I need to check Mitochondria volume in a muscle tissue and compare between 2 different fishes species, and I read checking CS activity has high correlation rate with mitochondrial area. I have never deal with this kind of techniques and need to learn from zero. Would appreciate to get a protocol and help. Thanks!
You might read the following references:
1. Morgunov I and Srere PA. Interacttion between citrate synthase and malate dehydrogenase substrate
channeling of oxaloacetate. 1998. J. Biol. Chem 273: 29540-29544
2. Trounce IA, Kim YL, Jun AS, Wallace DC. Assessment of mitochondrial oxidative phosphorylation in
patient muscle. biopsies, lymphoblasts, and transmitochondrial cell lines. 1996. Methods Enzymol 264: 484–
509
I found that Spinazzi et al., was a great help when I started using CS activity assay -
Spinazzi et al., Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nature Protocols 7, 1235–1246 (2012) doi:10.1038/nprot.2012.058
Not sure what you mean with correlation to mitochondrial area, but CS activity can be correlated to mitochondrial activity. You probably should include more assays if you want to investigate mitochondrial differences.
There are kits available for the determination of citrate synthase, for example: http://www.sigmaaldrich.com/catalog/product/sigma/cs0720
Cheers, Christian
Thank you all for your recommendations!
I Have actually read about the Kit but is very expensive "just to preliminar check" ... and that's why I was wondering if any other protocol simple and cheap is available. I will go to the references you recommend me.
Thank you all!
The assay was initiated by adding 10 mM oxaloacetate (20 lL) to the reaction mixture containing 100 mM Tris–HCl (pH 8.1), 0.2 mM DTNB (DTNB = 5,5'-dithiobis-(2-nitrobenzoic acid), 0.1% Triton X-100, 0.1 mM acetyl-CoA, and 2 ug protein (from fresh mitochondria) and the reaction was monitored at 412 nm. CS activity was expressed as nmol DTNB/min/mg protein (MEC = 13.6/mM/cm).
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