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A nitrocellulose filter binding assay for ribonucleotide reductase.

K Söderman, P Reichard

    Analytical Biochemistry
    |January 1, 1986
    PubMed
    Summary
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    Researchers developed a new, sensitive method to measure nucleotide binding to ribonucleotide reductase enzymes. This nitrocellulose filter assay provides results comparable to equilibrium dialysis for studying enzyme kinetics and purification.

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Enzymology

    Background:

    • Escherichia coli ribonucleotide reductase (RNR) is a crucial enzyme for DNA synthesis.
    • Protein B1, a subunit of E. coli RNR, possesses distinct nucleoside triphosphate binding sites (h-sites and l-sites) that regulate enzyme activity and substrate specificity.
    • Traditional methods like equilibrium dialysis are used to characterize these binding interactions.

    Purpose of the Study:

    • To introduce a sensitive, alternative method for measuring nucleotide binding to RNR.
    • To validate this new method against established techniques.
    • To highlight the utility of the new method for various RNR research applications.

    Main Methods:

    • Development of a nitrocellulose filter binding assay utilizing protein-specific binding of radioactive nucleotides.

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  • Comparison of results obtained from the nitrocellulose filter assay with those from equilibrium dialysis.
  • Application of the assay for studying pure reductases, characterizing mutants, and during purification.

    • Main Results:

    • The nitrocellulose filter binding assay demonstrated comparable results to equilibrium dialysis for measuring nucleotide binding.
    • The assay proved to be sensitive and effective for nucleotide binding studies.
    • The method successfully identified high-affinity (h-sites) and lower-affinity (l-sites) binding characteristics.

    Conclusions:

    • A novel, sensitive nitrocellulose filter assay provides a reliable alternative to equilibrium dialysis for studying nucleotide binding to RNR.
    • This method is broadly applicable for RNR research, including studies on purified enzymes from various sources, mutant characterization, and enzyme purification.
    • The assay facilitates a deeper understanding of RNR allosteric regulation and enzyme kinetics.