Recommended Preparation Of Phormia For Enzyme And Substrate Determination

Enzyme determination. Take 5 to 10 flies total or separate thorax and abdomen after decapitation.

Homogenize in 5-fold volume of ice cold TRA-buffer, 100 mM with 10 mM EDTA at pH 7.5 with a glass homogenizer. Centrifuge 20 minutes at 4°C with 5000 g. Take the supernatant and precipitate the dissolved proteins with 80% ammonium sulphate. Dissolve the precipitant with 1 ml homogenizing buffer after centrifu-gation as above.

Substrate determination. Place 10 flies into liquid nitrogen. This separates head, thorax, abdomen and extremities. Take the desired parts and grind them in liquid nitrogen after addition of 1 ml of 0.6 N HClO4. Transfer the mixture into centrifuge tubes and centrifuge after thawing 30 min at 4°C and 5000 g. Neutralize the supernatant with solid KHCO3 and store at —20°C until substrate determination.

Isolation of mitochondria. Put 10 thoraces (or abdomina) in 10 ml ice cold buffer (100 mM Tris/HCl, 10 mM EDTA, 320 mM sucrose, pH 7.5) and crush the material in a glass homogenizer with a Teflon pestle by raising and lowering it 20 times. Put the crude homo-genate onto a nylon filter to remove chitin, myofibrills and tracheae fragments and wash it with buffer up to an end volume of 50 ml. Centrifuge 20 min at 100 g in the cold. Take the supernatant and centrifuge once more for 10 min at 3000 g and 4°C. The mitochondria are now in the precipitate and can be redissolved in 4-ml buffer.

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