because natural contaminants may interfere with the difference absorbance at the minimum.

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