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T Wallimann

Publications and source records attributed to T Wallimann.

At least 73 records · Page 4Linked to original sources
PubMed1996

The in vitro kinetics of mitochondrial and cytosolic creatine kinase determined by saturation transfer 31P-NMR.

Michaelis- and dissociation constants of sarcomeric mitochondrial creatine kinase (Mi(b)-CK) in solution were determined by enzyme assay and compared to those of cytosolic MM-CK under identical conditions at pH 7.4 and 25 degrees C. Saturation transfer 31P-NMR was used to determine the steady state fluxes mediated by Mi-CK and MM-CK in solution. The NMR detected fluxes of both Mi-CK and MM-CK exhibited, as expected, a linear dependence on Vmax (Vmax range 0-9 mM.s-1). Interestingly, the oligomeric state of Mi-CK, with the Mi-CK octamer/dimer ratio ranging from 2 to 9, did not have a significant effect on the flux/Vmax ratio. Furthermore, the flux/Vmax ratio of Mi-CK was twice as high as that of MM-CK under similar conditions (flux/Vmax for Mi-CK was 0.31 and for MM-CK was 0.15). This difference was primarily due to a 4-fold higher apparent affinity for MgADP of Mi-CK compared to MM-CK (K(m)(MgADP) = 22 +/- 9 microM and 80 +/- 17 microM, resp.). The NMR observed fluxes were in agreement with the fluxes as calculated from the rate equation, using the appropriate metabolite concentrations and the kinetic constants from the spectrophotometric assays. Thus we conclude, that Mi-CK and MM-CK, when in solution, catalyse an exchange-reaction, the flux of which is fully observable by saturation transfer 31P-NMR.

Adenosine Diphosphate↗
PubMed1996

Reconstitution of active octameric mitochondrial creatine kinase from two genetically engineered fragments.

Creatine kinase (CK) has been postulated to consist of two flexibly hinged domains. A previously demonstrated protease-sensitive site in M-CK (Morris & Jackson, 1991) has directed our attempts to dissect mitochondrial CK (Mi-CK) into two protein fragments encompassing amino acids [1-167] and [168-380]. When expressed separately in Escherichia coli, the two fragments yielded large amounts of insoluble inclusion bodies, from which the respective polypeptides could be purified by a simple two-step procedure. In contrast, co-expression of the two fragments yielded a soluble, active, and correctly oligomerizing enzyme. This discontinuous CK showed nearly full specific activity and was virtually indistinguishable from native Mi-CK by far- and near-UV CD. However, the positive cooperativity of substrate binding was abolished, suggesting a role of the covalent domain linkage in the crosstalk between the substrate binding sites for ATP and creatine. The isolated C-terminal fragment refolded into a native-like conformation in vitro, whereas the N-terminal fragment was largely unfolded. Prefolded [168-380] interacted in vitro with [1-167] to form an active enzyme. Kinetic analysis indicated that the fragments associate rapidly and with high affinity (1/K1 = 17 microM) and then isomerize slowly to an active enzyme (k2 = 0.12 min-1; k-2 = 0.03 min-1). Our data suggest that the C-terminal fragment of Mi-CK represents an autonomous folding unit, and that the folding of the C-terminal part might precede the conformational stabilization of the N-terminal moiety in vivo.

Adenosine Triphosphate↗
PubMed1996

'Hot spots' of creatine kinase localization in brain: cerebellum, hippocampus and choroid plexus.

Creatine kinase (CK) isoenzymes, with emphasis on the mitochondrial CK isoenzymes, were characterized and localized in chicken cerebellum. Chicken cerebellum extracts analyzed by two-dimensional gels, using antipeptide antibodies specific for sarcomeric muscle-type mitochondrial CK (Mib-CK) and revealed the presence of a Mib-CK variant in avian cerebellum. This CK isoform was localized by immunofluorescence staining exclusively in the Purkinje neurons. The co-expression of this Mib-CK together with cytosolic muscle-type MM-CK, as observed in the same Purkinje neurons, may reflect the specific energy requirements associated with highly fluctuating Ca2+ levels (Ca2+ spiking) in these specialized neurons. Ubiquitous brain-type mitochondrial Mia-CK was found together with cytosolic BB-CK mainly in the glomeruli structures of the cerebellar granular layer. BB-CK, but much less so Mia-CK however, was also very prominent in Bergmann glial cells of the two mitochondrial Mi-CK isoenzymes in the chicken cerebellum is demonstrated. Other hot spots of CK localization were the granule and pyramidal cells of the hippocampus in rat. There, a developmental stage-dependent immunofluorescence staining, especially with antibodies against Mia-CK was noted. Epithelial cells of the choroid plexus were also highly enriched in CK. The possible implications of a CK/PCr circuit at these various cellular locations of the brain are discussed with respect to normal brain physiology and pathology.

Animals↗
PubMed1996

Identification of two distinctly localized mitochondrial creatine kinase isoenzymes in spermatozoa.

The creatine kinase (CK) isoenzyme system is essential for motility in rooster and sea urchin sperm. In the present study, biochemical characterization as well as immunofluorescence and confocal laser microscopy with highly specific antibodies against various chicken CK isoenzymes revealed that cytosolic brain-type CK isoenzyme (B-CK) is the only CK isoenzyme in rooster seminal plasma, while three isoenzymes, cytosolic B-CK, sarcomeric mitochondrial CK (Mib-CK), and a variant of ubiquitous Mi-CK ('Mia-CK variant'), are found in rooster spermatozoa. These three isoenzymes are localized in different regions of the sperm cell. B-CK and Mib-CK were localized along the entire sperm tail and in the mitochondria-rich midpiece, respectively. The 'Mia-CK variant', on the other hand, was found predominantly at the head-midpiece boundary, in a non-uniform manner in the midpiece itself and, surprisingly, at the distal end of the sperm tail as well as at the acrosome. Several lines of evidence show that the 'Mia-CK variant' shares some characteristics with purified Mia-CK from chicken brain, but also displays distinctive features. This is the first evidence for two different Mi-CK isoenzymes occurring in one cell and, additionally, for the co-expression of Mib-CK and cytosolic brain-type B-CK in the same cell. The relevance of these findings for sperm physiology and energetics is discussed.

Animals↗
PubMed1995

Autophosphorylation of creatine kinase: characterization and identification of a specifically phosphorylated peptide.

We report that several different chicken and rabbit creatine kinase (CK)1 isoenzymes showed an incorporation of 32P when incubated with [gamma-32P]ATP in an autophosphorylation assay. This modification was was shown to be of covalent nature and resulted from an intramolecular phosphorylation reaction that was not dependent on the CK enzymatic activity. By limited proteolysis and sequence analysis of the resulting peptides, the autophosphorylation sites of chicken brain-type CK could be localized within the primary sequence of the enzyme to a 4.5 kDa peptide, spanning a region that is very likely an essential part of the active site of creatine kinase. Homologous peptides were found to be autophosphorylated in chicken muscle-type CK and a mitochondrial CK isoform. Phosphopeptide as well as mutant enzyme analysis provided evidence that threonine-282(2), threonine-289 and serine-285 are involved in the autophosphorylation of CK. Thr-282 and Ser-285 are located close to the reactive cysteine-283. Thr-289 is located within a conserved glycine-rich region highly homologous to the glycine-rich loop of protein kinases, which is known to be important for ATP binding. Thus, it seems likely that the described region constitutes an essential part of the active site of CK.

Amino Acid Sequence↗
PubMed1995

Multiple-state equilibrium unfolding of guanidino kinases.

The denaturant-induced equilibrium unfolding of octameric mitochondrial creatine kinase, dimeric cytosolic muscle-type creatine kinase, and monomeric arginine kinase was investigated. Stable unfolding intermediates for all three enzymes were manifested by a strongly biphasic red shift of intrinsic protein fluorescence upon increasing denaturant concentrations. In the intermediate state, all proteins were monomeric and enzymatically inactive, but still retained a globular shape. Native tertiary structure interactions were largely disrupted, while at least 50% of the secondary structures were conserved, as suggested by near- and far-UV circular dichroism, respectively. A significantly increased surface hydrophobicity of the intermediate conformation, compared to both the native and the fully unfolded states, was observed by the binding of the hydrophobic fluorescent dye ANS. The observed properties agree formally with the definition of the molten globule state, but can be alternatively explained by a sequential unfolding of individual domains, involving a transient exposure of domain interfaces. Very similar unfolding profiles for all three proteins suggest that the formation of stable unfolding intermediates is not a consequence of the specific oligomeric structures of the CKs but rather due to a common, probably two-domain architecture of the guanidino kinase protomers.

Animals↗
PubMed1995

Re-evaluation of the structure and physiological function of guanidino kinases in fruitfly (Drosophila), sea urchin (Psammechinus miliaris) and man.

Purification and biophysical characterization of mitochondrial creatine kinase (Mi-CK) from sperm of the sea urchin Psammechinus miliaris, as well as gel-permeation chromatography of human heart Mi-CK demonstrate that these two Mi-CK isoenzymes form highly symmetrical octameric molecules with an M(r) of approx. 350,000, a value similar to that found for all other Mi-CK isoenzymes investigated so far. The absolute evolutionary conservation of this oligomeric form from sea urchins to mammals points both to its essentiality for Mi-CK function and to an important role of octameric Mi-CK in the energy metabolism of tissues and cells with high and fluctuating energy demands. To investigate whether a similar physiological principle also operates in an even more distantly related animal phylum, the arginine kinase (ArgK) isoenzyme system of Drosophila flight muscle was investigated with two independent subcellular fractionation procedures and subsequent analysis of the fractions by SDS/PAGE, immunoblotting and native isoenzyme electrophoresis. In contrast with a previous report [Munneke and Collier (1988) Biochem. Genet. 26, 131-141], strong evidence against the occurrence of a Mi-ArgK isoenzyme in Drosophila was obtained. The findings of the present study are discussed in the context of CK and ArgK function in general and of structural and bioenergetic differences between vertebrate striated muscles and arthropod flight muscles.

Animals↗
PubMed1995

Functional differences between dimeric and octameric mitochondrial creatine kinase.

Mitochondrial creatine kinase (Mi-CK) consists of octameric and dimeric molecules that are interconvertible. In the present study, the kinetic properties of purified chicken heart Mi-CK (Mib-CK) dimers and octamers were investigated separately under highly controlled conditions. Gel-permeation chromatography was performed before and after kinetic measurements in order to clearly define the proportions of octamers and dimers. 'Dimeric' Mi-CK solutions consisted of > or = 90% dimers throughout the experiment whereas 'octameric' Mi-CK solutions consisted in the beginning of 90% octamers, but upon measuring with the highest concentrations of creatine (Cr) and ATP approximately one-third of the octamers dissociated into dimers. These proper controls enabled us to pinpoint the observed kinetic differences between dimers and octamers solely to the oligomeric state of Mib-CK. Both dimeric and octameric Mi-CK displayed synergism in substrate binding (Kd values are higher than Km values), meaning that binding of the first substrate facilities subsequent binding of the second substrate. Most interestingly, Km(Cr) and Kd(Cr) values are both 2-3 times higher for octameric than for dimeric Mi-CK. Thus, at low Cr concentrations, the dimer is kinetically favoured for the forward direction of the reaction (phosphorylcreatine synthesis) compared with the octamer. The possible physiological significance of the lower Kd(Cr) value of dimeric versus octameric Mib-CK, as well as the apparent negative cooperativity of ATP binding at higher [Cr], are discussed within the context of a possible functional role for dimeric Mib-CK in vivo.

Animals↗
PubMed1995

Dimer-dimer interactions in octameric mitochondrial creatine kinase.

Mitochondrial creatine kinase (Mi-CK) forms octamers and dimers, which are readily interconvertible in vitro. The kinetic and thermodynamic octamer stability of wild-type and two mutant, octamer-destabilized forms of chicken sarcomeric Mi-CK was investigated at varying temperatures, pHs, and salt and substrate concentrations, in order to identify parameters which might regulate the octamer/dimer ratio in vivo and to assess the nature of octamer-stabilizing interactions. For wild-type Mi-CK, the rate of the transition state analogue complex (TSAC)-induced octamer decay increased with increasing temperature up to 28 degrees C; increasing pH markedly accelerated the decay in a biphasic manner. The substrate-dependent decay data suggest that also the productive enzymatic transition state of Mi-CK induces an octamer-destabilizing conformation. Thermodynamically, the octamers are stabilized by a combination of hydrophobic and polar contributions. Van't Hoff analysis showed that hydrophobic interactions dominate both in the absence of substrates and in the TSAC conformation, since the equilibrium octamer fractions increased with increasing temperatures, in spite of the accelerated decay kinetics. For the Mi-CK mutant E4Q, a similar temperature dependence was found; in contrast, mutant W264C exhibited an inverted temperature dependence, suggesting that hydrophobic interactions might be largely abolished in this mutant. Both the kinetic and the thermodynamic data seem to suggest that the octamer-dimer transitions of Mi-CK might not play a major role in a fast regulation of mitochondrial energy metabolism, but could rather be involved in slow long-term modulations.

Adenosine Diphosphate↗
PubMed1995

Equilibration and exchange of fluorescently labeled molecules in skinned skeletal muscle fibers visualized by confocal microscopy.

Confocal laser fluorescence microscopy was used to study in real time under nearly physiological conditions the equilibration and exchange characteristics of several different fluorescently labeled molecules into chemically skinned, unfixed skeletal muscle fibers of rabbit psoas. The time required for equilibration was found to vary widely from a few minutes up to several days. Specific interactions of molecules with myofibrillar structures seem to slow down equilibration significantly. Time for equilibration, therefore, cannot simply be predicted from diffusion parameters in solution. Specific interactions resulted in characteristic labeling patterns for molecules like creatine kinase (muscle type), pyruvate kinase, actin-binding IgG, and others. For the very slowly equilibrating Rh-NEM-S1, changes in affinity upon binding to actin in the absence of calcium and subsequent slow cooperative activation, beginning at the free end of the filament at the H-zone, were observed. In the presence of calcium, however, binding of Rh-NEM-S1 was homogeneous along the whole actin filament from the very beginning of equilibration. The dissociation properties of the dynamic interactions were analyzed using a chase protocol. Even molecules that bind with rather high affinity and that can be removed only by applying extreme experimental conditions like Rh-phalloidine or Rh-troponin could be displaced easily by unlabeled homologous molecules.

Actins↗
PubMed1994

In vitro complex formation between the octamer of mitochondrial creatine kinase and porin.

An interaction of mitochondrial creatine kinase with purified outer mitochondrial porin (voltage-dependent anion channel) was shown by co-sedimentation assays as well as by gel permeation chromatography. Porin formed high M(r) complexes with wild-type mitochondrial creatine kinase as well as with an N-terminal deletion mutant, lacking the first five N-terminal amino acids. The complexes were identified by creatine kinase activity in parallel with immunoblotting using specific antibodies against the two proteins. In addition, porin induced octamerization of the N-terminal creatine kinase mutant, which under the same conditions without porin, did not polymerize but remained more than 90% dimeric. Furthermore, binding of mitochondrial creatine kinase to porin affected the conductance of porin when reconstituted in "black membranes." At 10 mV the pore in the complex adopted a low conductance (1.5-2 nanosiemens) state, compared to the high conductance state (3-4 nanosiemens) of the free incorporated pores. The former state of the pore is known to be cationically selective. Thus, besides a specific structural interaction, a defined physiological function is assumed of the mitochondrial creatine kinase-porin complexes that are discussed here.

Animals↗
PubMed1994

The mitochondrial ATP/ADP carrier: interaction with detergents and purification by a novel procedure.

The interaction of several classes of detergents with mitochondrial ATP/ADP carrier (AAC) was studied. The detergents that were best suited for solubilization of active AAC differed in several physico-chemical properties, but contained relatively rigid or planar hydrophobic (sub)moieties. Based on specific binding of AAC to Blue Sepharose, a novel method for the purification of the AAC was developed. The new method gave AAC samples which were devoid of non-essential lipids and allowed to purify AAC isoenzymes from several species and tissues to a significantly higher degree of purity than that achieved up to now. Western blot analysis of purified AACs with an antiserum against chicken heart AAC confirmed that immunological variability is more important between tissues than between species. In contrast to liver and kidney AACs, brain AAC displayed similar antigenic properties to heart AAC.

Animals↗
PubMed1994

Mitochondrial creatine kinase: a major constituent of pathological inclusions seen in mitochondrial myopathies.

Overaccumulation of abnormally organized mitochondria in so-called "ragged-red" skeletal muscle fibers is a morphological hallmark of mitochondrial myopathies, in particular of mitochondrial encephalomyopathies. Characteristic for the abnormal mitochondria is the occurrence of highly ordered crystalline inclusions. Immuno-electron microscopy revealed that these inclusions react heavily with specific antibodies against mitochondrial creatine kinase (Mi-CK). Image processing of selected crystalline inclusions, sectioned along the crystallographic b, c planes, resulted in an averaged picture displaying an arrangement of regular, square-shaped particles with a central cavity. The overall appearance, dimensions, and symmetry of these building blocks are very reminiscent of single isolated Mi-CK octamers. Taking these findings together, it is concluded that Mi-CK octamers indeed represent the major, if not the only, component of these mitochondrial inclusions.

Creatine Kinase↗
PubMed1994

Creatine kinase isoenzymes in chicken cerebellum: specific localization of brain-type creatine kinase in Bergmann glial cells and muscle-type creatine kinase in Purkinje neurons.

Creatine kinase isoenzymes were localized in the chicken cerebellum by the use of isoenzyme-specific anti-chicken creatine kinase antibodies. Brain-type creatine kinase was found in high amounts in the molecular layer, particularly in Bergmann glial cells but also in other cells of the cerebellar cortex, e.g. in astrocytes and in the glomerular structures, as well as in cells of the deeper nuclei. A mitochondrial creatine kinase isoform was primarily localized to the glomerular structures in the granule cell layer and was also identified in Purkinje neurons. Surprisingly, a small amount of the muscle-type creatine kinase isoform was identified in cerebellar extracts by immunoprecipitation, immunoblotting and native enzyme electrophoresis, and was shown to be localized exclusively in Purkinje neurons. Cell type-specific expression of brain- and muscle-type creatine kinase in Bergmann glial cells and Purkinje neurons, respectively, may serve to adapt cellular ATP regeneration to the different energy requirements in these specialized cell types. The presence of brain-type creatine kinase in Bergmann glial cells and astrocytes is discussed within the context of the energy requirements for ion homeostasis (K+ resorption), as well as for metabolite and neurotransmitter trafficking. In addition, the presence of muscle-type creatine kinase in Purkinje neurons, which also express other muscle-specific proteins, is discussed with respect to the unique calcium metabolism of these neurons and their role in cerebellar motor learning.

Animals↗
PubMed1994

The N-terminal heptapeptide of mitochondrial creatine kinase is important for octamerization.

Mitochondrial creatine kinase (Mi-CK) isoenzymes, in contrast to cytosolic CKs, form octameric molecules composed of four stable dimers. Octamers and dimers are interconvertible. Removal of the N-terminal pentapeptide of chicken cardiac Mi-CK (Mib-CK) by limited proteolysis drastically destabilized the octamer. The role of the charged amino acids within the N-terminal heptapeptide was studied in detail by progressively substituting the four charged residues by uncharged ones. In these altered proteins, the octamer/dimer ratio at equilibrium conditions was shifted toward the dimer. Also, the in vitro dissociation rate of octamers into dimers was increased in correlation to the number of charged residues eliminated. Point mutant E4Q, with only one positive charged amino acid removed, already displayed a 50-fold higher equilibrium constant and a 13-fold increased dissociation rate compared to wild-type Mib-CK. Mutant 4-7, having all four charged residues in the N-terminal heptapeptide substituted, showed a 100-fold higher equilibrium constant and a 146-fold increased dissociation rate. The corresponding values for double mutant E4Q/K5L were intermediate between the single and quadruple mutants. This strongly suggests that the charged amino acids in the N-terminal heptapeptide of Mib-CK, and therefore ionic interactions mediated by the N-terminal moiety, play an important role in forming and stabilizing the octameric molecule. The role of dimer-octamer interconversion in vivo as a possible regulator of contact site formation and of mitochondrial oxidative phosphorylation is discussed.

Amino Acid Sequence↗
PubMed1994

The tryptophan residues of mitochondrial creatine kinase: roles of Trp-223, Trp-206, and Trp-264 in active-site and quaternary structure formation.

The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mib-CK) were individually replaced by phenylalanine or cysteine using site-directed mutagenesis. The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies. In the present work, Trp-223 is identified as an active-site residue whose replacement even by phenylalanine resulted in > or = 96% inactivation of the enzyme. Trp-223 is responsible for a strong (18-21%) fluorescence quenching effect occurring upon formation of a transition state-analogue complex (TSAC;Mib-CK.creatine.MgADP.NO3-), and Trp-223 is probably required for the conformational change leading to the TSAC-induced octamer dissociation of Mib-CK. Replacement of Trp-206 by cysteine led to a destabilization of the active-site structure, solvent exposure of Trp-223, and to the dissociation of the Mib-CK dimers into monomers. However, this dimer dissociation was counteracted by TSAC formation or the presence of ADP alone. Trp-264 is shown to be located at the dimer-dimer interfaces within the Mib-CK octamer, being the origin of another strong (25%) fluorescence quenching effect, which was observed upon the TSAC-induced octamer dissociation. Substitution of Trp-264 by cysteine drastically accelerated the TSAC-induced dissociation and destabilized the octameric structure by one-fourth of the total free interaction energy, probably by weakening hydrophobic contacts. The roles of the other 2 tryptophan residues, Trp-213 and Trp-268, could be less well assigned.

Animals↗