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. !!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.! !!!!!!!!!!!!!!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. !

!•  Ran six reactions, there were two sets for each. Each reaction solution contained glycine

(pH 10), dH2O, inhibitor, MgCl2 and PNPP.!!•  Analyzed each reaction under UV-VIS spectra at 405 nm for 60 secs.!!•  Recorded the slope for each of the reactions generated by the UV-VIS. !!•  Generated Michaelis-Menten and Lineweaver Burk plots using the rates obtained from

assays. ! !

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.!

!•  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 !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

 (cha

nge  in  [P

NP-­‐]/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.!

 !2.  Renoux, G. (1980) The general immunopharmacology of levamisole, PubMed. ! 20, 89-99.! !3. RCSB PDB. Illustration from Figure 3. Dec. 10, 2015! !4. Sigma-Aldrich. Illustration from Figure 2. Dec. 10, 2015!!!

Hydrophobic!Region!

Table 1. Inhibition Kinetics of Levamisole!

p-nitrophenylphosphate ! p-nitrophenol ! phosphate !

!Alkaline phosphatase!!