Continuous spectrometric assays for glutaminyl cyclase activity

被引:50
作者
Schilling, S
Hoffmann, T
Wermann, M
Heiser, U
Wasternack, C
Demuth, HU
机构
[1] Probiodrug AG, Biochem Lab, D-06120 Halle An Der Saale, Germany
[2] Leibniz Inst Plant Biochem, D-06120 Halle An Der Saale, Germany
关键词
D O I
10.1006/abio.2001.5560
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2002 Elsevier Science (USA).
引用
收藏
页码:49 / 56
页数:8
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