Puurand 2017 MiP2017
Compartmentalization of high energy phosphate carriers between cellular microcompartments is a phenomenon that ensures more efficient energy use and in turn depends on enzymes which are involved in phosphotransfer networks. Creatine kinase (CK) and adenylate kinase (AK) energy transfer systems which are functionally coupled to oxidative phosphorylation (OXPHOS) could serve as important regulators of cellular energy fluxes . We introduce some experimental approach to study the function of CK and AK phosphotransfer networks in regard to their capacity to regulate mitochondrial respiration.
Permeabilized cell technique and high-resolution respirometry were used to study functional coupling between CK and AK systems and OXPHOS in different cells and tissues. This technique preserves cellular microcompartments and mitochondrial morphology within the normal cytoarchitectural environment .
With these protocols the ability of creatine or adenosine monophosphate (AMP) to stimulate respiration through CK and AK reactions, respectively, is easily observable and quantifiable. Using the exogenous pyruvate kinase/phosphoenol pyruvate (PK/PEP) ADP trapping system enables us to clarify the functional regulation of energy transfer networks on the level of voltage dependent anion channels (VDAC) located within the mitochondrial outer membrane. Consequently, in the case of the CK system ADP produced by mitochondrial CK (MtCK) within the intermembrane space is re-imported into the matrix via adenine nucleotide translocase due its functional coupling with MtCK. Also, by using oxygraphy we can detect semi-quantitatively the ratio between mostly AK1- and AK2-related activities in mammalian tissues and cultured cells without extraction of tissue proteins . Functional coupling between hexokinase and mitochondria can be investigated by monitoring effect of glycose on respiration. The proposed protocols can be used in various tissues and cells in health and in pathology.
Cellular phosphotransfer networks are an integral part of management of the cellular energy metabolism. Alterations in their function can be found in case of several pathological conditions. Simple oxygraphic analysis can be performed to characterize functional coupling between CK and AK networks and OXPHOS.
Labels: MiParea: Respiration
Preparation: Permeabilized cells
- Lab Bioenergetics, National Inst Chemical Physics Biophysics, Tallinn, Estonia. – firstname.lastname@example.org
- Guzun R, Kaambre T, Bagur R, Grichine A, Usson Y, Varikmaa M, Anmann T, Tepp K, Timohhina N, Shevchuk I, Chekulayev V, Boucher F, Dos Santos6 P, Schlattner U, Wallimann T, Kuznetsov AV, Dzeja P, Aliev M, and Saks V (2015) Modular organization of cardiac energy metabolism: energy conversion, transfer and feedback regulation. Acta Physiol (Oxf) 213:84-106.
- Saks V, Guzun R, Timohhina N, Tepp K, Varikmaa M, Monge C, Beraud N, Kaambre T, Kuznetsov A, Kadaja L, Eimre M, Seppet E. (2010) Structure- function relationships in feedback regulation of energy fluxes in vivo in health and disease: Mitochondrial Interactosome. Biochim Biophys Acta 1797:678–97.
- Klepinin A, Ounpuu L, Guzun R, Chekulayev V, Timohhina N, Tepp K, Shevchuk I, Schlattner U, Kaambre T (2016) Simple oxygraphic analysis for the presence of adenylate kinase 1 and 2 in normal and tumor cells. J Bioenerg Biomembr 48:531–48.