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Prostaglandin Biosynthesis of Manduca Sexta Midgut

 

Introduction

 

The purpose of our research is to demonstrate that the midgut from Manduca sexta is competent to synthesize prostaglandins. Prostaglandins are oxygenated metabolites formed from Arachidonic Acid (C20:4n-6) that function in a variety of ways within insects.  We will refer to an article published in Insect Biochemistry and Molecular Biology: Prostaglandin biosynthesis by midgut tissue isolated from tobacco hornworm, Manduca sexta.  K. Büyükgüzel, et. al. 2002, as a resource. 

 
We hope to show that the midgut is capable of this biosynthesis and coupled with the fatty acid analysis show that the midgut needs a supply of arachidonic acid for this reaction.  

 
Procedure.

  1. Dissect & extract midgut from ice-chilled Manduca sexta larvae (4th instar).

  2. Suspend midgut in 1mL of ice-cold 0.05M potassium phosphate buffer, pH = 8.0 

  3. Sonciate midgut/buffer for 10 seconds at 30 watts

  4. Centrifuge for 10 min at 735g using microeppendorf centrifuge at 4oC.

  5. Remove the supernatant and centrifuge again for 20 minutes at 16,000g

  6. Remove the supernatant and assay for protein concentration using a microtiter plate reader at 562 nm using bicinchoninic acid reagent for assay and bovine serum albumin as protein standard.

  7. In vitro synthesis of prostaglandin:  

  1. 6 tubes (50 mL) were used

  2. Add 50mg (50mL of 1mg/mL) arachidonic acid into each tube and dry under N2 stream

  3. Add 25ml of cofactor* and buffer (8109.4 mL) to each tube, vortex

  4. Incubate for 10 min at 22 o C in shaker bath (preincubation)

  5. Add 1865.6 mL of sample ( protein source) into each tube, vortex

  6. Incubate for 0 .5 min at 22 o C in shaker bath

  7. To stop reaction with 1600 mL of 0.1 M HCl added to each tube, vortex

  8. Extract with 3 mL of ethyl acetate (x3), centrifuge @1200 rpm/ 2 min

  9. Extracts were combined and dried under N2

*Cofactor: 2.4mM reduced glutathione, 0.25mM hydroquinone and 25 mg hemoglobin

 

  1. Reaction products are purified using micro-chromatographic column.

  1. Set up a 1000 mL pipette tip plugged with glass wool, add slurry containing 750 mg of silica gel and 750 mL of ethyl acetate.

  2. Rinse column with 3 mL of ethyl acetate.

  3. Load column with reaction products suspended in ethyl acetate (rinsed x3).

  4. Resolve products by collecting fractions from 750 mL of each solvent (100% ethyl acetate, ethyl acetate/acetonitrile (1/1 v:v), 100% acetonitrile, acetonitrile/methanol (1/1 v:v), and 100% methanol).

  5. Collect fraction # 4 (acetonitrile/methanol) fraction in a 2 dram amber vial and retain for derivitization and analysis.

  1. Evaporate off  fraction #4 solvent under N2

  2. Add 100 mL of freshly prepared diazomethane* (CH2N2). Let stand at room temperature for 10 minutes

  3. Evaporate off diazomethane/ethyl ether and add 150 mL of BSTFA with 1%TMCS**. Heat at 60 oC for 20 minutes.

  4. Evaporate off BSTFA solvent under N2 and resuspend in 50 uL of isooctane for GC/MS analysis.

*Used to methylate the carboxyl group of the prostaglandins

**Used to add trimethylsilane groups to oxygen atoms of hydroxyl groups.

 

NOTE:  The reaction vial broke during the diazomethane reaction and therefore we lost our product.