Biomedical subjects

T Wallimann

Publications and source records attributed to T Wallimann.

At least 55 records · Page 3Linked to original sources

PubMed1998

Structural changes of creatine kinase upon substrate binding.

Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzyme) to 48.9 A (enzyme plus Mg-ATP) and to 48.2 A (enzyme plus TSAC). M-CK showed similar changes from 28.0 A (free enzyme) to 25.6 A (enzyme plus Mg-ATP) and to 25.5 A (enzyme plus TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK also showed a change of the radius of gyration from 21.5 A (free enzyme) to 19.7 A (enzyme plus Mg-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a Mg-nucleotide-induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In CK, however, additional movements have to be involved.

Adenosine Triphosphate↗

PubMed1998

The isoenzyme-diagnostic regions of muscle-type creatine kinase, the M-260 and M-300 box, are not responsible for its binding to the myofibrillar M-band.

Muscle-type creatine kinase is known for its unique interaction with the myofibrillar M-band, but the molecular origin for this structural relationship is not well understood. A systematic sequence comparison between the highly homologous cytosolic isoforms, muscle-type and brain-type creatine kinase, yielded two isoenzyme-specific regions in the muscle-type creatine kinases, the M-260 box (residues 258-270) and the M-300 box (residues 300-315). These particular regions were conspicuous for the specific interaction of this CK isoenzyme, but not of brain-type creatine kinase, with the sarcomeric M-band. In situ diffusion assays with fluorescently labeled native, as well as mutated muscle-type creatine kinase variants, were used to study by laser confocal microscopy their association with the M-band of chemically skinned muscle fibers. Neither a set of charge mutants of the M-260 box and/or the M-300 box nor a hybrid construct of both isoforms with the entire C-terminal region derived from the brain-type isoform showed any significant alteration in the in situ M-band-binding properties when compared to the wild-type form of muscle-type creatine kinase. This indicates that in the intact protein of muscle type creatine kinase, these C-terminal isoenzyme-specific regions are not important for M-band interaction and that the actual M-band interaction domain(s) lay mostly within the N-terminal half of the molecule. The highly conserved motives (M-260 box and M-300 box) may serve an isoenzyme-specific purpose yet to be identified.

Amino Acid Sequence↗

PubMed1998

Mutation of cis-proline 207 in mitochondrial creatine kinase to alanine leads to increased acid stability.

We show that the mutation of an uncharged residue far from the active site to another uncharged residue can have effects on the active site without disturbing the overall structure of the protein. Cis-proline 207 of mitochondrial creatine kinase was mutated to alanine. The mutant showed a decrease in the pH-optimum for ATP synthesis by 1.5 units while the maximum relative activity was lowered to 53% of the wild-type enzyme. In the direction of ATP consumption, the pH optimum was lowered by 1.3 units and the maximum relative activity was 49% of the wild-type enzyme. The enzyme kinetic parameters Km and Kd for the substrates did not change dramatically, indicating a largely unperturbed active site. Small-angle X-ray scattering was used to investigate the structural change concomitant with the mutation, yielding a scattering profile only slightly different from that of the wild-type enzyme. Neither the radius of gyration nor the molecular mass showed any significant differences, leading to the conclusion that quarternary organization and fold of the mutant and the wild-type enzymes were similar. Theoretical analysis suggests the most probable primary source of structural change to be a transition of residue 207 peptide bond torsional angle co from the cis to the trans configuration.

Creatine Kinase↗

PubMed1998

Brain ATP metabolism in hypoxia resistant mice fed guanidinopropionic acid.

Feeding beta-guanidinopropionic acid (GPA), a competitive inhibitor of creatine transport, decreases mortality and increases brain ATP stability in hypoxic mice. To study brain ATP metabolism in GPA-fed animals, respiratory rates were measured in grey matter and white matter slices as well as cerebral hemisphere mitochondria from GPA-fed mice and rats. Creatine kinase and adenylate kinase activities were measured in rat cerebral grey matter and white matter. Respiratory rates and oxidative phosphorylation were the same in GPA-fed mice and control mice. The adenylate kinase activity increased 50% and creatine kinase showed a small decrease in grey matter. In white matter, creatine kinase increased 50% while adenylate kinase decreased. Thus, GPA produces opposite adaptive changes in adenylate kinase and creatine kinase in grey matter and in white matter. These results suggest that the creatine kinase reaction in grey matter acts to regulate cellular ADP and ATP concentrations.

Adenosine Diphosphate↗

PubMed1998

Myofibrillar interaction of cytosolic creatine kinase (CK) isoenzymes: allocation of N-terminal binding epitope in MM-CK and BB-CK.

The molecular origin of the isoenzyme-specific interaction of cytosolic creatine kinase isoenzymes, muscle-type creatine kinase and brain-type creatine kinase, with myofibrillar structures has been studied by confocal microscopy in an functional in situ binding assay with chemically skinned, unfixed skeletal muscle fibers using wild-type and chimeric creatine kinase isoproteins. The specific interaction of both wild-type isoforms with the sarcomeric structure resulted in a stable, isoform-characteristic labeling pattern with muscle-type creatine kinase bound exclusively and tightly to the sarcomeric M-band while brain-type creatine kinase was confined to the I-band region. Chimeric proteins of both muscle-type and brain-type creatine kinases were constructed to localize the corresponding binding domain(s). Exchanged domains included the N-terminal part (residues 1-234), the region containing an isoenzyme 'diagnostic box' (residues 235-285) and the C-terminal part (residues 286-380). The purified recombinant proteins were all fully intact and enzymatically active. All chimeric proteins containing the N-terminal region (amino acid 1-234) of muscle-type or brain-type creatine kinase were always specifically targeted to the sarcomeric M-band or I-band, respectively. We therefore propose that the relevant epitope(s), determining the isoenzyme-specific targeting in skeletal muscle, are entirely located within the N-terminal regions of both cytosolic creatine kinase isoforms.

Animals↗

PubMed1998

Functional aspects of the X-ray structure of mitochondrial creatine kinase: a molecular physiology approach.

Mitochondrial creatine kinase (Mi-CK) is a central enzyme in energy metabolism of tissues with high and fluctuating energy requirements. In this review, recent progress in the functional and structural characterization of Mi-CK is summarized with special emphasis on the solved X-ray structure of chicken Mib-CK octamer (Fritz-Wolf et al., Nature 381, 341-345, 1996). The new results are discussed in a historical context and related to the characteristics of CK isoforms as known from a large number of biophysical and biochemical studies. Finally, two hypothetical functional aspects of the Mi-CK structure are proposed: (i) putative membrane binding motifs at the top and bottom faces of the octamer and (ii) a possible functional role of the central 20 A channel.

Amino Acid Sequence↗

PubMed1998

Oligomeric state and membrane binding behaviour of creatine kinase isoenzymes: implications for cellular function and mitochondrial structure.

The membrane binding properties of cytosolic and mitochondrial creatine kinase isoenzymes are reviewed in this article. Differences between both dimeric and octameric mitochondrial creatine kinase (Mi-CK) attached to membranes and the unbound form are elaborated with respect to possible biological function. The formation of crystalline mitochondrial inclusions under pathological conditions and its possible origin in the membrane attachment capabilities of Mi-CK are discussed. Finally, the implications of these results on mitochondrial energy transduction and structure are presented.

Binding Sites↗

PubMed1998

Creatine supplementation in health and disease. Effects of chronic creatine ingestion in vivo: down-regulation of the expression of creatine transporter isoforms in skeletal muscle.

Interest in creatine (Cr) as a nutritional supplement and ergogenic aid for athletes has surged over recent years. After cellular uptake, Cr is phosphorylated to phosphocreatine (PCr) by the creatine kinase (CK) reaction using ATP. At subcellular sites with high energy requirements, e.g. at the myofibrillar apparatus during muscle contraction, CK catalyzes the transphosphorylation of PCr to ADP to regenerate ATP, thus preventing a depletion of ATP levels. PCr is thus available as an immediate energy source, serving not only as an energy buffer but also as an energy transport vehicle. Ingestion of creatine increases intramuscular Cr, as well as PCr concentrations, and leads to exercise enhancement, especially in sprint performance. Additional benefits of Cr supplementation have also been noticed for high-intensity long-endurance tasks, e.g. shortening of recovery periods after physical exercise. The present article summarizes recent findings on the influence of Cr supplementation on energy metabolism, and introduces the Cr transporter protein (CreaT), responsible for uptake of Cr into cells, as one of the key-players for the multi-faceted regulation of cellular Cr homeostasis. Furthermore, it is suggested that patients with disturbances in Cr metabolism or with different neuro-muscular diseases may benefit from Cr supplementation as an adjuvant therapy to relieve or delay the onset of symptoms. Although it is still unclear how Cr biosynthesis and transport are regulated in health and disease, so far there are no reports of harmful side effects of Cr loading in humans. However, in this study, we report that chronic Cr supplementation in rats down-regulates in vivo the expression of the CreaT. In addition, we describe the presence of CreaT isoforms most likely generated by alternative splicing.

Amino Acid Sequence↗

PubMed1997

The role of creatine kinase in inhibition of mitochondrial permeability transition.

Cyclosporin A sensitive swelling of mitochondria isolated from control mouse livers and from the livers of transgenic mice expressing human ubiquitous mitochondrial creatine kinase occurred in the presence of both 40 microM calcium and 5 microM atractyloside which was accompanied by a 2.5-fold increase over state 4 respiration rates. Creatine and cyclocreatine inhibited the latter only in transgenic liver mitochondria. Protein complexes isolated from detergent solubilised rat brain extracts, containing octameric mitochondrial creatine kinase, porin and the adenine nucleotide translocator, were reconstituted into malate loaded lipid vesicles. Dimerisation of creatine kinase in the complexes and exposure of the reconstituted complexes to >200 microM calcium induced a cyclosporin A sensitive malate release. No malate release occurred with complexes containing octameric creatine kinase under the same conditions.

Animals↗

PubMed1997

Activation of sea-urchin sperm motility is accompanied by an increase in the creatine kinase exchange flux.

The kinetics of the creatine kinase (CK) reaction were studied in suspensions of quiescent and active, intact sea-urchin spermatozoa in artificial seawater, using 31P-NMR magnetization transfer. In inactive sperm, no CK-mediated exchange flux was detected, whereas in activated motile sperm, the forward pseudo-first-order rate constant was 0.13+/-0.04 s-1 at 10 degrees C, corresponding to a steady-state CK flux of 3.1+/-0.5 mM.s-1. Intracellular pH shifted from 6.6+/-0.1 to 7.6+/-0.1 upon activation. The phosphocreatine (PCr)/ATP and PCr/Pi ratios were only marginally reduced in activated sperm, whereas the estimated cytosolic free ADP concentration increased remarkably from 9 microM in quiescent, to 114 microM in activated spermatozoa. The elevation of CK flux upon sperm activation is discussed in the light of the proposition that in sea-urchin spermatozoa, which are fuelled entirely by oxidative phosphorylation, high-energy phosphate transport is mediated by a 'CK/PCr shuttle'.

Adenosine Diphosphate↗

PubMed1997

The active site histidines of creatine kinase. A critical role of His 61 situated on a flexible loop.

A histidine residue with a pKa of 7 has been inferred to act as a general acid-base catalyst for the reaction of creatine kinase (CK), catalyzing the reversible phosphorylation of creatine by ATP. The chicken sarcomeric muscle mitochondrial isoenzyme Mib-CK contains several histidine residues that are conserved throughout the family of creatine kinases. By X-ray crystal structure analysis, three of them (His 61, His 92, and His 186) were recently shown to be located close to the active site of the enzyme. These residues were exchanged against alanine or aspartate by in vitro mutagenesis, and the six mutant proteins were expressed in E. coli and purified. Structural integrity of the mutant proteins was checked by small-angle X-ray scattering. Kinetic analysis showed the mutant His 61 Asp to be completely inactive in the direction of ATP consumption while exhibiting a residual activity of 1.7% of the wild-type (wt) activity in the reverse direction. The respective His to Ala mutant of residue 61 showed approximately 1% wt activity in the forward and 10% wt activity in the reverse reaction. All other mutants showed near wt activities. Changes in the kinetic parameters K(m) or Vmax, as well as a significant loss of synergism in substrate binding, could be observed with all active mutants. These effects were most pronounced for the binding of creatine and phosphocreatine, whereas ATP or ADP binding were less severely affected. Based on our results, we assume that His 92 and His 186 are involved in the binding of creatine and ATP in the active site, whereas His 61 is of importance for the catalytic reaction but does not serve as an acid-base catalyst in the transphosphorylation of creatine and ATP. In addition, our data support the idea that the flexible loop bearing His 61 is able to move towards the active site and to participate in catalysis.

Animals↗

PubMed1997

Metabolic support of Na+ pump in apically permeabilized A6 kidney cell epithelia: role of creatine kinase.

The contribution of ATP-generating systems to Na+ pump (Na+-K+-ATPase) function was studied in Xenopus laevis A6 kidney epithelia apically permeabilized with digitonin. The ouabain-inhibitable Na+ pump current (I(P)) was measured in the presence of otherwise impermeant inhibitors and/or substrates at Na+ and K+ concentrations that allowed near-maximal pump function. Confocal fluorescence microscopy after apical addition of sulfosuccinimidobiotin (molecular weight of 443) showed that all cells were permeabilized. Less than 15% of the endogenous lactate dehydrogenase and creatine kinase (CK) were released into the apical medium. The I(P) was approximately 5 microA/cm2 in the presence of D-glucose. Blocking glycolysis with 2-deoxy-D-glucose or oxidative phosphorylation with antimycin A decreased it by > or = 50%. Exogenously added ATP prevented these decreases fully or partially, respectively. Two CK isoforms were detected, one likely being mitochondrial and the other corresponding to mammalian B isoform of CK. Phosphocreatine partially restored Na+ pump activity during inhibition of either ATP synthesis pathway. In conclusion, the ATP used by Na+ pumps of apically digitonin-permeabilized A6 epithelia is generated to a similar extent by glycolysis and oxidative phosphorylation. The CK system can partially support the ATP supply to the Na+ pumps.

Adenosine Triphosphate↗

PubMed1997

In vivo brain phosphocreatine and ATP regulation in mice fed a creatine analog.

Mitochondrial and cytosolic creatine kinase (CK) isozymes are active in cells with high and variable ATP metabolic rates. beta-Guanidinopropionic acid (GPA), a competitive inhibitor of creatine transport, was used to study the hypothesis that the creatine-CK-phosphocreatine (PCr) system is important in regulating brain ATP metabolism. The CK-catalyzed reaction rate and reactant concentrations were measured in vivo with 31P nuclear magnetic resonance spectroscopy during energy deficit (hypoxia) or high-energy turnover (seizures) states in urethane-anesthetized mice fed GPA, creatine, or standard chow (controls). Brain phosphagen (i.e., cellular energy reserves) or PCr plus phosphorylated GPA (GPAP) concentrations were equal. The phosphagen-to-NTP ratio was lower than in controls. In vivo CK reaction rate decreased fourfold, whereas ex vivo CK activity that was biochemically measured was doubled. During seizures, CK-catalyzed fluxes increased only in GPA-fed mice. Phosphagen increased in GPA-fed mice, whereas PCr decreased in controls. Survival was higher and brain phosphagen and ATP losses were less for hypoxic GPA-fed mice than for controls. In contrast to mice fed GPA, hypoxic survival and CK reactant concentrations during hypoxia and seizures were the same in creatine-fed mice and controls. Thus GPA, GPAP, or adaptive changes in ATP metabolism stabilize brain ATP and enhance survival during hypoxia in mice.

Adenosine Triphosphate↗

PubMed1997

Crystalline mitochondrial inclusion bodies isolated from creatine depleted rat soleus muscle.

Rats were fed a 2% guanidino propionic acid diet for up to 18 weeks to induce cellular creatine depletion by inhibition of creatine uptake by this creatine analogue. Ultrastructural analysis of creatine depleted tissues showed that mitochondrial intermembrane inclusion bodies appeared in all skeletal muscles analysed, after 11 weeks of feeding. Heart had relatively few even after 18 weeks of analogue feeding and none were evident in kidney, brain or liver. These structures were strongly immuno-positive for sarcomeric mitochondrial creatine kinase and upon removal from mitochondria, the inclusion bodies were shown to diffract to a resolution of 2.5 nm. Two-dimensional image analysis and three-dimensional reconstruction revealed arrays of creatine kinase octamers with additional components between the octameric structures. The same mitochondria had a 3-fold higher extractable specific creatine kinase activity than controls. Molecular mass gel filtration of inclusion body containing mitochondrial extracts from analogue fed rat solei revealed mitochondrial creatine kinase eluting as an aggregate of an apparent molecular mass > or = 2,000 kDa. Mitochondrial creatine kinase of control soleus mitochondrial extract eluted as an octamer, with a molecular mass of 340 kDa. Respiration measurements of control solei mitochondria displayed creatine mediated stimulation of oxidative phosphorylation that was absent in analogue-fed rat solei mitochondria. The latter also had 19% and 14% slower rates of state 4 and maximal state 3 respiration, respectively, than control mitochondria. These results indicate that mitochondrial creatine kinase co-crystallises with another component within the inter membrane space of select mitochondria in creatine depleted skeletal muscle, and is inactive in situ.

Adenylate Kinase↗

PubMed1997

Mitochondrial intermembrane inclusion bodies: the common denominator between human mitochondrial myopathies and creatine depletion, due to impairment of cellular energetics.

Mitochondrial inclusion bodies are often described in skeletal muscle of patients suffering diseases termed mitochondrial myopathies. A major component of these structures was discovered as being mitochondrial creatine kinase. Similar creatine kinase enriched inclusion bodies in the mitochondria of creatine depleted adult rat cardiomyocytes have been demonstrated. Structurally similar inclusion bodies are observed in mitochondria of ischemic and creatine depleted rat skeletal muscle. This paper describes the various methods for inducing mitochondrial inclusion bodies in rodent skeletal muscle, and compares their effects on muscle metabolism to the metabolic defects of mitochondrial myopathy muscle. We fed rats with a creatine analogue guanidino propionic acid and checked their solei for mitochondrial inclusion bodies, with the electron microscope. The activity of creatine kinase was analysed by measuring creatine stimulated oxidative phosphorylation in soleus skinned fibres using an oxygen electrode. The guanidino propionic acid-rat soleus mitochondria displayed no creatine stimulation, whereas control soleus did, even though the GPA solei had a five fold increase in creatine kinase protein per mitochondrial protein. The significance of these results in light of their relevance to human mitochondrial myopathies and the importance of altered cell energetics and metabolism in the formation of these crystalline structures are discussed.

Adult↗

PubMed1996

Membrane-binding and lipid vesicle cross-linking kinetics of the mitochondrial creatine kinase octamer.

Mitochondrial creatine kinase (Mi-CK; EC 2.7.3.2) is a positively charged enzyme located between the mitochondrial inner and outer membrane as well as along the cristae membranes. The octameric form of Mi-CK is able to cross-link membranes to form contact sites. The process of Mi-CK membrane binding and Mi-CK-induced cross-linking of model membrane vesicles containing different amounts of cardiolipin (CL) was investigated in vitro. First, the direct binding of octameric Mi-CK to immobilized lipid vesicles containing cardiolipin was monitored by plasmon resonance (BiaCore). The analysis of the pseudo-first-order on- and off-rate constants indicates that there are two binding sites with different affinity for Mi-CK on the membrane. The association equilibrium constants obtained at 25 degrees C were 813.7 (for 100% CL) and 343.6 (for 16% CL), respectively, for the high-affinity binding mode. Second, the Mi-CK-induced vesicle cross-linking kinetics were analyzed by fixed-angle light scattering. Only octameric Mi-CK induced bridged vesicle/protein complexes, whereas dimeric Mi-CK failed to induce vesicle cross-linking. For vesicles containing 100% cardiolipin, the pseudo-first-order association rate constant was 2.55 x 10(-3) s-1, while for membranes containing 16% cardiolipin and 84% PC a constant of 6.25 x 10(-3) s-1 was found. The examined kinetic properties of the system suggest a two-step model for Mi-CK-induced vesicle cross-linking which consists of a fast binding step of the enzyme to the membrane, followed by a remarkably slower cross-linking reaction between Mi-CK-covered vesicles. The data obtained by in vitro biophysical methods agree with earlier experiments done with mitoplasts and isolated mitochondrial membranes and explain the in vivo accumulation of Mi-CK at contact sites between the inner and outer mitochondrial membrane and the formation of Mi-CK-rich intramitochondrial inclusions observed in creatine-depleted animals as well as in patients with mitochondrial cytopathies.

Animals↗

PubMed1996

Differential effects of creatine depletion on the regulation of enzyme activities and on creatine-stimulated mitochondrial respiration in skeletal muscle, heart, and brain.

Guanidinopropionic acid (GPA), an analogue of creatine (Cr), is known to inhibit Cr uptake by cells. The metabolic effects of chronic Cr depletion on brain, heart and soleus muscle of rats were studied. In GPA hearts and soleus muscle, total specific creatine kinase (CK) activity was decreased by approx. 40% compared to controls, whereas in brain this same activity was elevated by a factor of two. Immunoblot analysis of soleus mitochondria from GPA rats showed an approximate 4-fold increase in Mi-CK protein and a concomitant 3-fold increase in adenine nucleotide translocator (ANT) protein, when compared to control. In GPA-fed rats, the specific activities of adenylate kinase (ADK) and succinate dehydrogenase were significantly higher in brain and soleus (2-fold), but heart remained the same. However, hexokinase (HK) decreased by approx. 50% both in heart and soleus, indicating that muscle and brain follow different strategies to compensate the energy deficit caused by creatine depletion. Skinned muscle fibres from Cr-depleted soleus attained approx. only 70% maximum state 3 respiration with 0.1 M ADP in the presence of 10 mM Cr compared to 100% in control fibres. This defect in Cr stimulated respiration was also seen in isolated heart mitochondria, but was normal in those from brain. The observed deficit of Cr-stimulated respiration, the significant accumulation of Mib-CK and ANT, concomitant with the formation of Mib-CK rich intra-mitochondrial inclusions shown by electron microscopy, indicate that Mib-CK function and coupling to oxidative phosphorylation (OXPHOS), is impaired in these abnormal mitochondria. In addition, our results show tissue-specific metabolic compensations to Cr depletion.

Adaptation, Physiological↗

PubMed1996

Structure of mitochondrial creatine kinase.

Creatine kinase (CK, EC 2.7.3.2), an enzyme important for energy metabolism in cells of high and fluctuating energy requirements, catalyses the reversible transfer of a phosphoryl goup from phosphocreatine to ADP. We have solved the structure of the octameric mitochondrial isoform, Mib-CK, which is located in the intermembrane compartment and along the cristae membranes. Mib-CK consumes ATP produced in the mitochondria for the production of phosphocreatine, which is then exported into the cytosol for fast regeneration of ATP by the cytosolic CK isoforms. The octamer has 422 point-group symmetry, and appears as a cube of side length 93 angstrom with a channel 20 angstrom wide extending along the four-fold axis. Positively charged amino acids at the four-fold faces of the octamer possibly interact with negatively charged mitochondrial membranes. Each monomer consists of a small alpha-helical domain and a large domain containing an eight-stranded antiparallel beta-sheet flanked by seven alpha-helices. The conserved residues of the CK family form a compact cluster that covers the active site between the domains.

Amino Acid Sequence↗