The mitochondrial creatine phosphokinase is associated with inner membrane cardiolipin.
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Animals↗
Biomedical subjects
E Carafoli
Publications and source records attributed to E Carafoli.
The calcium pumping ATPase of heart plasma membrane.
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Adenosine Triphosphate↗
ATPase activity and Ca2+ transport by reconstituted tryptic fragments of the Ca2+ pump of the erythrocyte plasma membrane.
The purified Ca2+ ATPase of the erythrocyte plasma membrane has been submitted to controlled trypsin proteolysis under conditions that favor either its (putative) E1 or E2 configurations. The former configuration has been forced by treating the enzyme with Ca2+-saturated calmodulin, the latter with vanadate and Mg2+. The E1 conformation leads to the accumulation of a polypeptide of Mr 85 KDa which still binds calmodulin, the E2 conformation to the accumulation of one of Mr 81 KDa which does not. Both fragments arise from the hydrolysis of a transient 90 KDa product which has Ca2+-calmodulin dependent ATPase activity, and which retains the ability to pump Ca2+ in reconstituted liposomes. Highly enriched preparations of the 85 and 81 KDa fragments have been obtained and reconstituted into liposomes. The former has limited ATPase and Ca2+ transport ability and is not stimulated by calmodulin. The latter has much higher ATPase and Ca2+ transport activity. It is proposed that the Ca2+ pumping ATPase of erythrocytes plasma membrane contains a 9 KDa domain which is essential for the interaction of the enzyme with calmodulin and for the full expression of the hydrolytic and transport activity. This putative 9 KDa sequence contains a 4 KDa "inhibitory" domain which limits the activity of the ATPase. In the presence of this 4 KDa sequence, i.e., when the enzyme is degraded to the 85 KDa product, calmodulin can still be bound, but no longer stimulates ATPase and Ca2+ transport.
Adenosine Triphosphate↗
Membrane transport in the cellular homeostasis of calcium.
The messenger function of Ca2+ requires its maintenance at a very low concentration level within cells. In this way, significant changes in concentration can be achieved by mobilizing relatively minor amounts of the cation. The reversible complexation by specific membranous and nonmembranous binding proteins is the basic mechanism for the control of cellular Ca2+. Specific membrane proteins transport Ca between cells and the environment, and between cytoplasm and the space within organelles. Numerous Ca2+ transport systems have been described, but they correspond to only four transport modes: ATPases, (Na+/Ca2+) exchangers, channels, and electrophoretic uniporters. The transport systems interact with Ca2+ with either low or high affinity, thus playing different roles in the functional cycle of cells. From the standpoint of the division of labor among cell organelles, endo(sarco)plasmic reticulum can be considered as the structure presiding over the fine (high-affinity) and rapid tuning of cell Ca2+, and the mitochondria as long-term, low-affinity Ca2+ buffering structures. Their most important task may be the regulation of Ca2+ in their matrix, rather than in the cytosol.
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Mitochondrial pathology: an overview.
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Adenosine Triphosphate↗
Ca-ATPase in erythrocyte membrane ghost cells of MHS rats.
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Animals↗
Solubilization and reconstitution of the Na+/Ca2+ exchanger of cardiac sarcolemma. Properties of the reconstituted system and tentative identification of the protein(s) responsible for the exchange activity.
The cardiac sarcolemma Na+/Ca2+ exchanger has been solubilized in Triton X-100 and reconstituted into asolectin liposomes by removing the detergent with Amberlite XAD-2 beads. The specific Na+/Ca2+ exchange activity of the proteoliposomes was increased about 10 times with respect to the original sarcolemma. The exchange activity was strongly dependent on the amount of acidic phospholipids present in the liposomes. Phosphatidylserine and phosphatidylinositol increased the initial rate of the reconstituted exchanger. Fractionation of the solubilized sarcolemma by rate zonal centrifugation produced fractions in which the specific activity of the exchanger was enriched up to 130-fold with respect to the original membranes. The fractionation scheme used has shown that the exchange activity correlated best with a 33-kDa protein present in the fractionated extract. No correlation has been found between exchange activity and other proteins of higher molecular mass (70 or 82 kDa) which have previously been suggested to correspond to the Na+/Ca2+ exchanger. Thus, the 33-kDa protein may represent the Na+/Ca2+ exchanger of cardiac sarcolemma.
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Adriamycin as a probe for the transversal distribution of cardiolipin in the inner mitochondrial membrane.
The ability of adriamycin to complex cardiolipin was used to determine the distribution of cardiolipin across the inner membrane of rat liver and heart mitochondria. In both mitochondrial types, about 57 +/- 5% of the total cardiolipin was found to be located in the cytoplasmic face of the inner membrane. Mitochondria and mitoplasts were used to study the cytoplasmic face of the inner membrane, purified submitochondrial vesicles with inverted membrane orientation for the matrix face. The cardiolipin amount titrated by adriamycin in the latter was found to be complementary to the amount titrated in the cytoplasmic face. The adriamycin association constant determined for the first saturation level of mitochondria was in good agreement with the value published by Goormaghtigh et al. (Goormaghtigh, E., Chatelain, P., Caspers, J., and Ruysschaert, J. M. (1980) Biochim. Biophys. Acta 597, 1-14) for cardiolipin in artificial membranes. Two binding plateaus were observed when increasing amounts of adriamycin were added to mitochondria. The plateau at higher concentrations is conveniently explained by the penetration of adriamycin into mitochondria and the titration of cardiolipin in the matrix face. Scatchard plot analysis of the binding curves leading to the two plateaus produced almost identical association constants. The total amount of cardiolipin in mitochondria calculated from curves of this type corresponded to the total amount of cardiolipin determined by phosphate analysis of extracts, analyzed by thin layer chromatography.
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Phosphorylation of the Ca2+-pumping ATPase of heart sarcolemma and erythrocyte plasma membrane by the cAMP-dependent protein kinase.
The Ca2+ ATPase of heart sarcolemma was stimulated by the exposure of sarcolemma vesicles to ATP and the catalytic subunit of the cAMP-dependent protein kinase. The effect of the phosphorylation system was primarily on the Km(Ca2+) of the pumping ATPase. The ATPase purified from heart sarcolemma or erythrocytes became phosphorylated under the conditions mentioned above. Hydroxylamine treatment of the labeled ATPase has shown that the phosphorylation was additive to be acylphosphate formed on the ATPase during the reaction cycle. The stoichiometry of the kinase-promoted phosphorylation (i.e. the fraction of the ATPase molecules that became labeled) approached 30% with both the heart and the erythrocyte enzyme.
Animals↗
The inhibitor peptide of the mitochondrial F1.F0-ATPase interacts with calmodulin and stimulates the calmodulin-dependent Ca2+-ATPase of erythrocytes.
The binding of calmodulin to the mitochondrial F1.F0-ATPase has been studied. [125I]Iodoazidocalmodulin binds to the epsilon-subunit and to the endogeneous ATPase inhibitor peptide in a Ca2+-dependent reaction. The effect of the mitochondrial ATPase inhibitor peptide on the purified Ca2+-ATPase of erythrocytes has also been analyzed. The inhibitor peptide stimulates the ATPase when pre-incubated with the enzyme. The activation of the Ca2+-ATPase by calmodulin is not influenced by the inhibitor peptide, indicating that the two mechanisms of activation are different. These in vitro effects of the two regulatory proteins may reflect a common origin of the two ATPases considered and/or of the regulatory proteins.
Adenosine Triphosphatases↗
Spectroscopic and functional properties of subunit III-depleted cytochrome oxidase.
Beef heart cytochrome c oxidase has been depleted of subunit III by treatment with chymotrypsin. The removal of subunit III has been evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel fluorography of preparations of the oxidase labeled with [14C]dicyclohexylcarbodiimide prior to proteolysis. Removal of subunit III resulted in a perturbation of the visible spectrum of reduced cytochrome oxidase. Subunit III-depleted oxidase is spectroscopically very similar to the oxidase from Paracoccus denitrificans. When reconstituted into liposomes, the depleted enzyme still pumped protons in response to a pulse of reduced cytochrome c. The H+/e- stoichiometry averaged 0.5. Redox-linked proton translocation could be observed only when respiratory control ratios were higher than 3 and the reductant pulse was of a magnitude that allowed for no more than 5 turnovers of the oxidase.
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Cardiolipin is the membrane receptor for mitochondrial creatine phosphokinase.
Treatment of rat heart mitochondria with phosphate or mersalyl releases a number of proteins, including the mitochondrial creatine kinase (mt-CK). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the released proteins showed that phosphate is more selective than mersalyl in releasing mt-CK. The rebinding of mt-CK to mitochondria was selectively inhibited by adriamycin, which complexes membrane-bound cardiolipin. mt-CK activity and binding experiments have shown that intact mitochondria are able to bind approximately twice the amount of mt-CK they originally contain. Liver mitochondria bound heart mitochondria mt-CK to the same extent as creatine kinase-depleted heart mitochondria. mt-CK was bound by liposomes but only if they contained cardiolipin. The binding of mt-CK to cardiolipin-containing liposomes was inhibited by adriamycin. Phosphatidylcholine liposomes reconstituted with the purified ADP/ATP translocator failed to bind mt-CK.
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ATP-dependent Ca2+ transport in vesicles isolated from the bile canalicular region of the hepatocyte plasma membrane.
Three plasma membrane subfractions have been isolated and characterized from rat liver cells. The high affinity Ca2+-stimulated ATPase is highly enriched in the bile canalicular subfraction. Taking into account cross-contamination by the blood sinusoidal and lateral membranes it is suggested that the high-affinity Ca2+-ATPase is located exclusively in this fraction. The high-affinity Ca2+-ATPase is coupled to Ca2+ transport, is calmodulin-insensitive, sensitive to vanadate under appropriate experimental conditions and is strongly inhibited by La3+. In the presence of Ca2+ and ATP the ATPase forms a phosphorylated intermediate of molecular mass about 200 kDa.
Adenosine Triphosphate↗
Calmodulin in the regulation of calcium fluxes in cardiac sarcolemma.
Three systems mediate the fluxes of calcium across heart sarcolemma: the slow calcium channel (influx), the ATP-dependent calcium pump (efflux), and the Na+/Ca2+ exchanger (efflux, but possibly also influx). Calmodulin regulates the pumping ATPase by direct interaction and also by activating a protein kinase. The Na+/Ca2+ exchanger is modulated by calmodulin via a phosphorylation-dephosphorylation cycle. Both the kinase and the phosphatase are membrane-bound and calmodulin-sensitive. The kinase has higher Ca2+ affinity than the phosphatase.
Adenosine Triphosphatases↗
The homeostasis of calcium in heart cells.
The signalling function of Ca2+ demands a very low ionic concentration of the cation within heart cells. This is achieved by two mechanisms, the reversible complexation of calcium by non membranous (protein) ligands, and its binding and transport by transmembrane proteins. The second mechanism is more efficient, since the complexation by soluble proteins is limited by their amount in heart sarcoplasm or within heart organelles, whereas membrane proteins can regulate calcium efficiently, even if present in low amounts, if they 'return' rapidly in the uncomplexed from after each binding and transport cycle. Seven systems for the transport of calcium have been documented in heart membranes. There are located in sarcolemma, two in mitochondria, two in sarcoplasmic reticulum. These seven systems can be simplified to four basic transport modes: ATPases, Na+/Ca2+ exchangers, channels, electrophoretic uniporters. They have either low or high calcium affinity, thus serving different purposes in the various phases of the functional cycle of heart cells. On an integrated level, sarcoplasmic reticulum can be considered as the organelle presiding over the rapid and fine regulation of Ca2+ linked to the contraction/relaxation cycle. Sarcolemma regulates Ca2+ with both low and high affinity, but handles a quantitatively minor amount of Ca2+ (trigger Ca2+). Mitochondria are low-affinity organelles, whose primary role probably is the regulation of Ca2+ in their matrix, rather than in the sarcoplasm.
Animals↗
The calcium signal.
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Adenosine Triphosphate↗
Calcium ATPase in erythrocytes of spontaneously hypertensive rats of the Milan strain.
Calcium (Ca)-dependent ATPase activity was determined in erythrocyte membrane ghosts from normal and spontaneously hypertensive rats of the Milan strain in order to detect changes in enzyme activity which had previously been shown in the spontaneously hypertensive rat strain. Activity was similar in control and hypertensive rats in the absence of calmodulin. In contrast, activity in the presence of saturating amounts of calmodulin was significantly lower in the hypertensive rats. At the Vmax (free Ca2+ concentration 10 mumol/l) the decrease was about 30% (70.1 +/- 8.94 versus 49.1 +/- 4.75 nmol of ATP split/mg of ghost proteins per min; P less than 0.05). The affinity of the ATP-dependent Ca2+ pump for Ca2+ (Km about 1 mumol/l) was not altered in the hypertensive rats. It is possible that deficient Ca ATPase activity sustains an increase of intracellular free Ca in cells of hypertensive rats concomitant with the intracellular sodium (Na) decrease typical of this strain.
Animals↗
The Ca2+-pumping ATPase in skeletal muscle sarcolemma. Calmodulin dependence, regulation by cAMP-dependent phosphorylation, and purification.
A Ca2+-pumping ATPase has been shown to be an integral component of skeletal muscle sarcolemma. The enzyme is stimulated by calmodulin and inhibited by micromolar concentrations of vanadate. The presence of calmodulin results in the transition of the AT-Pase to a high Ca2+ affinity state; removal of calmodulin reverses this effect. The Ca2+-ATPase and the Ca2+ uptake associated with it are also regulated by a specific cAMP-dependent phosphorylation system. The phosphorylation of the membrane enhances the Ca2+-ATPase activity. The effect is reversible. Three sarcolemmal proteins (Mr 35,000, 28,000, and 26,000) were identified as specific substrates of the cAMP-dependent phosphorylation system. The sarcolemmal Ca2+-ATPase was isolated by means of a calmodulin affinity chromatography column as a protein band of Mr of about 140,000, which could be eluted from the column by EDTA. The band possesses Ca2+-ATPase activity and is phosphorylated with [gamma-32P]ATP in a Ca2+-dependent manner. The membrane of the transverse tubules does not contain the calmodulin-sensitive Ca2+-ATPase. It contains only one cAMP-dependent phosphoprotein (Mr 30,000).
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