Biology Reference
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250
1
200
0.8
150
0.6
100
0.4
cdk7/cyclin H
cdk2/cyclin A
cdc2/cyclin A
cdk2
cdk7
cdc2
50
0.2
0
0
0
0.5
1
1.5
2
2,5
0
0.2
0.4
0.6
0.8
1
1.2
cdks (µM)
cdk/cyclin (µM)
Figure 4.7 Binding of Mant-ATP to cyclin-dependent kinase (Cdk) and Cdk/cyclin com-
plex. Mant-ATP, the N-methyl-anthraniloyl moiety, is linked to 2 0 - and/or 3 0 -hydroxyl of
the ribose. The fluorescence of the Mant-group is excited at 350 nm and emission mon-
itored at 450 nm. The high sensitivity of the Mant-ATP was used to quantify the binding
of nucleotide to different Cdks in the free form (A) or in complex with their Cyclin part-
ner (B). Upon binding to Cdk and Cdk/cyclin complex, Mant fluorescence increased by
2.5- and 5-fold, respectively. In order to determine dissociation constant, the curves
were fitted using quadratic equation (Eq. 4.3 ) (adapted from Morris et al.). 27
the binding of Mant-ATP to the cell cycle protein kinase cyclin-dependent
kinase (Cdk2) results in a 2.5-fold increase in fluorescence ( Fig. 4.7 ) and
allows for the determination of the dissociation constant at the steady-state
level using either hyperbolic or quadratic equations, which take into
account the concentration of the enzyme and probe: 27,28
n
o
1
=
2
2
F
¼
F ini
DF
ðÞ
ð
E t
þ
L
þ
K d
Þ
ð
E t
þ
L
þ
K d
Þ
4 E t L
=
2 E t
½
4
:
3
where F is the observed relative fluorescence intensity, F ini is the fluores-
cence intensity at the start of the titration, DF is the variation of the fluores-
cence intensity between the initial value and at a saturating concentration of
substrate, E t is the total concentration of enzyme, L is the total concentration
of ligand, and K d is the dissociation constant of the enzyme-ligand complex.
Bisubstrate nucleotides (Ap5A and Ap4A) have been designed to character-
ize enzymes harboring multiple nucleotide binding sites. An interesting
system was described to measure the affinities of nucleotide kinases
(NDP-, ADP-, AMP-, or TMP-kinases) for their substrates and inhibitors,
based on a fluorescent analog of the bisubstrate inhibitor diadenosine
pentaphosphate (AP5A): a , g -di[(3 0 -or2 0 -)- O -( N -methylanthraniloyl)
adenosine-5 0 ] pentaphosphate (mAP5Am). 29-31
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