I am finding difficult to measure the ACE activity in liver tissue homogenates by using the flouroscent substrate. Because of the large amount of other metalloproteases (zinc) present in the liver in addition to ACE, fluorescence substrate undergoing non-specific hydrolysis. Even though, I put ACE inhibitor (Captopril), it's not working.
Could anyone help to solve the problem?
Dear Ravinder, I have no idea!v Sorry, we shall ask Bruce if he knows at the clinical meeting. LTA Robin.
do you need to measure soluble ACE or membrane ACE? If you want to get rid of the metalloprotease activity, I would add a non specific inhibitor such as TAPI to inhibit the MMP and ADAM family members. I don't think TAPI will inhibit ACE. If you can measure just membrane activity, then your problem becomes simpler. You can make a membrane prep by homogenizing liver in 0.25M sucrose, 50mM Tris, pH8 and protease inhibitor cocktails. Then just spin at high speed to pellet the membranes. Then resuspend in the sucrose buffer. As a suspension, the activity is more manageble. I would try this, minus and plus captopril
Hi Marcia,
Thanks for adding your answer. Yes, I am measuring soluble ACE activity. Should I add TAPI while doing the Homogenisation or doing ACE activity assay. To what ratio should I add TAPI?
To get ACE activity check the following
1. Ratio of tissue and buffer. for homogenization.2. Dialysis of homogenate.3. Concentration of substrate
4.Incubation time 5. Concentration of captopril
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