Title: Technical note: Assessing the functional capacity of mitochondria isolated from lactating mammary tissue: Choose your chelating agent wisely Authors
|Hadsell, Darryl -|
|George, Jessy -|
|Abraham, P -|
|Collier, R -|
|Lambert, B -|
Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 2, 2008
Publication Date: May 1, 2009
Citation: Hadsell, D.L., George, J., Abraham, P.A., Collier, R.J., Lambert, B.D. 2009. Technical note: Assessing the functional capacity of mitochondria isolated from lactating mammary tissue: Choose your chelating agent wisely. Journal of Dairy Science. 92:2038-2045. Interpretive Summary: The mitochondrion is the power house of the cell. In the lactating mammary gland, mitochondria have been isolated and characterized, but these preparations have typically exhibited reduced functional capacity in comparison to mitochondria from other organs within the body. This technical note report that we have discovered a key factor that limits the energy-conserving activity of mammary mitochondria isolated from lactating animals. We also describe optimized conditions for a high throughput method of analysis that will facilitate further studies aimed at elucidating the importance of mitochondria to milk production.
Technical Abstract: Previous work has indicated that respiratory activity of mitochondrial preparations prepared from lactating mammary tissue is often much lower than that of mitochondria isolated from other organs such as the liver. Initial studies in our own laboratory also found that mammary mitochondria prepared from lactating mice had much lower ATP synthesis activity than those isolated from liver tissue obtained from the same animals. In this paper, we describe an improved procedure for obtaining coupled mitochondria from the mammary tissue of lactating mice. Using a high-throughput assay for mitochondrial ATP synthesis, we demonstrated that mammary mitochondria, unlike liver mitochondria, are sensitive to the concentration of bovine serum albumin and to the choice of chelating agent used in the preparation and assay buffers. Mammary mitochondria prepared and assayed in buffers containing 1 mM ethylene glycol-bis-(beta-aminoethyl ether)-N,N' tetraacetic acid (EGTA) and 0.4% bovine serum albumin have a similar ATP synthesis activity as liver mitochondria. In addition, we show that the chelating agent EDTA ablates the ATP synthesis capacity of mammary mitochondria through a mechanism that does not involve the release of cytochrome c. We also demonstrate that these improved isolation and assay procedures are both scalable and applicable to bovine mammary tissue, and we describe optimal conditions for cryopreservation and recovery of functionally active mitochondria. This work will facilitate future studies aimed at determining the importance of mammary mitochondria to milk production.