Continuous spectrometric assays for glutaminyl cyclase activity

Analytical Biochemistry
Stephan SchillingHans-Ulrich Demuth

Abstract

The enzymatic conversion of one chromogenic substrate, l-glutamine-p-nitroanilide, and two fluorogenic substrates, l-glutaminyl-2-naphthylamide and l-glutaminyl-4-methylcoumarinylamide, into their respective pyroglutamic acid derivatives by glutaminyl cyclase (QC) was estimated by introducing a new coupled assay using pyroglutamyl aminopeptidase as the auxiliary enzyme. For the purified papaya QC, the kinetic parameters were found to be in the range of those previously reported for other glutaminyl peptides, such as Gln-Gln, Gln-Ala, or Gln-tert-butyl ester. The assay can be performed in the presence of ammonia up to a concentration of 50 mM. Increasing ionic strength, e.g., potassium chloride up to 300 mM, resulted in an increase in enzymatic activity of about 20%. This is the first report of a fast, continuous, and reliable determination of QC activity, even in the presence of ammonium ions, during the course of protein purification and enzymatic analysis.

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Citations

Aug 27, 2013·PloS One·Cristiana CastaldoPaola Turano
Jan 15, 2004·Biological Chemistry·Stephan SchillingHans-Ulrich Demuth
May 13, 2014·Toxicon : Official Journal of the International Society on Toxinology·Ying-Ming WangInn-Ho Tsai
Jun 3, 2014·International Journal of Developmental Neuroscience : the Official Journal of the International Society for Developmental Neuroscience·Corinna HöflingMaike Hartlage-Rübsamen
Jun 19, 2013·Insect Biochemistry and Molecular Biology·Steven W AdamsonShahid Karim
Oct 7, 2009·The FEBS Journal·Anett StephanStephan Schilling
Jan 12, 2008·Clinica Chimica Acta; International Journal of Clinical Chemistry·Anastassia GontsarovaHeinrich Brinkmeier
Apr 8, 2015·Nature Chemical Biology·Maria Jimenez-SanchezDavid C Rubinsztein
Apr 6, 2004·FEBS Letters·Stephan SchillingHans-Ulrich Demuth
Apr 28, 2017·The Journal of Pharmacology and Experimental Therapeutics·Torsten HoffmannStephan Schilling
Jul 3, 2020·Journal of Neurochemistry·Adam P GunnUNKNOWN AIBL Research Group
Oct 3, 2015·Alzheimer's & Dementia : Translational Research & Clinical Interventions·Inge LuesKonrad Glund
May 19, 2021·Journal of Medicinal Chemistry·Chenshu XuHaiqiang Wu
Oct 30, 2009·Journal of Medicinal Chemistry·Mirko BuchholzUlrich Heiser
Jul 27, 2021·Acta Neuropathologica·Maike Hartlage-RübsamenSteffen Roßner
Jan 20, 2006·Journal of Medicinal Chemistry·Mirko BuchholzHans-Ulrich Demuth

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