scholarly journals Mitochondrial creatine kinase isoform expression does not correlate with its mode of action

1997 ◽  
Vol 322 (1) ◽  
pp. 73-78 ◽  
Author(s):  
Keltoum ANFLOUS ◽  
Vladimir VEKSLER ◽  
Philippe MATEO ◽  
Françoise SAMSON ◽  
Valdour SAKS ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Smooth Muscle ◽  
Adenylate Kinase ◽  
Adenine Nucleotide ◽  
Electrophoretic Analysis ◽  
Intermembrane Space ◽  
Mitochondrial Creatine Kinase ◽  
Cardiac Tissues ◽  
Atp Production ◽  
Mitochondrial Inner Membrane

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.

P1, P5-Bis-(5′-adenosyl)pentaphosphate: Is this Adenylate Kinase Inhibitor Substrate for Mitochondrial Processes?

1977 ◽  
Vol 32 (9-10) ◽  
pp. 786-791 ◽  
Author(s):  
Josef Köhrle ◽  
Joachim Lüstorff ◽  
Eckhard Schlimme
Keyword(s):  
Adenylate Kinase ◽  
Kinase Inhibitor ◽  
Adenine Nucleotide ◽  
Nucleoside Diphosphate Kinase ◽  
Liver Mitochondria ◽  
Inhibitory Effect ◽  
Rat Liver Mitochondria ◽  
Intermembrane Space ◽  
Rabbit Muscle ◽  
Inner Mitochondrial Membrane

Abstract 1. P1, P5-Bis-(5′-adenosyl)pentaphosphate (Ap5A) inhibits “soluble” adenylate kinase even when this enzyme is an integral part of the complete mitochondrion. The Ki is 10-5м , i. e. about two orders of magnitude higher than the inhibitor constants determined for the purified adenylate kinase of rabbit muscle and an enzyme preparation separated from the mitochondrial intermembrane space. The weaker inhibitory effect is due to a lower accessibility of the enzyme.2. As to be expected Ap5A which is of the “multisubstrate analogue”-type does not affect mito­ chondrial nucleoside diphosphate kinase.3. Though Ap5A owns the structural elements of both ATP and ADP it is not a substrate of the adenine nucleotide carrier, i.e. neither it is exchanged across the inner mitochondrial membrane nor specifically bound.4. Ap5A is not metabolized by rat liver mitochondria.

Evaluation of adenosine 5'-monophosphate and fluoride as adenylate kinase inhibitors in the creatine kinase assay.

1976 ◽  
Vol 22 (7) ◽  
pp. 1078-1083 ◽  
Author(s):  
T G Rosano ◽  
K J Clayson ◽  
P E Strandjord
Keyword(s):  
Creatine Kinase ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Creatine Kinase Activity ◽  
Serum Creatine Kinase ◽  
Assay System ◽  
Kinase Assay ◽  
Rabbit Muscle ◽  
Atp Production

Abstract Adenylate kinase (EC 2.7.4.3) interferes positively in the serum creatine kinase (EC 2.7.3.2) assay when the rate of ATP production is monitored by a coupled enzyme system. A dual assay, measuring creatine kinase and adenylate kinase activity, was used to evaluate AMP and other possible adenylate kinase inhibitors that would permit specific measurement of creatine kinase activity in the presence of adenylate kinase. We found that AMP, routinely included in the creatine kinase assay system to inhibit adenylate kinase, partially inhibits both human serum creatine kinase and purified creatine kinase from rabbit muscle. The amount of creatine kinase inhibition is related directly to the AMP concentration and inversely to the substrate (ADP) concentration. We found that 25 mmol/liter of fluoride inhibits adenylate kinase without measurable effect on creatine kinase activity. We developed a serum creatine kinase assay including fluoride, and compared it with the dual assay system and with two commercial assay kits. Other halides or adenosine 2'-monophosphate did not selectively inhibit adenylate kinase.

Adenine nucleotide-creatine-phosphate module in myocardial metabolic system explains fast phase of dynamic regulation of oxidative phosphorylation

2007 ◽  
Vol 293 (3) ◽  
pp. C815-C829 ◽  
Author(s):  
Johannes H. G. M. van Beek
Keyword(s):  
Oxidative Phosphorylation ◽  
Time Constant ◽  
Computational Models ◽  
Adenine Nucleotide ◽  
Mitochondrial Outer Membrane ◽  
Small Subset ◽  
Intermembrane Space ◽  
Metabolic System ◽  
Skeleton Model ◽  
Atp Production

Computational models of a large metabolic system can be assembled from modules that represent a biological function emerging from interaction of a small subset of molecules. A “skeleton model” is tested here for a module that regulates the first phase of dynamic adaptation of oxidative phosphorylation (OxPhos) to demand in heart muscle cells. The model contains only diffusion, mitochondrial outer membrane (MOM) permeation, and two isoforms of creatine kinase (CK), in cytosol and mitochondrial intermembrane space (IMS), respectively. The communication with two neighboring modules occurs via stimulation of mitochondrial ATP production by ADP and Pi from the IMS and via time-varying cytosolic ATP hydrolysis during contraction. Assuming normal cytosolic diffusion and high MOM permeability for ADP, the response time of OxPhos ( tmito; generalized time constant) to steps in cardiac pacing rate is predicted to be 2.4 s. In contrast, with low MOM permeability, tmito is predicted to be 15 s. An optimized MOM permeability of 21 μm/s gives tmito = 3.7 s, in agreement with experiments on rabbit heart with blocked glycolytic ATP synthesis. The model correctly predicts a lower tmito if CK activity is reduced by 98%. Among others, the following predictions result from the model analysis: 1) CK activity buffers large ADP oscillations; 2) ATP production is pulsatile in beating heart, although it adapts slowly to demand with “time constant” ∼14 heartbeats; 3) if the muscle isoform of CK is overexpressed, OxPhos reacts slower to changing workload; and 4) if mitochondrial CK is overexpressed, OxPhos reacts faster.

scholarly journals Proteinase treatment of intact hepatic mitochondria has differential effects on inhibition of carnitine palmitoyltransferase by different inhibitors

1992 ◽  
Vol 282 (3) ◽  
pp. 909-914 ◽  
Author(s):  
K Kashfi ◽  
G A Cook
Keyword(s):  
Outer Membrane ◽  
Adenylate Kinase ◽  
Citrate Synthase ◽  
Physiological State ◽  
Carnitine Palmitoyltransferase ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Mitochondrial Inner Membrane ◽  
Malonyl Coa ◽  
No Release

Proteolysis of intact mitochondria by Nagarse (subtilisin BPN') and papain resulted in limited loss of activity of the outer-membrane carnitine palmitoyltransferase, but much greater loss of sensitivity to inhibition by malonyl-CoA. In contrast with a previous report [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382], we found that trypsin had no effect on malonyl-CoA sensitivity. Even when 80% of activity was destroyed by trypsin, there was no difference in the malonyl-CoA sensitivity of the enzyme remaining. Trypsin caused release of the intermembrane-space enzyme adenylate kinase, indicating loss of integrity of the mitochondrial outer membrane, whereas Nagarse and papain caused no release of that enzyme. Citrate synthase was not released by any of the three proteinases, indicating no damage to the mitochondrial inner membrane. When we examined the effects of proteolysis on the inhibition of carnitine palmitoyltransferase by a wide variety of inhibitors having different mechanisms of inhibition, we found differential proteolytic effects that were specific for those inhibitors (malonyl-CoA and hydroxyphenylglyoxylate) that have their inhibitory potencies diminished by changes in physiological state. Both of those inhibitors protected carnitine palmitoyltransferase from the effects of proteolysis, but did not inhibit the proteinases directly. Inhibition by two other inhibitors (DL-2-bromopalmitoyl-CoA and N-benzyladriamycin 14-valerate) was not altered by proteinase treatment, even when most of the enzyme activity had been destroyed. Inhibition by glyburide, which is minimally affected by physiological state, was affected only to a slight extent at the highest concentration of trypsin tested. Proteolysis by Nagarse appeared to produce loss of co-operativity in malonyl-CoA inhibition. The effects of proteolysis are discussed and compared with changes in Ki occurring with changing physiological states.

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