Molecular weight of Escherichia coli beta-galactosidase in concentrated solutions of guanidine hydrochloride.
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ABSTRACT:
SUBMITTER: Erickson RP
PROVIDER: S-EPMC1179595 | biostudies-other | 1970 Nov
REPOSITORIES: biostudies-other
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Project description:Two sets of iso-1-cytochrome c variants have been prepared with N-terminal insertions of pure polyglutamine, i.e., PolyQ variants, or polyglutamine interrupted with lysine every sixth residue, i.e., Gln-rich variants. The polymer properties of these pure polyGln or Gln-rich sequences have been evaluated using equilibrium and kinetic His-heme loop formation methods for loop sizes ranging from 22 to 46 in 1.5, 3.0, and 6.0 M guanidine hydrochloride (GdnHCl). In 6.0 M GdnHCl, the scaling exponent, ν(3), for the pure polyGln sequences, is ~1.7--significantly less than ν(3) ≈ 2.15 for the Gln-rich sequences. The stability of the His-heme loops becomes progressively greater for the pure polyGln sequences relative to the Gln-rich sequences as GdnHCl concentration decreases from 6.0 to 1.5 M. Thus, the context of the sequence effects the polymer properties of Gln repeats even in denaturing concentrations of GdnHCl. Comparison of data for the Gln-rich variants with previous results for Gly-rich and Ala-rich variants shows that ν(3) ~ 2.2 for the Gln-rich, Gly-rich, and Ala-rich sequences in 6.0 M GdnHCl, whereas ν(3) remains unchanged at 3.0 M GdnHCl concentration for the Gln-rich and Ala-rich sequences but decreases to ~1.7 for the Gly-rich sequences. Thus, the polymer properties of Gln-rich and Ala-rich sequences are less sensitive to solvent quality in denaturing solutions of GdnHCl than Gly-rich sequences. Evaluation of Flory's characteristic ratio, C(n), for the Gln-rich and Ala-rich sequences relative to the Gly-rich sequences shows that Gln-rich sequences are stiffer than Ala-rich sequences at both 3.0 and 6.0 M GdnHCl.
| S-EPMC3491724 | biostudies-literature
Project description:The intrinsic polymer properties of glycine-rich sequences are evaluated with a set of iso-1-cytochrome c variants with N-terminal inserts of the sequence (GGGGGK)(n) for n = 1-5. The thermodynamics and kinetics of His-heme loop formation are measured as a function of guanidine hydrochloride (GdnHCl) concentration for loop sizes ranging from 22 to 46 residues. The scaling exponent for loop formation, ?(3), evaluated using the Jacobson-Stockmayer equation is near 1.8, at 1.5 and 3.0 M GdnHCl, but it increases to 2.2 in 6.0 M GdnHCl. Previous work on a set of iso-1-cytochrome c variants with (AAAAAK)(n) inserts gave ?(3) = 2.2 for alanine-rich sequences in both 3.0 and 6.0 M GdnHCl. Chain stiffness was evaluated from the relative magnitude of Flory's characteristic ratio, C(n), for alanine-rich versus glycine-rich sequences. In 3.0 M GdnHCl, C(n)(Ala)/C(n)(Gly) is 1.6, decreasing to 1.3 in 6.0 M GdnHCl. The data suggest that solvent-backbone interactions dominate polypeptide conformational properties under good solvent conditions whereas side-chain-dependent properties are more important under poor solvent conditions. The results provide a direct experimental assessment in terms of polymer properties of the distinct roles of Gly versus Ala in the folding code.
| S-EPMC3328712 | biostudies-literature
Project description:1. Repression by glucose of beta-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i(-)) and permease-less (y(-)) cells as well as in the corresponding i(+) and y(+) strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-beta-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of beta-galactosidase synthesis (e.g. isopropyl thio-beta-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-beta-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.
| S-EPMC1265142 | biostudies-other
Project description:1. 5-Phosphorylribose 1-pyrophosphate, in the presence of beta-mercaptoethanol, protected beta-galactosidase from heat inactivation. Many other substances, including 3':5'-cyclic-AMP, were without effect. 2. The efficiency of complementation in vitro of beta-galactosidase segments was decreased by 5-phosphorylribose 1-pyrophosphate but not by 3':5'-cyclic-AMP. Neither substance affected the activity of the complete enzyme. 3. Some indications as to the possible identity of the catabolite repression effector are presented.
| S-EPMC1179217 | biostudies-other
Project description:Carboxymethylated beta-galactosidase from Escherichia coli was dissociated at 100 degrees C to form carboxymethylated fragments A and B. The mol.wts. of carboxymethylated fragments A and B were determined by gel filtration to be 64300 and 22400 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of carboxymethylated fragments A and B that had been pretreated with 2-mercaptoethanol and sodium dodecyl sulphate yielded mol.wts. of 64000 and 22100 respectively. Carboxymethylated fragments A and B had arginine as their C-terminal amino acid. When a crude extract of E. coli M15 was filtered through a column of Sepharose 6B, it was found that carboxymethylated fragment B could restore beta-galactosidase activity when added to fractions having mol.wts. estimated to be 123000, 262000 and 506000. These fractions are referred to as ;complementable fractions'. Similarly, it was found that carboxymethylated fragment A could restore enzyme activity to tractions having mol.wts. estimated to be 63000, 253000 and 506000. Estimates of the molecular weights of the beta-galactosidase activity obtained by restoration with carboxymethylated fragments A and B were made by filtering the active enzyme through another column of Sepharose 6B. The enzyme obtained by complementation with carboxymethylated fragment B, i.e. the complemented enzyme, had mol.wt. 525000, and that obtained with carboxymethylated fragment A had mol.wts. of 525000, 646000 and 2000000. The latter finding suggests that multiple forms of complemented beta-galactosidase can exist.
| S-EPMC1164922 | biostudies-other
Project description:BETA-Galactosidase (EC 3.2.1.23), prepared from strains ML 308 and K12 3300 of Escherichia coli, dissociated into an inactive monomer in the presence of Ag+. When such a monomer preparation is treated with excess of thiol an enzymically active dimer is formed in addition to an active tetramer. It is suggested that Ag+ may be of value in studies on other multimeric proteins as a mild dissociating agent.
| S-EPMC1172374 | biostudies-other
Project description:Rapid and accurate sensing of β-galactosidase (β-gal) activity is particularly critical for the early detection of many diseases and has become a topic of interest in recent years. However, most traditional probes for β-gal sensing often suffer from the disadvantages of narrow dynamic range, low reaction efficiency and are only employed with either colorimetric or fluorescence sensing. Furthermore, β-galactosidase sensing based assay for efficient detection and antibiotic resistance analysis of Escherichia coli (E.coli) is not available. Here, an enzyme-induced probe assay was reported for dual sensitive fluorescence and colorimetric measurement of β-gal activity, and was further employed for detection of Escherichia coli and their antibiotic resistance analysis. The DCM-βgal probe was virtually non-emissive in aqueous solution, while it could be activated by β-gal to produce bright emission. Under optimized conditions, DCM-βgal displayed high sensitivity, selectivity and rapid response to β-gal with a low detection limit of 1.5 × 10-3 U ml-1. Importantly, this assay was successfully applied to sensitive detection of E. coli cells with a fast detection process within 5 h and a low detection concentration of 1 × 103 CFU ml-1. Furthermore, the enzyme-activatable assay was also successfully applied for high throughput E. coli antibiotic resistance analysis. The DCM-βgal strategy is applied for the first time on the detection of E. coli cells and their antibiotic resistance analysis. It is provided with the advantages of high selectively, a simple operation, low cost and rapid detection. The detection platform can also be extended to analyze the level of β-gal in other types of cells or biological samples. Overall, the simple, effective and dual-readout assay holds promise for efficient sensing of β-gal activity and provides a potential tool for E. coli detection and their antibiotic resistance analysis.
| S-EPMC9659582 | biostudies-literature
Project description:BACKGROUND: Sequence periodicity with a period close to the DNA helical repeat is a very basic genomic property. This genomic feature was demonstrated for many prokaryotic genomes. The Escherichia coli sequences display the period close to 11 base pairs. RESULTS: Here we demonstrate that practically only ApA/TpT dinucleotides contribute to overall dinucleotide periodicity in Escherichia coli. The noncoding sequences reveal this periodicity much more prominently compared to protein-coding sequences. The sequence periodicity of ApC/GpT, ApT and GpC dinucleotides along the Escherichia coli K-12 is found to be located as well mainly within the intergenic regions. CONCLUSIONS: The observed concentration of the dinucleotide sequence periodicity in the intergenic regions of E. coli suggests that the periodicity is a typical property of prokaryotic intergenic regions. We suppose that this preferential distribution of dinucleotide periodicity serves many biological functions; first of all, the regulation of transcription.
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Project description:Genetically encoded reporter proteins are important and widely used tools for the identification and capture of a promoter, tracking the dynamic behavior of transcription, and the quantification of promoter activity. The sensitivity of the reporter gene is a critical factor for an ideal reporter system because weak transcriptional signal has usually failed to be detected using classical reporters. In this study, we present a novel reporter system for improved monitoring of transcription in E. coli based on β-galactosidase α-complementation. In this reporter system, the β-galactosidase activity resulting from the assembly of a reporter lacZα and an existing α-acceptor in advance serves as a measure of transcriptional activity in vivo. To validate the potential of the lacZα-derived reporter system, a series of artificial operons were constructed, and the moderately strong lac promoter, ara promoter, and weak pbr promoter were chosen as the detection promoters. The response profiles of lacZα was similar to that of wild type lacZ in artificial lac operons. Due to its small size and efficient expression profile, the detection sensitivity of a lacZα-derived reporter system was significantly higher than that of the traditional full-length β-galactosidase and the fluorescent protein mCherry reporter system in artificial ara operons. As expected, the response sensitivity of the lacZα-derived reporter system was also demonstrated to be significantly higher than that of the β-galactosidase and mCherry reporter systems in lead-sensitive artificial pbr operons. The lacZα-derived reporter system may prove to be a valuable tool for detecting promoter activity, especially low-level transcription in vivo.
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Project description:It is generally accepted that the hydroxide ion (OH-) is a strong hydrogen bond acceptor and that its anomalously high diffusion constant in water results from a Grotthuss-like structural diffusion mechanism. However, the spatial extent over which OH- ions influence the dynamics of the hydrogen-bond network of water remained largely unclear. Here, we measure the ultrafast dynamics of OH groups of HDO molecules interacting with the deuterated hydroxide ion OD-. For solutions with OD- concentrations up to 4 M, we find that HDO molecules that are not directly interacting with the ions have a reorientation time constant of ∼2.7 ps, similar to that of pure liquid water. When the concentration of OD- ions is increased, the reorientation time constant increases, indicating a strong slowing down of the structural dynamics of the solution.
| S-EPMC7520889 | biostudies-literature