Novel Method for Investigation of Interactions between Mitochondrial Creatine Kinase and Adenine Nucleotide Translocase

In adult mammalian ventricular tissue, mitochondrial creatine kinase (mi-CK), which is bound to the outer surface of the mitochondrial inner membrane, is functionally coupled to oxidative phosphorylation. This is shown, in saponin-permeabilized rat ventricular fibres, by a shift in the apparent Km of mitochondrial respiration for ADP from 300ŷ56 ƁM to 111ŷ40 ƁM (P < 0.001) on the addition of 25 mM creatine, due to a local accumulation of ADP close to the ATP/ADP translocator. We have found that, in atrial fibres, the apparent Km for ADP is high, but is not decreased by creatine, suggesting an absence of coupling in this tissue, as has previously been observed in smooth muscle. mi-CK is encoded by two different genes, which are expressed in a tissue-specific manner: the sarcomeric isoform is expressed in ventricular and skeletal muscles, while the ubiquitous isoform is expressed in smooth muscle, brain and other tissues. In order to determine whether a specific function can be attributed to the expression of a specific isoform, we investigated mi-CK mRNA expression by Northern blot analysis. Hybridization with synthetic oligonucleotides specific for each mi-CK isoform showed the expression of only the sarcomeric isoform in rat atria. This result was confirmed by PCR using primers specific for each isoform. In addition, electrophoretic analysis of CK isoforms showed no difference in the octamer/dimer ratio of mi-CK in the atria and ventricles. In atria, unlike the soleus or ventricles, the maximum potential rate of mitochondrial phosphocreatine synthesis was lower than the maximal rate of ATP production by the mitochondria. The total CK/adenylate kinase ratio was also lower in atria than in the other tissues, suggesting a greater contribution of adenylate kinase to adenine nucleotide compartmentation in this tissue. The functional differences between mi-CK in the two cardiac tissues seem to imply a specific arrangement of the proteins in the intermembrane space rather than the expression of specific isoforms.

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Creatine kinase of heart mitochondria. Functional coupling of ADP transfer to the adenine nucleotide translocase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)68284-0 ◽

1982 ◽

Vol 257 (2) ◽

pp. 899-905 ◽

Cited By ~ 2

Author(s):

R.W. Moreadith ◽

W.E. Jacobus

Keyword(s):

Creatine Kinase ◽

Adenine Nucleotide ◽

Adenine Nucleotide Translocase ◽

Heart Mitochondria ◽

Functional Coupling

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The function of complexes between the outer mitochondrial membrane pore (VDAC) and the adenine nucleotide translocase in regulation of energy metabolism and apoptosis.

Acta Biochimica Polonica ◽

10.18388/abp.2003_3693 ◽

2003 ◽

Vol 50 (2) ◽

pp. 389-404 ◽

Cited By ~ 117

Author(s):

Mikhail Y Vyssokikh ◽

Dieter Brdiczka

Keyword(s):

Creatine Kinase ◽

Cytochrome C ◽

Mitochondrial Membrane ◽

Adenine Nucleotide ◽

Permeability Transition ◽

Adenine Nucleotide Translocase ◽

Outer Mitochondrial Membrane ◽

Cytochrome C Release ◽

Membrane Pore ◽

Contact Sites

The outer mitochondrial membrane pore (VDAC) changes its structure either voltage-dependently in artificial membranes or physiologically by interaction with the adenine nucleotide translocase (ANT) in the c-conformation. This interaction creates contact sites and leads in addition to a specific organisation of cytochrome c in the VDAC-ANT complexes. The VDAC structure that is specific for contact sites generates a signal at the surface for several proteins in the cytosol to bind with high capacity, such as hexokinase, glycerol kinase and Bax. If the VDAC binding site is not occupied by hexokinase, the VDAC-ANT complex has two critical qualities: firstly, Bax gets access to cytochrome c and secondly the ANT is set in its c-conformation that easily changes conformation into an unspecific channel (uniporter) causing permeability transition. Activity of bound hexokinase protects against both, it hinders Bax binding and employs the ANT as anti-porter. The octamer of mitochondrial creatine kinase binds to VDAC from the inner surface of the outer membrane. This firstly restrains interaction between VDAC and ANT and secondly changes the VDAC structure into low affinity for hexokinase and Bax. Cytochrome c in the creatine kinase complex will be differently organised, not accessible to Bax and the ANT is run as anti-porter by the active creatine kinase octamer. However, when, for example, free radicals cause dissociation of the octamer, VDAC interacts with the ANT with the same results as described above: Bax-dependent cytochrome c release and risk of permeability transition pore opening.

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Estimation of heart mitochondrial creatine kinase flux using magnetization transfer NMR spectroscopy

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1992.262.4.h1022 ◽

1992 ◽

Vol 262 (4) ◽

pp. H1022-H1028 ◽

Cited By ~ 3

Author(s):

R. Zahler ◽

J. S. Ingwall

Keyword(s):

Creatine Kinase ◽

Nmr Spectroscopy ◽

Adenine Nucleotide ◽

Magnetization Transfer ◽

Atp Synthesis ◽

Synthesis Rate ◽

Saturation Transfer ◽

Mitochondrial Creatine Kinase ◽

Atp Pool ◽

Order Of Magnitude

In heart, both enzyme and substrate of the creatine kinase (CK) reaction are compartmentalized: 0-25% of total CK activity is associated with the mitochondrial CK isoenzyme (mito-CK); 2-30% of the total ATP pool is in the mitochondria. Because most ATP is produced by oxidative phosphorylation, this ATP may be the preferred substrate for the mito-CK reaction. Thus flux through the mito-CK reaction should increase in proportion to the amount of mito-CK, until the enzyme becomes saturated. We previously developed a model of saturation-transfer nuclear magnetic resonance (NMR) spectroscopy that permits calculation of mito-CK flux. Here, we test the model for consistency in two ways: 1) we compare fluxes in rabbit hearts with 0% mito-CK and with 6% mito-CK at two rates of ATP synthesis and 2) we analyze six groups of rat and rabbit hearts with differing amounts of mito-CK and differing work loads. Hearts with no detectable mito-CK activity do not increase their baseline low level of mito-CK flux in response to the increased demand of contraction, but mito-CK flux increases with increased work in hearts with measurable amounts of mito-CK. Furthermore, mito-CK flux increases monotonically with increasing ATP synthesis rates but increases and then saturates with increasing mito-CK activity. Calculated mito-CK flux is of the same order of magnitude as ATP synthesis rate, as would be expected from the coupling of the mito-CK reaction to adenine nucleotide translocase. Thus the model predicts the appropriate relationship between mito-CK activity and mito-CK flux.

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