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Related Experiment Videos

Hypersensitive substrate for ribonucleases.

B R Kelemen1, T A Klink, M A Behlke

  • 1Department of Biochemistry, University of Wisconsin-Madison, WI 53706, USA.

Nucleic Acids Research
|September 3, 1999
PubMed
Summary
This summary is machine-generated.

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A novel, highly sensitive fluorescent substrate, 6-FAM-dArUdAdA-6-TAMRA, was developed for detecting ribonucleolytic activity. This assay enables rapid determination of enzyme inhibition constants, crucial for understanding enzyme function.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Assay Development

Background:

  • Ribonucleolytic activity is crucial in various biological processes.
  • Existing assays for ribonucleolytic activity often lack sensitivity.
  • Developing sensitive assays is key for studying enzyme kinetics and inhibition.

Purpose of the Study:

  • To develop a novel, hypersensitive substrate for assaying ribonucleolytic activity.
  • To characterize the substrate's performance with specific enzymes like Ribonuclease A and angiogenin.
  • To demonstrate the substrate's utility in determining enzyme inhibition constants.

Main Methods:

  • Systematic development of a fluorescence-based substrate.
  • Utilizing fluorescence quenching of fluorescein by rhodamine, modulated by a ribonucleotide.

Related Experiment Videos

  • Characterization of substrate cleavage by Bovine pancreatic ribonuclease A and human angiogenin.

    • Main Results:

    • The optimal substrate, 6-FAM-dArUdAdA-6-TAMRA, exhibits a 180-fold fluorescence increase upon cleavage.
    • Achieved high catalytic efficiency (kcat/Km) for Ribonuclease A (3.6 x 10^7 M^-1s^-1).
    • Demonstrated >10-fold increased sensitivity for angiogenin cleavage compared to existing substrates.

    Conclusions:

    • The developed substrate is the most sensitive known for detecting ribonucleolytic activity.
    • This hypersensitive assay allows for rapid determination of inhibition constants (Ki).
    • The substrate is valuable for studying enzyme kinetics and screening inhibitors.