NAD binding by human CD38 analyzed by Trp189 fluorescence
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NAD binding by human CD38 analyzed by Trp189 fluorescence. / Wolters, Valerie; Rosche, Anette; Bauche, Andreas; Kulow, Frederike; Harneit, Angelika; Fliegert, Ralf; Guse, Andreas H.
In: BBA-MOL CELL RES, Vol. 1866, No. 7, 07.2019, p. 1189-1196.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - NAD binding by human CD38 analyzed by Trp189 fluorescence
AU - Wolters, Valerie
AU - Rosche, Anette
AU - Bauche, Andreas
AU - Kulow, Frederike
AU - Harneit, Angelika
AU - Fliegert, Ralf
AU - Guse, Andreas H
N1 - Copyright © 2018 Elsevier B.V. All rights reserved.
PY - 2019/7
Y1 - 2019/7
N2 - The NAD-glycohydrolase/ADP-ribosyl cyclase CD38 catalyzes the metabolism of nicotinamide adenine dinucleotide (NAD) to the Ca2+ mobilizing second messengers ADP-ribose (ADPR), 2'-deoxy-ADPR, and cyclic ADP-ribose (cADPR). In the present study, we investigated binding and metabolism of NAD by a soluble fragment of human CD38, sCD38, and its catalytically inactive mutant by monitoring changes in endogenous tryptophan (Trp) fluorescence. Addition of NAD resulted in a concentration-dependent decrease in sCD38 fluorescence that is mainly caused by the Trp residue W189. Amplitude of the fluorescence decrease was fitted as one-site binding curve revealing a dissociation constant for NAD of 29 μM. A comparable dissociation constant was found with the catalytically inactive sCD38 mutant (KD 37 μM NAD) indicating that binding of NAD is not significantly affected by the mutation. The NAD-induced decrease in Trp fluorescence completely recovered in case of sCD38. Kinetics of recovery was slowed down with decreasing temperature and sCD38 concentration and increasing NAD concentration demonstrating that recovery in fluorescence is proportional to the enzymatic activity of sCD38. Accordingly, recovery in fluorescence was not observed with the catalytically inactive mutant. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
AB - The NAD-glycohydrolase/ADP-ribosyl cyclase CD38 catalyzes the metabolism of nicotinamide adenine dinucleotide (NAD) to the Ca2+ mobilizing second messengers ADP-ribose (ADPR), 2'-deoxy-ADPR, and cyclic ADP-ribose (cADPR). In the present study, we investigated binding and metabolism of NAD by a soluble fragment of human CD38, sCD38, and its catalytically inactive mutant by monitoring changes in endogenous tryptophan (Trp) fluorescence. Addition of NAD resulted in a concentration-dependent decrease in sCD38 fluorescence that is mainly caused by the Trp residue W189. Amplitude of the fluorescence decrease was fitted as one-site binding curve revealing a dissociation constant for NAD of 29 μM. A comparable dissociation constant was found with the catalytically inactive sCD38 mutant (KD 37 μM NAD) indicating that binding of NAD is not significantly affected by the mutation. The NAD-induced decrease in Trp fluorescence completely recovered in case of sCD38. Kinetics of recovery was slowed down with decreasing temperature and sCD38 concentration and increasing NAD concentration demonstrating that recovery in fluorescence is proportional to the enzymatic activity of sCD38. Accordingly, recovery in fluorescence was not observed with the catalytically inactive mutant. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
KW - Journal Article
U2 - 10.1016/j.bbamcr.2018.11.011
DO - 10.1016/j.bbamcr.2018.11.011
M3 - SCORING: Journal article
C2 - 30472140
VL - 1866
SP - 1189
EP - 1196
JO - BBA-MOL CELL RES
JF - BBA-MOL CELL RES
SN - 0167-4889
IS - 7
ER -
