1. Letitia A. Hill and Tony F. Rivera
Department of Biology and Chemistry, Moravian College, 1200 Main Street, Bethlehem, PA 18018
Inhibitory Properties of Levamisole
Introduction
The question we are investigating is whether or not L-levamisole (levamisole) can show the
same inhibition activity on shrimp alkaline phosphatase (SAP) that it does on other alkaline
phosphatase enzymes studied from mammalian organ tissue. The inhibitory properties of
levamisole on various rat tissues have been previously reported by M. Borgers, 1973.
Borgers research shows that several phosphatase complexes remain unchanged after
inhibition, and is found to be substrate independent, which suggests that the chemical
nature of levamisole induces uncompetitive inhibition. !
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SAP was chosen for study because of its high resolution crystal structure. Figure 3 is a 3D
representation of the SAP enzyme that contains an active site for p-nitrophenylphosphate
(PNPP) substrate. Alkaline phosphatase is an active enzyme found in many animals. SAP
is an active enzyme found in artic shrimp, Pandalus borealis. This enzyme converts PNPP
into p-nitrophenylate ion (PNP-), Scheme 1. Hydrolysis of PNPP yields PNP-, and can be
studied using UV-VIS spectroscopy. PNP- has an absorbance of 405 nm and will produce
a yellow color; therefore, upon inhibition we can expect to find a change in the absorbance
rate.!
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Levamisole acts as an immunostimulant agent and this activity has been suggested to be
facilitated by the aromatic ring in its chemical structure [Renoux, 1980]. In Figure 3, the
hydrophobic binding pockets for levamisole are represented in a cluster of blue dots. The
blue dots represent hydrophobic dense regions, where the aromatic group of levamisole
can potentially form nonpolar interactions with the protein. Enzyme interactions with
levamisole can change the structure of SAP and negatively effect substrate binding.!
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Experimental Methods
• Prepared a 5 mM stock concentration of L-levamisole in dH2O, and diluted stock to a final
working concentration of 1.6 mM. !
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• Ran six reactions, there were two sets for each. Each reaction solution contained glycine
(pH 10), dH2O, inhibitor, MgCl2 and PNPP.!
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• Analyzed each reaction under UV-VIS spectra at 405 nm for 60 secs.!
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• Recorded the slope for each of the reactions generated by the UV-VIS. !
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• Generated Michaelis-Menten and Lineweaver Burk plots using the rates obtained from
assays. !
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Results
• The Vmax of the uninhibited substrate was 0.68 + 0.2 uM/min.!
• The Vmax of the inhibited substrate was 0.40 + 0.4 uM/min.!
• In Table 1 the inhibited substrate velocity remained relatively constant during the
course of the six reactions.!
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• In the Michaelis-Menten plot in Figure 4, the graph for levamisole does not plateau
at the same velocity of the uninhibited assays. !
• In the Lineweaver Burk graph, Levamisole and uninhibited do not intersect, and the
two line graphs are relatively parallel to each other. !
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Conclusion
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Data supports our claim that levamisole acts as an uncompetitive inhibitor for SAP.
This is shown by the Lineweaver Burk plot (Fig.1). The linear line that corresponds to
inhibition by levamisole does not intersect with the line for uninhibited substrate in
quadrant 2. Inhibition of substrate by levamisole is not similar to the inhibition activity of
inorganic phosphate. Moreover, the Michaelis-Menten plot (Fig 4) provides further
warrant of uncompetitive inhibition, because the uninhibited substrate and the inhibited
substrate do not plateau in the same area. Instead, the inhibited substrate plateaus
over the course of the six reactions. In addition, the inhibitory rates found in Table 1 are
constant despite the change in substrate concentration. Together our data supports the
arguments reported in the M. Borger publication. L- levamisole uncompetively inhibits
SAP.!
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0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0
5
10
15
20
25
Vo
(µM/min)
[PNPP]
mM
Michaelis-­‐Menten
Uninhibied
vs
Inhibited
Vo
Inhibited
Vo
Uninhibited
Uninhibited
Fit
Inhibited
Fit
-­‐2.0
0.0
2.0
4.0
6.0
8.0
10.0
-­‐0.6
-­‐0.4
-­‐0.2
0
0.2
0.4
0.6
0.8
1
1.2
1/Vo
(change
in
[PNP-­‐]/sec)
1/[PNPP]
mM
Lineweaver
Burk
Inhibited
(Levamisole)
Uninhibited
Inhibited
(Inorganic
Phosphate)
Linear
(Inhibited
(Levamisole))
Linear
(Uninhibited)
Linear
(Inhibited
(Inorganic
Phosphate))
Figure 2: Structure of L-levamisole!
Figure 1: Lineweaver Burk Plot!
Figure 3: Shrimp Alkaline Phosphatase !
Figure 4: Michaelis-Menten Plot!
Scheme 1: Enzymatic hydrolysis of p-nitrophenylphosphate.!
References
1. Borgers, M. (1973) The Cytochemical Application of New Potent Inhibitors of
Alkaline Phosphatases, J Histochem Cytochem. 21, 812-824.!
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2. Renoux, G. (1980) The general immunopharmacology of levamisole, PubMed. !
20, 89-99.!
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3. RCSB PDB. Illustration from Figure 3. Dec. 10, 2015!
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4. Sigma-Aldrich. Illustration from Figure 2. Dec. 10, 2015!
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Hydrophobic!
Region!
Table 1. Inhibition Kinetics of Levamisole!
p-nitrophenylphosphate ! p-nitrophenol ! phosphate !
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Alkaline phosphatase!
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