Mg-nucleotides induced dissociation of liposome-bound creatine kinase: reversible changes in its secondary structure and in the fluidity of the bilayer

MgADP binding to mitochondrial creatine kinase (mtCK) adsorbed on liposomes was induced by the photorelease of caged ADP. The nucleotide binding produced two types of structural changes. One was related to the well-established release of mtCK from the liposomes. The other corresponded to reversible structural changes induced by nucleotide binding to mtCK as demonstrated here. Infrared spectroscopy data show that the MgADP-induced desorption of mtCK from vesicles led to a slight increase in alpha-helix structures in mtCK at the expense of a small decrease in beta-sheet structures and a concomitant increase in the fluidity of the membranes. The desorption of mtCK induced by MgADP and MgATP was almost complete, as shown by centrifugation and enzymatic activity measurements. The photorelease of MgADP in a reactive medium containing phosphocreatine and mtCK associated with liposomes led to nucleotide binding and to the formation of MgATP and creatine. Addition of phosphocreatine also desorbed mtCK from liposomes, while addition of creatine did not. Interpretation of these results would suggest that ADP, ATP or phosphocreatine induce the release of mtCK from membranes, increase the phospholipid bilayer fluidity, and may also decrease the number of contact sites between inner and outer mitochondrial membranes, thus affecting the activity of other mitochondrial enzymes. It is tempting to propose that membrane mtCK binding regulation by nucleotide and PCr concentrations may serve as a physiological adaptation for energy supply.     

Structural changes of mitochondrial creatine kinase upon binding of ADP, ATP, or Pi, observed by reaction-induced infrared difference spectra

Structural modifications of rabbit heart mitochondrial creatine kinase induced by the binding of its nucleotide substrates and Pi were investigated. Reaction-induced difference spectra (RIDS), resulting from the difference between infrared spectra recorded before and after the photorelease of a caged ligand, allow us to detect very small variations in protein structure. Our results indicated that the protein secondary structure remained relatively stable during nucleotide binding. Indeed, this binding to creatine kinase affected only a few amino acids, and caused small peptide backbone deformations and alterations of the carbonyl side chains of aspartate or glutamate, reflecting modifications within preexisting elements rather than a net change in secondary structure. Nonetheless, MgADP and MgATP RIDS were distinct, whereas the MgPi RIDS presented some similarities with the MgATP one. The difference between MgADP and MgATP RIDS could reflect a distinct configuration of the two metal-nucleotide complexes inducing a different positioning and/or a distinct binding mode to the creatine kinase active site. Comparison of the MgATP and MgPi RIDS suggests that Pi binding took place at the same binding site as the gamma-phosphoryl group of ATP. Thus, the difference between MgADP and MgATP RIDS would mainly be due to the effect of the gamma-P of ATP. The differences observed when comparing the RIDS resulting from the binding of nucleotides to octameric mitochondrial creatine kinase or dimeric cytosolic isoform could reflect the distinct oligomerization states and physicochemical or kinetic properties of the two isoenzymes.     

Mitochondrial creatine kinase binding to phospholipids decreases fluidity of membranes and promotes new lipid-induced beta structures as monitored by red edge excitation shift, laurdan fluorescence, and FTIR

Structural modifications induced by the binding of mitochondrial creatine kinase (mtCK) to saturated and unsaturated phospholipids were monitored by using Laurdan, a membrane probe sensitive to the polarity of the environment. The abrupt change characteristic of a phase transition of lipids alone was attenuated by addition of mtCK. Generalized polarization spectra indicated that mtCK surface binding changed the phospholipid liquid-crystalline state to a more rigid state. Infrared spectra of lipids further strengthened these results: upon mtCK binding, the phospholipid methylene chains had a more rigid conformation than that observed without mtCK at the same temperature. After mtCK binding to vesicles of perdeuterated dimyristoylphosphatidylcholine and nondeuterated dimyristoylphosphatidylglycerol, no lateral phase separation was observed, suggesting that both lipids were rigidified. Moreover, mtCK bound to liposomes exhibited an uncommon red edge excitation shift of 19 nm, while that of the soluble enzyme was only 6 nm. These results indicated that the environment of some mtCK tryptophan residues was motionally restricted. Strong stabilization of the enzyme structure against heat denaturation was observed upon lipid binding. In addition, lipids promoted a new reversible protein-protein or protein-lipid interaction, as evidenced by infrared data showing a slight modification of the beta sheet over alpha helix ratio with formation of a new 1632-cm(-)(1) beta sheet instead of the soluble protein 1636-cm(-)(1) one. Such modifications, inducing a decrease in the fluidity of the mitochondrial membranes, may play a role in vesicle aggregation; they could be implicated in the appearance of contact sites between internal and external mitochondrial membranes.     

Tension transients initiated by photogeneration of MgADP in skinned skeletal muscle fibers

Addition of MgADP to skinned skeletal muscle fibers causes a rise in Ca(2+)-activated isometric tension. Mechanisms underlying this tension increase have been investigated by rapid photogeneration of ADP within skinned single fibers of rabbit psoas muscle. Photolysis of caged ADP (P2-1(2-nitrophenyl)ethyladenosine 5'-diphosphate) resulted in an exponential increase in isometric tension with an apparent rate constant, kADP, of 9.6 +/- 0.3 s-1 (mean +/- SE, n = 28) and an amplitude, PADP, of 4.9 +/- 0.3% Po under standard conditions (0.5 mM photoreleased MgADP, 4 mM MgATP, pH 7.0, pCa 4.5, 0.18 M ionic strength, 15 degrees C). PADP depended upon the concentration of photoreleased MgADP as well as the concentration of MgATP. A plot of 1/PADP vs. 1/[MgADP] at three MgATP concentrations was consistent with competition between MgADP and MgATP for the same site on the crossbridge. The rate of the transient, kADP, also depended upon the concentration of MgADP and MgATP. At both 4 and 1 mM MgATP, kADP was not significantly different after photorelease of 0.1-0.5 mM MgADP, but was reduced by 28-40% when 3.5 mM MgADP was added before photorelease of 0.5 mM MgADP. kADP was accelerated by about twofold when MgATP was varied from 0.5 to 8 mM MgATP. These effects of MgATP and MgADP were not readily accounted for by population of high force-producing states resulting from reversal of the ADP dissociation process. Rather, the results suggest that competition between MgADP and MgATP for crossbridges at the end of the cycle slows detachment leading to accumulation of force-generating crossbridges. Elevation of steady- state Pi concentration from 0.5 to 30 mM caused acceleration of kADP from 10.2 +/- 0.5 to 27.8 +/- 1.8 s-1, indicating that the tension rise involved crossbridge flux through the Pi dissociation step of the cycle.     

Creatine kinase in regulation of heart function and metabolism. II. The effect of phosphocreatine on the rigor tension of EGTA-treated rat myocardial fibers

Bundles of rat cardiac fibers were treated with EGTA to increase the permeability of the sarcolemma to ions and small molecules. In the medium without calcium, the EGTA-treated fibers developed rigor tension dependent on the concentration of MgATP in the bathing solution: half-maximal tension was recorded at 2.5 mM MgATP and maximal tension at 0.1 mM MgATP in the medium. However, in the presence of 15 mM phosphocreatine without added creatine kinase a decrease of MgATP concentration to 0.1 mM did not result in any development of rigor tension. Phosphocreatine prevented rigor tension development in the absence of added MgATP when MgADP was added. In the presence of MgADP, phosphocreatine decreased rigor tension more rapidly and to a higher extent than added MgATP. At 5 mM MgADP, half-maximal rigor tension was observed in the presence of 2 mM phosphocreatine which is close to the Km value for phosphocreatine in the creatine-kinase reaction. These results demonstrate that the intact creatine kinase in the EGTA-treated fibers with increased sarcolemmal permeability is able to ensure rapid replenishment of MgATP in the myofibrillar compartment at the expense of phosphocreatine. The data obtained conform completely to the concept of adenine-nucleotide compartmentation in cardiac cells and of energy channelling by the phosphocreatine-creatine shuttle mechanism.     

Ability of a phosphocreatine-myofibrillar creatine kinase system to prevent the rigor tension of myocardial fibers

In the calcium-free medium the EGTA-treated rat myocardial fibres developed rigor tension dependent on the concentration of MgATP in the bathing solution: half-maximal tension was recorded at 2.5 mM MgATP and the maximal tension at 0.1 mM. However, in the presence of 15 mM phosphocreatine without added creatine kinase a decrease of MgATP concentration to 0.1 mM did not result in any development of rigor tension. In the presence of MgADP phosphocreatine decreased rigor tension more rapidly and to the higher extent than MgATP. At 5 mM MgADP half-maximal rigor tension was observed in the presence of 2 mM phosphocreatine which is close to the km value for phosphocreatine in the creatine kinase reaction. These results demonstrate that the native creatine kinase in the EGTA-treated fibres is able to create high local ATP concentration in the myofibrillar compartment at the expense of phosphocreatine under the conditions of deficiency or even absence of ATP. It appears that at the energy supply disturbances the myocardial contracture develops at least partially due to low activity of the myofibrillar creatine kinase because of phosphocreatine deficiency.     

Role of creatine kinase in force development in chemically skinned rat cardiac muscle

The influence of phosphocreatine in the presence or absence of MgATP and MgADP was studied in Triton X-100-treated thin papillary muscles and ventricular strips of the rat heart. The pCa/tension relationships, the pMgATP/tension relationships, and the tension responses to quick length changes were analyzed. The results show three major consequences of the reduction of the phosphocreatine concentration in the presence of millimolar concentrations of the MgATP. (a) The resting tension and the maximal Ca2+-activated tension were increased, and the pCa/tension relationship was shifted toward higher pCa values and its steepness was decreased; these effects were enhanced by the inclusion of MgADP. (b) The time constant of tension recoveries after quick stretches applied during maximal activation was increased, while the extent of these recoveries was decreased. (c) The study of pMgATP/tension relationships in low Ca concentrations showed that the decrease in phosphocreatine induced a shift toward higher MgATP values with no changes in maximal rigor tension or the slope coefficient; these effects were increased by the increase in MgADP and were independent of the preparation diameter. Thus, modifications of the apparent Ca sensitivity and resting and maximal tension when phosphocreatine is decreased seem to be due to an increasing participation of rigor-like or slowly cycling cross-bridges spending more time in the attached state. These results suggest that endogenous creatine kinase is able to ensure maximal efficiency of myosin ATPase by producing a local high MgATP/MgADP ratio.     

Transition state stabilization by six arginines clustered in the active site of creatine kinase

Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP.nitrate.creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.     

Acyl chain composition determines cardiolipin clustering induced by mitochondrial creatine kinase binding to monolayers

It has been recently shown that mitochondrial creatine kinase (mtCK) organizes mitochondrial model membrane by modulating the state and fluidity of lipids and by promoting the formation of protein-cardiolipin clusters. This report shows, using Brewster angle microscopy, that such clustering is largely dependent on the acyl chain composition of phospholipids. Indeed, mtCK-cardiolipin domains were observed not only with unsaturated cardiolipins, but also with the cardiolipin precursor phosphatidylglycerol. On the other hand, in the case of saturated dimyristoylphosphatidylglycerol and tetramyristoylcardiolipin, mtCK was homogeneously distributed underneath the monolayer. However, an overall decrease in membrane fluidity was indicated by infrared spectroscopy as well as by extrinsic fluorescence spectroscopy using Laurdan as a fluorescent probe, both for tetramyristoylcardiolipin and bovine heart cardiolipin containing liposomes. The binding mechanism implicated the insertion of protein segments into monolayers, as evidenced from alternative current polarography, regardless of the chain unsaturation for the phosphatidylglycerols and cardiolipins tested.     

Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly

Combined mutation of "catalytic carboxylates" in both nucleotide binding domains (NBDs) of P-glycoprotein generates a conformation capable of tight binding of 8-azido-ADP (Sauna, Z. E., Müller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry 41, 13989-14000). Here we characterized this conformation using pure mouse MDR3 P-glycoprotein and natural MgATP and MgADP. Mutants E552A/E1197A, E552Q/E1197Q, E552D/E1197D, and E552K/E1197K had low but real ATPase activity in the order Ala > Gln > Asp > Lys, emphasizing the requirement for Glu stereochemistry. Mutant E552A/E1197A bound MgATP and MgADP (1 mol/mol) with K(d) 9.2 and 92 microm, showed strong temperature sensitivity of MgATP binding and equal dissociation rates for MgATP and MgADP. With MgATP as the added ligand, 80% of bound nucleotide was in the form of ATP. None of these parameters was vanadate-sensitive. The other mutants showed lower stoichiometry of MgATP and MgADP binding, in the order Ala > Gln > Asp > Lys. We conclude that the E552A/E1197A mutation arrests the enzyme in a conformation, likely a stabilized NBD dimer, which occludes nucleotide, shows preferential binding of ATP, does not progress to a normal vanadate-sensitive transition state, but hydrolyzes ATP and releases ADP slowly. Impairment of turnover is primarily due to inability to form the normal transition state rather than to slow ADP release. The Gln, Asp, and Lys mutants are less effective at stabilizing the occluded nucleotide, putative dimeric NBD, conformation. We envisage that in wild-type the occluded nucleotide conformation occurs transiently after MgATP binds to both NBDs with associated dimerization, and before progression to the transition state.     

Spectroscopic and thermodynamic measurements of nucleotide-induced changes in the human 70-kDa heat shock cognate protein

Hsp70 alternates between an ATP-bound state in which the affinity for substrate is low and an ADP-bound state in which the affinity for substrate is high, as a result Hsp70 assists the protein folding process through nucleotide-controlled cycles of substrate binding and release. In this work, we describe the cloning and purification of the human 70-kDa heat shock cognate protein, Hsc70, and the use of circular dichroism, intrinsic emission fluorescence, and isothermal titration calorimetry to characterize conformational changes induced by ADP and ATP binding. Binding of either ADP or ATP were not accompanied by a net change in secondary structure suggesting that the conformational rearrangement caused by nucleotide binding is localized. MgADP or MgATP had a greater effect in the stability at stress temperatures than ADP or ATP did. Isothermal titration calorimetry data pointed out that Hsc70 had a lower affinity for ATP (KD=710 nM) than for ADP (KD=260 nM).     

Studies on the mitochondrially bound form of rat brain creatine kinase

1. The development of the total rat brain creatine kinase was studied in brain homogenates. Until approx. 14-15 days after birth, the activity remains less than one-third that of the adult activity (207+/-6 units/g wet wt. s.d.; n=3). Over the next 10 days the activity increases markedly to the adult value and thereafter remains essentially constant. 2. In the adult brain, approx. 5% (11.9+/-2.2 units/g wet wt. s.d.; n=5) of the total creatine kinase is associated with the mitochondrial fraction. This creatine kinase could not be solubilized by sodium acetate solutions of up to 0.8m concentration, whereas 66% of the hexokinase associated with brain mitochondria was released under these conditions. 3. Rat brain mitochondria incubated in the presence of various concentrations of creatine (1, 5 and 10mm) and ADP (100mum) synthesized phosphocreatine at rates of approx. 4.5, 11 and 17.5nmol/min per mg of mitochondrial protein. Atractyloside (50mum) or oligomycin (1.5mug/mg of mitochondrial protein) completely inhibited the synthesis of phosphocreatine. 4. The apparent K(m) and V(max.) values of the mitochondrially bound rat brain creatine kinase were determined in both directions. The V(max.) in the direction of phosphocreatine synthesis is 237nmol/min per mg of mitochondrial protein, with an apparent K(m) for creatine of 1.67mm and for MgATP(2-) of 0.1mm, and in the reverse direction V(max.) is 489nmol/min per mg of mitochondrial protein, with an apparent K(m) for phosphocreatine of 0.4mm and for MgADP(-) of 27mum. 5. The results are discussed with reference to the role that the mitochondrially bound creatine kinase may play in the development of brain energy metabolism.     

Effect of the epsilon-subunit on nucleotide binding to Escherichia coli F1-ATPase catalytic sites.

The influence of the epsilon-subunit on the nucleotide binding affinities of the three catalytic sites of Escherichia coli F1-ATPase was investigated, using a genetically engineered Trp probe in the adenine-binding subdomain (beta-Trp-331). The interaction between epsilon and F1 was not affected by the mutation. Kd for binding of epsilon to betaY331W mutant F1 was approximately 1 nM, and epsilon inhibited ATPase activity by 90%. The only nucleotide binding affinities that showed significant differences in the epsilon-depleted and epsilon-replete forms of the enzyme were those for MgATP and MgADP at the high-affinity catalytic site 1. Kd1(MgATP) and Kd1(MgADP) were an order of magnitude higher in the absence of epsilon than in its presence. In contrast, the binding affinities for MgATP and MgADP at sites 2 and 3 were similar in the epsilon-depleted and epsilon-replete enzymes, as were the affinities at all three sites for free ATP and ADP. Comparison of MgATP binding and hydrolysis parameters showed that in the presence as well as the absence of epsilon, Km equals Kd3. Thus, in both cases, all three catalytic binding sites have to be occupied to obtain rapid (Vmax) MgATP hydrolysis rates.     

Direct measurement of energy fluxes from mitochondria into cytoplasm in permeabilized cardiac cells <Emphasis Type="Italic">in situ:</Emphasis> some evidence for mitochondrial interactosome

The aim of this study was to measure energy fluxes from mitochondria in isolated permeabilized cardiomyocytes. Respiration of permeabilized cardiomyocytes and mitochondrial membrane potential were measured in presence of MgATP, pyruvate kinase – phosphoenolpyruvate and creatine. ATP and phosphocreatine concentrations in medium surrounding cardiomyocytes were determined. While ATP concentration did not change in time, mitochondria effectively produced phosphocreatine (PCr) with PCr/O₂ ratio equal to 5.68 ± 0.14. Addition of heterodimeric tubulin to isolated mitochondria was found to increase apparent Km for exogenous ADP from 11 ± 2 μM to 330 ± 47 μM, but creatine again decreased it to 23 ± 6 μM. These results show directly that under physiological conditions the major energy carrier from mitochondria into cytoplasm is PCr, produced by mitochondrial creatine kinase (MtCK), which functional coupling to adenine nucleotide translocase is enhanced by selective limitation of permeability of mitochondrial outer membrane within supercomplex ATP Synthasome-MtCK-VDAC-tubulin, Mitochondrial Interactosome.     

Mitochondrial creatine kinase in contact sites: interaction with porin and adenine nucleotide translocase, role in permeability transition and sensitivity to oxidative damage

The creatine/phosphocreatine circuit provides an efficient energy buffering and transport system in a variety of cells with high and fluctuating energy requirements. It connects sites of energy production (mitochondria, glycolysis) with sites of energy consumption (various cellular ATPases). The cellular creatine/phosphocreatine pool is linked to the ATP/ADP pool by the action of different isoforms of creatine kinase located at distinct subcellular compartments. Octameric mitochondrial creatine kinase (MtCK), together with porin and adenine nucleotide translocase, forms a microcompartment at contact sites between inner and outer mitochondrial membranes and facilitates the production and export into the cytosol of phosphocreatine. MtCK is probably in direct protein-protein contact with outer membrane porin, whereas interaction with inner membrane adenine nucleotide translocase is rather mediated by acidic phopholipids (like cardiolipin) present in significant amounts in the inner membrane. Octamer-dimer transitions of MtCK as well as different creatine kinase substrates have a profound influence on controlling mitochondrial permeability transition (MPT). Inactivation by reactive oxygen species of MtCK and destabilization of its octameric structure are factors that contribute to impairment of energy homeostasis and facilitated opening of the MPT pore, which eventually lead to tissue damage during periods of ischemia/reperfusion.     

Creatine kinase in regulation of heart function and metabolism. I. Further evidence for compartmentation of adenine nucleotides in cardiac myofibrillar and sarcolemmal coupled ATPase-creatine kinase systems

In isolated and purified cardiac myofibrillar and sarcolemmal preparations, the route of movement of ADP produced in the Mg2+-ATPase reactions was studied by investigating the efficiency of competition between the endogenous creatine kinase and exogenous pyruvate kinase reactions. In the homogeneous control system composed of hexokinase and glucose as ATPase, soluble creatine kinase rapidly rephosphorylated ADP produced in the presence of 1 mM ATP, but the addition of pyruvate kinase in an increasing amount inhibited the reaction of creatine release from phosphocreatine and symmetrically increased the rate of pyruvate production from phosphoenol pyruvate. At a pyruvate-kinase/creatine-kinase activity ratio (PK/CK) of 50, all ADP was used by the pyruvate kinase. In myofibrillar and sarcolemmal preparations containing particulate creatine kinase, the creatine kinase reaction was much less efficiently suppressed by pyruvate kinase, and at PK/CK = 50 half-maximal release of creatine was still observed. The rate of immediate myofibrillar MgADP rephosphorylation in the endogenous creatine-kinase reaction was observed to be governed by the concentration of phosphocreatine in accordance with the kinetics of this enzyme. The physiological significance of these findings is discussed.     

Nitrogenase IX. Effect of the MgATP generator on the catalytic and EPR properties of the enzyme in vitro.

Nitrogenase(nitrogen:(acceptor) oxidoreduction, EC 1.7.99.2) of Clostridium pasteuranium is very sensitive to the ratio of MgADP/MgATP in dithionite oxidation assays. Variation of concentration of creatine kinase, an ATP-regenerating enzyme, can be used to control the ratio of ADP/ATP and thereby the dithionite oxidation activity of nitrogenase. The in vitro properties of nitrogenase support the suggestion of Haaker (Haaker, H., deKok, A. and Veeger, C. (1974) Biochim. Biophys. Acta 357, 344-357) that in vivo the nucleotide ratio and not the electron supply normally regulates nitrogenase activity. In EPR experiments it has been shown that the "steady state" varies as a function of the concentration of creatine kinase. The spectral differences are interpreted as being a function of the ratio of MgADP/MgATP obtained in the pseudo steady-state condition, which occurs as a result of variation in relative rates of ATP-utilizing and ATP-generating reactions, that is, the relative nitrogenase and creatine kinase activities. Implications of these finding for interpretation of previously reported kinetic and EPR studies are discussed.     

Regulation of respiration in brain mitochondria and synaptosomes: restrictions of ADP diffusion in situ, roles of tubulin, and mitochondrial creatine kinase

The role of ubiquitous mitochondrial creatine kinase (uMtCK) reaction in regulation of mitochondrial respiration was studied in purified preparations of rat brain synaptosomes and mitochondria. In permeabilized synaptosomes, apparent Km for exogenous ADP, Km (ADP), in regulation of respiration in situ was rather high (110 +/- 11 microM) in comparison with isolated brain mitochondria (9 +/- 1 microM). This apparent Km for ADP observed in isolated mitochondria in vitro dramatically increased to 169 +/- 52 microM after their incubation with 1 muM of dimeric tubulin showing that in rat brain, particularly in synaptosomes, mitochondrial outer membrane permeability for ADP, and ATP may be restricted by tubulin binding to voltage dependent anion channel (VDAC). On the other hand, in synaptosomes apparent Km (ADP) decreased to 25 +/- 1 microM in the presence of 20 mM creatine. To fully understand this effect of creatine on kinetics of respiration regulation, complete kinetic analysis of uMtCK reaction in isolated brain mitochondria was carried out. This showed that oxidative phosphorylation specifically altered only the dissociation constants for MgATP, by decreasing that from ternary complex MtCK.Cr.MgATP (K (a)) from 0.13 +/- 0.02 to 0.018 +/- 0.007 mM and that from binary complex MtCK.MgATP (K (ia)) from 1.1 +/- 0.29 mM to 0.17 +/- 0.07 mM. Apparent decrease of dissociation constants for MgATP reflects effective cycling of ATP and ADP between uMtCK and adenine nucleotide translocase (ANT). These results emphasize important role and various pathophysiological implications of the phosphocreatine-creatine kinase system in energy transfer in brain cells, including synaptosomes.     

Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Determining ligand dissociation constants of binary and ternary complexes from the kinetics of enzyme inactivation

Adenosine 5-phosphosulfate (APS) kinase from Penicillium chrysogenum is irreversibly inactivated by trinitrobenzene sulfonate in a pseudo-first order process. Under standard assay conditions kapp was 1.9 X 10(-3) s-1. Saturating MgATP or MgADP decreased Kapp to a limit of 4.1 X 10(-4) s-1. There are several explanations for the partial protection, including the presence of two essential lysyl side chains, only one of which is at the active site. Analysis of the inactivation kinetics by means of linear plots derived for partial protection yielded dissociation constants for E X MgATP (Kia) and E X MgADP (Kiq) of 2.9 mM and 1.8 mM, respectively. Low concentrations of APS alone provided no protection against trinitrobenzene sulfonate inactivation, but in the presence of 1 mM MgADP, as little as 2 microM APS provided additional protection while 100 microM APS reduced kapp to the limit of 4.1 X 10(-4) s-1. The results confirm the formation of a dead end E X MgADP X APS proposed earlier as the cause of the potent substrate inhibition by APS. Linear plots of 1/delta k versus 1/[MgADP] at different fixed [APS] and of 1/delta k versus 1/[APS] at different fixed [MgADP] were characteristic of the ordered binding of MgADP before APS (or the highly synergistic random binding of the two ligands). The true APS dissociation constant of the dead end E X MgADP X APS complex (K'ib) was determined to be 1.9 microM. From the value of K'ib and the previously reported value of KIB (apparent inhibition constant of APS as a substrate inhibitor of the catalytic reaction at saturating MgATP), the ratio of the MgADP and PAPS release rate constants (k4/k3) was calculated to be 11. Inactivation kinetics was used to study the effects of Mg2+ and high salt on ADP and APS binding. The results indicated that free ADP binds to the enzyme more tightly than does MgADP at low ionic strength. High salt decreased free ADP binding, but had little effect on MgADP binding. APS binds more tightly to E X MgADP in the absence or presence of salt than to E X ADP.     

Specific limitations for intracellular diffusion of ADP in cardiomyocytes

Isolated cardiomyocytes and bundles of cardiac fibers were studied after lysis of their sarcolemma by saponin (40-50 micrograms/ml). 60-70% of cardiomyocytes were rod-like and Ca2(+)-tolerant. The kinetics of stimulation of oxidative phosphorylation by ADP and creatine via the mitochondrial creatine kinase reaction: MgATP + creatine----MgADP + phosphocreatine, was investigated after perforation of sarcolemma. The criterion for sarcolemmal perforation was an almost complete (80-100%) leakage of lactate dehydrogenase. It was shown that the Km values for ADP during stimulation of oxidative phosphorylation in cardiomyocytes are 250 +/- 39 microM (264 +/- 57 microM in cardiac bundles) which exceeds by one order of magnitude the Km value for ADP in isolated mitochondria (18 +/- 5 microM). On the contrary, Km for creatine is the same for all preparations studied (6-6.9 mM). The data obtained suggest the absence of diffusion difficulties for creatine inside the cells. In contrast, intracellular diffusion of ADP is restricted, most probably, dye to its binding to intracellular structures. These data emphasize the crucial role of the creatine kinase system in energy transfer processes. In the presence of 25 mM creatine Km for ADP is decreased to 36 +/- 6 mM due to a manyfold use of ADP in the coupled creatine kinase-oxidative phosphorylation reaction occurring in mitochondria.